Title of Thesis
Studies on the sexually dimorphic gastrin-releasing
peptide system in the lumbosacral spinal cord that controls
male reproductive function
2016, March
Takumi Oti
Graduate School of
Natural Science and Technology
(Doctor’s Course)
OKAYAMA UNIVERSITY
Contents
General Introduction -------------------------------------------------------------------------------------- 1
Chapter 1. Analysis of the local neuronal circuit mediates the male sexual function using the
HVEM tomography -------------------------------------------------------------------------- 5
Summary ------------------------ 6
Introduction --------------------- 7
Materials and Methods -------- 9
Results ------------------------- 12
Discussion --------------------- 13
Figures ------------------------- 15
Chapter 2. Identification of the spinal gastrin-releasing peptide system projecting into the spinal
nucleus of the bulbocavernosus in male rats -------------------------------------------- 17
Summary ----------------------- 18
Introduction -------------------- 19
Materials and Methods ------- 21
Results -------------------------- 24
Discussion ---------------------- 27
Table and Figures ------------- 32
Chapter 3. The role of androgen receptor in the sexually dimorphic gastrin-releasing peptide
system in the lumbosacral spinal cord during development -------------------------- 40
Summary ----------------------- 41
Introduction -------------------- 42
Materials and Methods ------- 43
Results -------------------------- 46
Discussion ---------------------- 47
Figures -------------------------- 51
Chapter 4. Postnatal development of the gastrin-releasing peptide system in the lumbosacral
spinal cord ----------------------------------------------------------------------------------- 54
Summary ----------------------- 55
Introduction -------------------- 56
Materials and Methods ------- 58
Results -------------------------- 61
Discussion ---------------------- 63
Figures -------------------------- 65
Chapter 5. Perinatal development of the sexually dimorphic gastrin-releasing peptide system in
the lumbosacral spinal cord that mediates male reproductive function ------------- 68
Summary ----------------------- 69
Introduction -------------------- 70
Materials and Methods ------- 72
Results -------------------------- 77
Discussion ---------------------- 80
Figures -------------------------- 84
General Discussion -------------------------------------------------------------------------------------- 91
Acknowledgements -------------------------------------------------------------------------------------- 95
References ------------------------------------------------------------------------------------------------ 96
General Introduction
Neural circuits underlying the male reproductive function are composed of several
nuclei located in the brain and spinal cord (Sakamoto 2012, 2014). Lower spinal cord
injuries frequently cause reproductive dysfunction in men, including erectile
dysfunction and ejaculation disorder (Brown et al. 2006; Sipski 1998). This indicates
that important neural centers for male reproductive function are located within the lower
spinal cord (Sakamoto 2012, 2014). However, the molecular and neuroanatomical
mechanisms controlling male reproductive function at the lower spinal cord level
remain unclear.
It is previously demonstrated that neurons that express gastrin-releasing
peptide (GRP neurons) in the lumbosacral spinal cord mediate the function of spinal
centers that promotes penile reflexes during the copulatory behavior of male rats
(Sakamoto 2014; Sakamoto et al. 2008; Sakamoto and Oti 2015) (Fig. 1). These GRP
neurons project axons into the more caudal lumbosacral spinal cord (L5–S1 level),
including the sacral parasympathetic nucleus (SPN) and somatic motor neurons in the
spinal nucleus of the bulbocavernosus (SNB) (Sakamoto 2012, 2014; Sakamoto et al.
2010; Sakamoto and Kawata 2009; Sakamoto et al. 2008; Sakamoto et al. 2009a;
Sakamoto et al. 2009b) (Fig. 1). Co-localization of GRP and neuronal nitric oxide
synthase (nNOS), a marker for autonomic ganglionic neurons (Vizzard et al. 1995a) in
this region of the spinal cord in male rats showed that GRP-containing fibers densely
surround SPN neurons, expressing nNOS (Sakamoto et al. 2008). SPN neurons have
been implicated in the control of ejaculation, also showing to express high levels of
nNOS (Studeny and Vizzard 2005). This system of GRP neurons and projections to the
SPN is sexually dimorphic, being prominent in male rats, but vestigial or absent in
females (Sakamoto and Kawata 2009; Sakamoto et al. 2008). In addition, the SNB is a
cluster of motor neurons located in the fifth and sixth lumbar and first sacral segments
of the spinal cord (L5-S1 level) (Sakamoto 2014; Breedlove and Arnold 1980; Forger
and Breedlove 1986; Sengelaub and Forger 2008). These motor neurons and their target
muscles, the bulbocavernosus and levator ani, which are attached to the base of the
penis, play an important role in male copulatory behavior (Forger and Breedlove 1986;
Sengelaub and Forger 2008; Breedlove and Arnold 1983a, b). Sakamoto et al. (2008)
also found evidence for the presence of a GRP receptor (GRPR) in the lumbar and
1
sacral spinal cord of rats based on specific binding of GRP. A higher GRPR expression
level in nNOS-positive neurons of the lumbar and sacral autonomic SPN was evident
from reverse transcription-PCR, Western blotting and immunohistochemistry in rats
(Sakamoto et al. 2008; Sakamoto et al. 2009b). Remarkably, pharmacological
stimulation of GRPR restores penile reflexes and ejaculation rate in castrated male rats,
and antagonistic blockage to this spinal region attenuates penile reflexes and ejaculation
rate in normal male rats (Sakamoto et al. 2008). Thus, the lumbar spinal cord locally
contains several neural circuits that are important in regulating male sexual behaviors in
rodents (Matsuda et al. 2008), although the chemical neuroanatomy involved in these
intra-spinal mechanisms remains elusive.
In Chapter 1, using a high-voltage electron microscopy (HVEM), we describe
the chemical 3-D neuroanatomy of the rat lower spinal cord revealed by double-labeling
(GRP and nNOS) immunohistochemistry at the ultrastructural level (Oti et al. 2012).
Subsequently, in Chapter 2, the synaptic contacts from GRP neurons to the SNB
neurons were studied using a viral trans-synaptic retrograde tracing technique
(Dobberfuhl et al. 2014).
In adulthood, the number of GRP neurons in the upper lumbar spinal region
(L3–L4 level) is greater in males than that in females (Sakamoto et al. 2008; Sakamoto
et al. 2009b). The GRP-expressing neurons in the lumbar spinal cord of male rats also
express androgen receptor (AR) but do not express estrogen receptor alpha (Sakamoto
and Kawata 2009; Sakamoto et al. 2008). Androgens play a significant role in
ejaculation in male rats (Hull and Dominguez 2007) and humans (Meston and Frohlich
2000); therefore, the presence of AR in GRP-expressing neurons in the ejaculation
center offers a locus for androgenic modulation of ejaculation and other sexual reflexes.
To determine if ARs direct control sexual dimorphism of these neurons,
a genetically
male (XY) rats carrying the testicular feminization mutation (Tfm) of the Ar gene was
used (Sakamoto et al. 2008; Sakamoto et al. 2009b). The Tfm mutation se rats
embryologically develop testes and secrete testosterone prenatally; however, because
they express dysfunctional AR protein, these rats develop a wholly feminine exterior
phenotype, including a clitoris and defective vagina, rather than a penis and prostate
(Bardin et al. 1971; Yarbrough et al. 1990; Zuloaga et al. 2008b). The spinal cord of
genetically male rats carrying the Tfm allele is hyperfeminine and exhibits even fewer
GRP-positive neurons than does the spinal cord of wild-type females. The spinal cord of
2
Tfm males showed fewer GRP-positive neurons than that of wild-type males, as was
expected (Sakamoto et al. 2008; Sakamoto et al. 2009b). In Chapter 3, therefore, to
elucidate whether central AR is important for the sex differentiation of the spinal GRP
system, a conditional mouse line selectively lacking the AR gene in the nervous system
was used (Sakamoto et al. 2014). In addition, while Sakamoto et al. (2008) previously
demonstrated the male-dominant sexual dimorphism of the spinal GRP system, which
plays an important role in the male reproductive function in adult rats, little is known
about the developmental changes in this spinal center during ontogeny. Subsequently, in
Chapter 4, this system at several stages from pre-puberty to post-puberty (sexual
maturation) was investigated to assess the effects of the escalated circulation sex
steroids during puberty, and was compared the development of the spinal GRP system
between sexes (Katayama et al. 2016).
Early in life, androgens do play a key role in the sexual differentiation of the
central nervous system through a mechanism that is mediated by signaling through the
nuclear ARs (Breedlove and Arnold 1983a, b; Matsuda et al. 2008; Morris et al. 2004).
In males, a considerable amount of androgens secreted transiently from the testes
represents the “androgen surge” that masculinizes the developing brain as well as the
external and internal genitalia (Matsuda et al. 2008; Morris et al. 2004; Phoenix et al.
1959). Sex differences caused by the androgen surge are restricted to a limited period;
so-called the “critical period” (Morris et al. 2004; Phoenix et al. 1959). For example, a
critical period in rats occurs 18–27 days after fertilization when masculinization of the
brain and spinal cord typically depends on transiently higher levels of circulating
androgens (Matsuda et al. 2008; Morris et al. 2004). However, little is known about the
effects of the androgen surge during perinatal development of this sexually dimorphic
spinal GRP system. Finally, in Chapter 5, to study the mechanisms underlying the
development of the sexually dimorphic spinal GRP system in rats, the effects of
administering the anti-androgen flutamide to neonatal male rats were examined (Oti et
al. 2016). Further, newborn male rats were castrated to prevent the production of
endogenous androgens. In addition, androgen treatments to neonatal female rats were
performed to evaluate their effect on the development of the male-specific spinal GRP
system during a critical period (Oti et al. 2016).
3
Figure 1
Schematic drawing summarizing the gastrin-releasing peptide (GRP) system in the lumbosacral spinal
cord that controls male sexual function. A sexually dimorphic spinal cord system of GRP-containing
neurons in the upper lumbar spinal cord “ejaculation center” projects axons to both the autonomic (sacral
parasympathetic nucleus; SPN) and somatic (spinal nucleus of the bulbocavernosus; SNB) centers in the
lower lumbosacral spinal cord, which mediate penile reflexes and trigger ejaculateion. The figure was
reproduced from Sakamoto and Oti (2015).
4
Chapter 1
Analysis of the local neuronal circuit mediates the male sexual function using the
HVEM tomography
5
Summary
Three-dimensional (3-D) analysis of anatomical ultrastructures is important in
biological research. However, 3-D image analysis on exact serial sets of ultra-thin
sections from biological specimens is very difficult to achieve, and limited information
can be obtained by 3-D reconstruction from these sections due to the small area that can
be reconstructed. On the other hand, the high penetration power of electrons by an
ultra-high accelerating voltage enables thick sections of biological specimens to be
examined. High-voltage electron microscopy (HVEM) is particularly useful for 3-D
analysis of the central nervous system because considerably thick sections can be
observed at the ultrastructure level. Here, HVEM tomography assisted by light
microscopy was applied to a study of the 3-D chemical neuroanatomy of the rat lower
spinal cord annotated by double-labeling immunohistochemistry. This powerful
methodology is useful for studying molecular and/or chemical neuroanatomy at the 3-D
ultrastructural level.
6
Introduction
Three-dimensional (3-D) analysis of anatomical ultrastructures is tremendously
important in most fields of biological research. However, it is technically hard to carry
out a 3-D image analysis of exact serial sets of ultra-thin sections from biological
specimens. Confocal laser scanning microscopy allows imaging and quantification of
the 3-D organization of such specimens at present. However, the detailed morphology
of cells remains obscure due to the insufficient spatial resolution of light microscopy
including fluorescent visualization methods (Belichenko and Dahlstrom 1995;
Miyawaki 2003; Nishi and Kawata 2006). Additionally, unstained (non-fluorescing)
domains cannot be recognized in fluorescent histochemistry (Belichenko and Dahlstrom
1995; Miyawaki 2003). In contrast, conventional transmission electron microscopy
(TEM) provides extremely detailed and fine structural information, although the images
obtained are mostly 2-D due to the physical properties of this technique (usage of
ultra-thin sections: ~100 nm in thickness) (Hama and Kosaka 1981). Consequently, the
TEM images cannot use to analyze the 3-D structures of cells. On the other hand, the
high penetration power of electrons at an ultra-high accelerating voltage enables thick
sections of biological specimens to be examined. This property of high-voltage electron
microscopy (HVEM) is particularly useful to analyze the 3-D structure of the central
nervous system because considerably thick sections (semi-thin sections: ~5 µm in
thickness) can be observed at the ultrastructure level (Hama and Kosaka 1981).
It is reported that the gastrin-releasing peptide (GRP) system mediates spinal
centers promoting male copulatory reflexes in rats (Sakamoto et al. 2008). Remarkably,
pharmacological stimulation of GRP receptors restores penile reflexes and ejaculation
rate in castrated male rats, and antagonistic blockage to this spinal region attenuates
penile reflexes and ejaculation rate in normal male rats (Sakamoto 2012; Sakamoto et al.
2008). Thus, this system of neurons in the upper lumbar spinal cord uses this specific
new peptide (GRP) to drive lower spinal autonomic centers that coordinate male
reproductive functions such as erection and ejaculation (Sakamoto 2012; Sakamoto et al.
2008). Locally, the lumbar spinal cord contains several neural circuits that are important
in regulating male sexual behaviors in rodents (Matsuda et al. 2008), although the
chemical neuroanatomy involved in these intra-spinal mechanisms remains elusive.
Using an HVEM, the present study describe the chemical 3-D neuroanatomy of the rat
7
lower spinal cord revealed by double-labeling immunohistochemistry at the
ultrastructural level.
8
Materials and Methods
Animals
Sprague-Dawley rats (Shimizu Laboratory Supplies Co., Ltd., Kyoto, Japan) were used
in this study. All rats were maintained on a 12-h light/dark cycle (lights on at 06:00 and
off at 18:00), and provided with unlimited access to water and rodent chow. All
experimental procedures have been authorized by the Committee for Animal Research,
Okayama University and Kyoto Prefectural University of Medicine, Japan.
Double immunofluorescence for GRP and neuronal nitric oxide synthase (nNOS)
Three male and three female rats were overdosed with sodium pentobarbital (100 mg/kg
body weight), and perfusion fixed with 4% paraformaldehyde in 0.1 M phosphate buffer
(pH 7.4). Spinal cords were immediately removed and immersed in the same fresh
fixative for 3 h. After immersion in 25% sucrose in 0.1 M phosphate buffer for 48 h at 4
°C for cryoprotection, lumbar spinal cords were quickly frozen using powdered dry ice
and cut into 30-µm thick cross sections on a cryostat (CM3050 S, Leica, Nussloch,
Germany). The double immunofluorescence analysis according to the established
methods was performed (Sakamoto et al. 2008). To determine the projection site of
GRP-immunoreactive (ir) axons, double-immunofluorescence staining of GRP
(anti-GRP rabbit polyclonal antibody; 1:5,000 dilution) (Phoenix Pharmaceuticals,
Burlingame, CA, USA) and nNOS (anti-nNOS mouse monoclonal antibody, A-11;
1:5,000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), a marker protein
for neurons in the sacral parasympathetic nucleus (SPN) (Studeny and Vizzard, 2005),
was performed. Alexa Fluor 546-linked anti-mouse IgG raised in goats (Molecular
Probes, Eugene, OR, USA) and Alexa Fluor 488-linked anti-rabbit IgG raised in goats
(Molecular Probes) were used for detection at a 1:1,000 dilution. Immunostained
sections were imaged with a confocal laser scanning microscopy (Fluoview 1000,
Olympus, Tokyo, Japan). The magnification and numeric aperture of the objective lens
were: 10×, 20×, 40×, and 60×.
Double-labeling immunohistochemistry of GRP and nNOS using HVEM
Three male rats were overdosed with sodium pentobarbital and perfusion fixed with 4%
paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer. Lumbar spinal
9
cords were immediately removed and immersed in the same fresh fixative for 3 h.
Spinal sections (L5–L6 and S1 level; 30 µm in thickness) were prepared with a
microslicer (Dosaka EM, Kyoto, Japan). The free-floating sections were placed in 0.1
M phosphate buffer containing 25% sucrose and 10% glycerol for 1 h for cryoprotection
and freeze-thawed three times using liquid nitrogen to enhance the penetration of
antibodies. The sections were thoroughly washed with phosphate-buffered saline (PBS;
pH 7.4) and preincubated in PBS containing 0.05% Triton X-100, 1% normal goat
serum and 1% BSA for 2 h at room temperature to block nonspecific reactions. Sections
were then incubated in a mixture of rabbit anti-GRP and mouse anti-nNOS antibodies
(1:5,000 dilution in each) for 5 days at 4 °C. After being washed with PBS, the sections
were treated with a mixture of 1.4-nm gold particle-conjugated anti-rabbit IgG raised in
goats (Nanoprobes, Stony Brook, NY, USA) at a dilution of 1:200 and biotinylated
anti-mouse IgG raised in goats at a dilution of 1:1,000 for 3 h at room temperature.
After being washed with PBS, the sections were post-fixed for 20 min with 1%
glutaraldehyde in 0.1 M phosphate buffer at 4 °C, again washed thoroughly in PBS and
distilled water, and then silver-developed in the dark with an HQ Silver Kit
(Nanoprobes). After the sections were washed with distilled water and PBS, nNOS-ir
was developed by the avidin-biotin complex method and diaminobenzidine (DAB)
reaction as described previously (Sakamoto et al., 2008). After being washed with PBS,
the sections were placed in 1% OsO4 in 0.1 M phosphate buffer for 30 min, dehydrated,
and flat-embedded in epoxy resin as described previously (Sakamoto et al. 2010).
Semi-thin sections (1 µm in thickness) containing nNOS-ir neurons contacting the
GRP-ir fibers in the SPN were collected on nickel grids coated with collodion film.
Each specimen was first selected using a light microscope (Olympus; BH-2) for
reference purposes. Selected grids were electron-stained with uranyl acetate and lead
citrate, and viewed under an HVEM (Hitachi H-1250M; National Institute for
Physiological Sciences, Okazaki, Japan) at an accelerating voltage of 1,000 kV.
Tomography
The specimen was tilted from -59.0° to +59.0°, and the images were captured at tilt
steps (37 images per view; 0.0, ±4.0, ±8.0, ±12.0, ±15.9, ±19.8, ±23.5, ±27.2, ±30.8,
±34.2, ±37.5, ±40.7, ±43.8, ±46.7, ±49.4, ±52.0, ±54.5, ±56.8, ±59.0) which were
10
!
calculated by the formula: Δθ = d/L · cosθ∆θ = ! cosθ (d: resolution, L: thickness of
sample, θ: tilt step) according to Saxton et al. (1984). The images were digitized and
3-D models were constructed using Chimera and IMOD software (Kremer et al., 1996;
Pettersen et al., 2004).
11
Results
The projection of GRP-ir fibers to the SPN in the lower spinal cord (L5–L6 and S1
level; containing axon terminals of the GRP neurons) of male rats was first examined
because their SPN provides autonomic preganglionic fibers to genitalia (Morgan et al.,
1981). Double immunofluorescence of GRP and nNOS clearly showed that GRP-ir
fibers densely projected into the SPN (Fig. 1a–d). These GRP-ir fibers surrounding cell
bodies and dendrites of the SPN neurons were observed in only males (n = 3) (Fig. 1e)
but vestigial or absent in females (n = 3) (data not shown).
Furthermore, by means of HVEM, the dual immunoelectron microscopic
technique was applied for double labeling of the chemical neuroanatomy. A total of 37
images per view were obtained by tilting the specimen from -59.0° to +59.0°
continuously. Figure 2 presents 18 representative HVEM serial tilting images (extracted
only plus-directed rotations) as well as an origin (untilted) image. 3-D reconstruction
was performed from these 37 serial tilting images. The DAB reaction products were
visualized as fine fuzzy material that was distributed homogeneously in the dendrites of
nNOS neurons. DAB labeling was clearly distinguished from that of GRP labeling with
the nanogold/silver enhancement method (Fig. 3a and b). Nanogold/silver-developed
reaction products were specifically localized around a single dendrite distributed in the
SPN with higher electron density (observed in the axons or terminals of GRP neurons)
than the DAB reaction products (Fig. 3a). Asterisks in Fig. 3b indicate the representative
parts of the terminals of GRP neurons. The 3-D analysis via a polarizing lens sterically
revealed that some populations of GRP-ir terminals formed axonal terminals with the
dendrites of SPN neurons (Fig. 3a and b). The 3-D reconstruction from HVEM images
using dual immune-labeling described many projections of GRP neurons forming
axonal contacts with the dendrites of SPN neurons (Fig. 3c) (for more details, see
Supplementary Movie 1).
12
Discussion
By means of a double immunofluorescence for GRP and nNOS, GRP-containing inputs
formed numerous close appositions (possible sites of synaptic contacts) with dendrites
of SPN neurons. Here, we applied HVEM tomography assisted by light microscopy to a
study of the 3-D chemical neuroanatomy of the rat lower spinal cord annotated by
double-labeling immunohistochemistry. HVEM tomography from a single semi-thin
section revealed that the terminals of GRP neurons formed 3-D axonal projections with
the dendrites of SPN neurons. These structures might be possible sites of synaptic
contacts. However, a definite synaptic specialization could not be fully demonstrated by
this approach. On the other hand, two-photon laser scanning microscopy is an excellent
fluorescence nanoscopy technique, which is characterized by lower phototoxicity and
higher penetration depth—approximately 1 mm deeper than the conventional confocal
microscopy (Denk and Svoboda 1997). Stimulated emission depletion microscopy
(STED) is a newly developed fluorescence microscopy technique that uses the
non-linear de-excitation of fluorescent dyes to overcome the resolution limitation
imposed by diffraction with standard confocal laser scanning microscopes and
conventional far-field optical microscopes (Hell 2007). Accordingly, STED is
considered a form of super-resolution fluorescent microscopy. For nanoscale
examination, fluorescence nanoscopy is an excellent methodology, and is indispensable
with fluorescing domains. Recent techniques using fluorescent nanoscopy can visualize
cell physiology in living tissue using real-time imaging techniques, but cannot be
applied to non-fluorescing domains. TEM provides an alternative that allows extremely
detailed and fine structural information under high-vacuum and processing by chemical
fixation or rapid freezing. In the present study, the resulting 3-D reconstruction of a
SPN dendrite with contacting terminals by HVEM links the information from confocal
laser scanning microscopy and the 3-D reconstruction from TEM ultrathin serial
sections. Thus, the present histochemical 3-D methodology using HVEM could bridge
fluorescence nanoscopy and conventional TEM analyses. To our knowledge, this is the
first demonstration of 3-D chemical neuroanatomy revealed by HVEM using a dual
immunoelectron microscopic technique. Taken together, these results suggest that
GRP-ir afferents to SPN neurons may control copulatory reflexes via these synapses,
since the activation of parasympathetic system is involved in penile erection (Giuliano
13
and Rampin 2000). 3-D tomography by HVEM could examine multiple 3-D axonal
projections of GRP neurons to nNOS-ir dendrites in a wide yet discrete area using a
single semi-thin section (1 µm thickness). Sakamoto et al. (2008) previously reported
that conventional TEM immunohistochemistry using a set of ultra-thin serial sections
(60 nm thickness) also revealed a single synaptic input in spite of the broad distribution
of both GRP-ir fibers and SPN neuron dendrites in the SPN. By a combination of first
visualizing semi-thin sections by light microscopy and then analyzing the same sections
using HVEM, it is concluded that HVEM is superior in detecting multiple synaptic
formations than conventional TEM.
In conclusion, our new 3-D methodology that links HVEM to light microscopy is a
useful and powerful tool for studying molecular and/or chemical neuroanatomy at the
3-D ultrastructure level. The methodology used in this study can be applied in multiple
ways. Therefore, this is a critical contribution to 3-D ultrastructural investigations of the
central nervous system in the present post-genomic age.
14
Figure 1
Double immunofluorescence for GRP [(a) and (c, e; green)] and nNOS [(b) and (c, e; magenta)] shows
that GRP-containing axons surrounding the autonomic SPN neurons are prominent in male rats. (d) The
schematic drawing indicates the localization of SPN in the lumbar and sacral spinal cord (L5–L6 and S1
level). The blocked area in (c) is enlarged in (e). Arrowheads indicate possible synaptic contacts (e).
Images were captured with 40× (objective lens), 1.0× (optical zoom) (a-c) and 60× (objective lens), 2.5×
(optical zoom) (e). Scale bar = 50 µm (c) and 10 µm (e). The figure was reproduced from Oti et al.
(2012).
Figure 2
Eighteen representative HVEM serial tilting images (extracted only plus-directed rotations) as well as an
origin (untilted) image. These HVEM images were obtained by tilting the specimen from 0.0° to +59.0°.
Scale bar = 2 µm. The figure was reproduced from Oti et al. (2012).
15
Figure 3
3-D chemical neuroanatomy using the dual immunoelectron microscopic technique. Stereo-paired HVEM
images obtained by tilting the specimen stage ±8° revealed the 3-D axonal projections (asterisks) into a
single SPN dendrite (magenta) (a and b). The 3-D reconstruction of HVEM images using a dual
immunoelectron microscopic technique for GRP (green) and nNOS (magenta) (c). Scale bar = 2 µm. The
figure was reproduced from Oti et al. (2012).
16
Chapter 2
Identification of the spinal gastrin-releasing peptide system projecting into the
spinal nucleus of the bulbocavernosus in male rats
17
Summary
The pelvic striated muscles play an important role in mediating erections and
ejaculation, and together these muscles compose a tightly coordinated neuromuscular
system that is androgen sensitive and sexually dimorphic. To identify spinal and brains
neurons involved in the control of the levator ani (LA) and bulbocavernosus (BC)
muscles in the male adult and preadolescent rat. Rats were anesthetized, and the
trans-synaptic retrograde tracer pseudorabies virus (PRV) was injected into the LA
muscle of adults or the ventral BC muscle in 30-day-old rats. After 3–5 days rats were
sacrificed, and PRV-labeled neurons in the spinal cords and brains were identified using
immunohistochemistry. The presence of gastrin-releasing peptide (GRP) in the lumbar
spinal neurons was examined. The location and number of PRV-labeled neurons in the
spinal cord and brain and GRP colocalization in the lumbar spinal cord. PRV-labeled
spinal interneurons were observed distributed throughout T11-S1 of the spinal cord,
subsequent to dorsal medial motor neuron infection. The majority of spinal interneurons
were found in the lumbosacral spinal cord in the region of the dorsal gray commissure
and parasympathetic preganglionic neurons. Preadolescent rats had more PRV-labeled
spinal interneurons at L5-S1 where the motor neurons were located but relatively less
spread rostrally in the spinal cord compared with adults. Lumbar spinothalmic neurons
in medial gray of L3-L4 co-localized PRV and GRP. In the brain consistent labeling
was seen in areas known to be involved in male sexual behavior including the
ventrolateral medulla, hypothalamic paraventricular nucleus, and medial preoptic area.
Common spinal and brain pathways project to the LA and BS muscles in the rat
suggesting that these muscles act together to coordinate male sexual reflexes.
Differences may exist in the amount of synaptic connections/neuronal pathways in
adolescents compared with adults.
18
Introduction
In humans, the puborectalis muscle forms a complete sling around the rectum, and
along with the pubococcygeus and iliococcygeus muscles, these three muscles form the
human pelvic diaphragm, collectively known as the levator ani (LA). In humans, the
levator complex plays a primary role in continence (Thor and de Groat 2010; Wallner et
al. 2009). In the male rat, the LA muscle grossly resembles the human puborectalis
muscle, but only partially surrounds the rectum, and attaches to the ventral bulb of the
bulbocavernosus (BC) muscle, which is attached to the proximal penile bulb (Holmes et
al. 1991; Poortmans and Wyndaele 1998). The LA muscle in the rodent has been
denominated by some investigators the dorsal BC muscle because of its anatomical
location, motoneuron innervation (Breedlove and Arnold 1980; Holmes et al. 1991;
Holmes and Sachs 1994), and as a sexually dimorphic muscle, is virtually absent in the
adult female rat (Bremer et al. 2003; Pacheco et al. 1989; Poortmans and Wyndaele
1998).
Ejaculation is comprised of emission and expulsion. Emission is mediated
through the hypogastric nerve innervated by sympathetic preganglionic neurons in the
intermediolateral and medial gray and the pelvic nerve that carries signals from
parasympathetic preganglionic neurons residing in the sacral parasympathetic nucleus
(Giuliano 2011; Nadelhaft and Booth 1984). Contractions of the ventral BC and
ischiocavernosus muscles augment erection of the glans penis (Hart and
Melese-D'Hospital 1983; Sachs 1983). Expulsion of seminal fluids is mediated by the
coordinated contraction of the ventral BC and ischiocavernosus muscles (Gerstenberg et
al. 1990; McKenna et al. 1991; Schmidt and Schmidt 1993). There is less evidence for a
role of the LA in ejaculation. One study by Sachs and Holmes provided evidence that
contractions of the LA muscle act in coordination with the ventral BC muscle to
augment penile erection and that both LA and BC muscle activity is tightly coordinated
during copulation (Holmes and Sachs 1994).
The BC muscle complex (including the LA muscle) is a commonly measured
end point in reproductive toxicology studies (McIntyre et al. 2001; Owens et al. 2006).
Like penile reflexes, the size of these muscles is sensitive to developmental
anti-androgens and testosterone exposure and likewise the pudendal motor neurons
express a sexually dimorphic distribution of androgen receptors (Fishman and
19
Breedlove 1992; Johansen et al. 2007; Zuloaga et al. 2007). The motor neurons
innervating the LA and BC muscles are located in the ventral horn of the lumbosacral
cord within Onuf’s nucleus (Roppolo et al. 1985; Schroder 1981, 1985; Thor et al.
1989). In the rat, Onuf’s nucleus is divided into a spinal nucleus of bulbocavernosus
(SNB), which innervates the ventral and distal lobes of the BC muscle, the LA muscle
and the anal sphincter, and the dorsolateral nucleus (DLN), which innervates the
ischiocavernosus muscle and external urethral sphincter (Breedlove and Arnold 1980;
Collins et al. 1991; McKenna and Nadelhaft 1986; Peshori et al. 1995). These muscles
are primarily innervated by the pudendal motor nerve and the sacral spinal nerves that
originate in the lumbosacral region of the spinal cord (Bremer et al. 2003; McKenna and
Nadelhaft 1986; Pierce et al. 2005; Thor and de Groat 2010).
The SNB motor neurons innervating the LA muscle have been studied in
rodents (Guan et al. 2007; Ranson et al. 2007; Ranson et al. 2005). However, the spinal
and brain neurons projecting to the LA muscle have not been closely examined.
Furthermore, the CNS pathways innervating the perineal muscles in preadolescent male
pups have not been reported. This study examines the spinal and brain neurons that
innervate the LA muscle (i.e., the dorsal BC muscle lobe) in adult male rats and ventral
BC muscle in preadolescent male rats using retrograde neuronal tracing techniques. The
usefulness and specificity of trans-neuronal tracers for the identification of CNS
pathways has been previously published (Card et al. 1993; Card et al. 1990). The
advantage of using viruses as neuronal tracers is their ability to replicate in the neurons
and thus amplify the visualization signal in addition to labeling first, second, and third
order neurons in a connecting neuronal pathway.
20
Materials and Methods
Animals
All methods were performed in strict compliance with the Institutional Animal Care and
Use Committee at the University of North Carolina at Chapel Hill. Rats (adult males n =
16, Sprague-Dawley, 7–10 weeks of age, 300–400 g, Charles River Labs, Raleigh NC;
or male pups, 30-days old n = 4, Wistar-Kyoto, Charles River Labs) were deeply
anesthetized with ketamine/xylazine (50:4 mg/kg intramuscular). The perineal muscles
were accessed via a ventral surgical approach. A midline incision was made in the skin
overlying the scrotum to expose the perineal muscle complex (Figure 1). Connective
tissue overlying the muscles was removed and the BC and LA muscle lobes were
separated gently using saline soaked cotton tip applicators. Pseudorabies virus (PRV)
(Bartha’s K strain, 2–3 °ø 107 plaque-forming units, a gift from Lynn Enquist,
Princeton NJ) was injected (5–10 µL) using a Hamilton syringe into the left side of the
LA muscle in adults or into the left ventral BC muscle (2 µL) in preadolescent pups.
During each injection, the needle remained in situ for ∼1-minute and when removed
pressure was applied to the injection site using a cotton tip applicator to prevent leakage
of the virus.
At the end of survival period, (3–5 days for adults, 3 days for the pups) rats
were deeply anesthetized with urethane (1.25 g/kg i.p.) and perfused trans-cardially
with phosphate-buffered saline (PBS) followed with 4% paraformaldehyde. In the adult
3 days survival group (n = 4) very little PRV labeling in the spinal cord was observed,
therefore the data presented was focused on 4 and 5 days survival times. Brains and
spinal cords were collected and transferred to a 30% sucrose solution until sectioned.
The dorsal spinal roots were identified and coronal sections of spinal cords (T10-S3)
and brains were cut (40 µm) on a freezing microtome. A series of one in six sections (i.e.
every ∼0.24 mm) was processed for immunohistochemical staining.
Immunohistochemistry
Free-floating sections were incubated in rabbit anti-PRV (spinal cord 1:2,000, brain
1:1,000, Affinity Bioreagents) for 15–20 hours and then washed and incubated in
biotinylated goat anti-rabbit immunoglobulin (IgG, 1:500,Vector Laboratories,
Burlingame, CA) for 1 hour. The sections were then incubated in avidin-biotin complex
21
and then in DAB (3′3-diaminobenzidine tetra hydrochloride) for 10 minutes. After
washing, the sections were mounted onto Fisher Plus glass slides (Fisher Scientific, El
Paso TX), dehydrated, coverslipped, and examined under brightfield illumination.
In adult animals with a 4 day survival time (n = 6), a series of sections from
the spinal cord was also processed using double immunofluorescence [34]. After
blocking nonspecific binding components with 1% normal goat serum and 1% BSA in
PBS (pH 7.4) containing 0.3% Triton X-100, the sections were incubated with the
cocktail of primary rabbit antiserum against gastrin-releasing peptide (GRP) (anti-GRP
rabbit polyclonal antibody; 1:5,000 dilution, Phoenix Pharmaceuticals, Burlingame CA)
and the primary mouse antiserum against PRV (anti-PRV mouse monoclonal antibody,
3G9F3; 1:5,000 dilution; VMRD, Pullman WA). After rinsing with PBS, sections were
incubated with Alexa Fluor 488-linked anti-rabbit IgG raised in goat and Alexa Fluor
546-linked anti-mouse IgG raised in goat (Molecular Probes, Grand Island NY, 1:1,000
dilution). For double labeling of PRV and cholinergic neurons, sections were blocked
with donkey serum and then incubated in a cocktail of goat anti-choline acetyl
transferase (ChAT, 1:1,000; Chemicon/Millipore Billerica MA) and rabbit anti-PRV
(spinal cord 1:2,000, Affinity Bioreagents, Golden CO). After rinsing sections were
incubated in Alexa Fluor 488- linked anti-goat IgG raised in donkey and CY3 linked
anti-rabbit IgG raised in donkey (1:500, Jackson Immuno Research Labs, West Grove
PA). Immunostained sections were imaged with a confocal laser scanning microscopy
(FluoView 1000, Olympus, Japan).
Data Analysis
Cells were counted as PRV positive if a reasonable portion of the cell body was easily
visible and stained in the section. The number of PRV-labeled cells in T11-S2 of the
spinal cord were counted for each rat in the 4 days survival group for adults (n = 6) and
the 3 days survival for pups (n = 4). The location of the cells within each spinal segment
were classified into five regions: dorsal horn (laminae I, II and III); lateral gray (lateral
region of laminae VI, VII, including the intermediolateral cell column); intermediate
gray (laminae V, VI, VII); medial gray (lamina X, intermediomedial nucleus and medial
regions of laminae IV, V); and ventral horn (laminae VIII, IX) (Molander et al. 1984).
Figure 2 illustrates the regions examined. Labeled cells were counted on both sides of
the spinal cord and the left and right sides were added together. The average number of
22
cells counted/ animal is presented. The brain regions containing PRV-labeled cells were
examined. The number of cells were counted in a 1 in 6 series through the brain, (n = 4
for each group). For each brain region the average number of cells/animal was
calculated. The relative distribution is shown in Table 1. 0 = no cells; + = 1–9 cells; ++
10–25 cells; +++ 26–49 cells; ++++50–99; +++++ >101 cells.
23
Results
Innervation of the LA Muscle in Adult Male Rats
Spinal Cord
Injections of PRV into the LA muscle resulted in PRV retrogradely labeled neurons in
the SNB of L5-S1, primarily in the ipsilateral L5-L6 (Figure 3A). Bilateral labeling of
SNB and labeling of DLM motor neurons was seen after longer survival times. The
SNB motor neuron labeling after LA injections was similar to that previously reported
using more conventional tracing techniques (Ranson et al. 2007; Ranson et al. 2005).
PRV-labeled spinal interneurons were consistently observed after 4 and 5
days survival. The average number of cells found in each segment and the location of
PRV-labeled cells in T11-S2 were counted in the 4 day survival group. The majority
(∼50% of the total counted) of labeled neurons were found in L5-S1. Around 33% of
the total PRV-labeled cells were found in T11-L2 (Figure 4) with ∼13% of the total
PRV-labeled cells located in L3-L4. Many PRV cells were located in the medial region
of L4-S1in lamina X and the dorsal gray commissure (Figures 4, 5A and B).
PRV-labeled cells were also found in the lateral gray of L5-S1in the region including
the parasympathetic preganglionic neurons (Figures 4 and 5B). In addition,
PRV-labeled neurons were found in the intermediate gray of L4-L6 and T11-L1 and in
the lateral gray of T11-L1 (Figure 4). Many PRV-labeled neurons in the lateral gray of
L5-S1 and T13-L1 contained ChAT (Figure 6A), suggesting they were the sympathetic
and parasympathetic preganglionic neurons.
The presence of GRP in the PRV positive neurons of the medial region of
L3-L4 was examined in sections from adult rats. This region contains the lumbar
spinothalamic (LSt) cells that have been proposed to be part of the spinal pattern
generator for ejaculation (Allard et al. 2005). Both PRV and GRP neurons were found
in the medial region of L3-L4 after LA injections (Figure 7). Figure 7D indicates a
schematic drawing of the upper lumbar spinal cord (L3–L4 level) showing the location
of GRP cell bodies and the region in which the numbers of GRP and PRV cells were
quantified. Around half the PRV neurons in this region were found to contain GRP
(50.1 °æ 10.5%; n = 5), other neurons in this region express either GRP or PRV only
(Figure 7).
24
Brain
After 4 days survival PRV-labeled neurons were found primarily in the brain stem in
the ventrolateral medulla, parapyramidal (PP) region of the medulla and A5
noradrenergic cell group (Figure 8A and Table 1). In addition, PRV-labeled cells were
found in the lateral paragigantocellularis (LPGi), raphe obscurus, raphe pallidus, raphe
magnus, locus coeruleus/subcoeruleus, and Barrington’s nucleus (Bar). In the forebrain
PRV-labeled cells were found in the lateral hypothalamus and paraventricular nucleus
(PVN). After 5 days survival more PRV-labeled neurons were found in these areas with
additional brain regions labeled. These additional brain regions included the central gray
where PRV-labeled cells were primarily in the ventrolateral region, ventral tegmental
area at the level of the substantia nigra, anterior hypothalamus, medial preoptic area
(MPOA), lateral preoptic area and bed nucleus of the stria terminals (BNST) (Table 1
and Figure 8B–D).
Innervation of the BC Muscle in Adolescent Male Rats
Spinal Cord
PRV labeling of SNB motor neurons in the adolescent pup spinal cord was similar to
adults that received LA muscle injections (Figure 3B) and adults receiving ventral BC
muscle injections [37]. Bilateral labeling of SNB motor neurons and PRV-labeled
DLN motor neurons was observed in all 4 rats.
PRV-labeled spinal interneurons were consistently found after injection of
PRV into the ventral BC (Figures 5 and 9). PRV-labeled cells were mainly found in
L5-S1 (62%), with the majority of spinal interneurons distributed in the medial,
intermediate and lateral gray of L5-S1including the dorsal gray commissure and
parasympathetic preganglionic cell region (Figure 9). A proportion (25%) of the total
PRV-labeled cells was located in T11-L2 with labeled neurons in the lateral gray of
T11-L1 (Figure 5C) including the sympathetic preganglionic cell area. Very few (6% of
the total counted) PRV-labeled cells were found in L3-L4 spinal cord (Figures 5D and
9).
Almost twice as many PRV-labeled spinal interneurons were labeled in the
pups compared to the 4 day survival time in adults, which may be due in part to the
survival times examined, the injection site and the size differences. As expected, both
adults and pups had the highest relative percentage distribution of cells in L5-S1 (50%
25
adults; 62% pups), where the pudendal motor neurons are located as well as the
parasympathetic preganglionic neurons. In addition, PRV-labeled cells were found in
the spinal cord segments (T13-L2) containing the sympathetic preganglionic neurons
(19% adults, 15% pups). However, adults had a higher percentage of PRV-labeled cells
in L3-L4 compared to the pups (13% adults, 6% pups). In previous studies examining
PRV pathways after ventral BC muscle injections in adults, labeled neurons were found
more highly distributed in rostral segments including T13-L2 and L3-L4 compared to
the present distribution in preadolescent rats (Marson and McKenna 1996; Xu et al.
2006).
Brain
PRV-labeled neurons were found primarily in the brain stem in the ventrolateral
medulla, A5 noradrenergic cell group and Bar (Table 1). In addition, PRV-labeled cells
were found in the reticular formation, PP region of the medulla, LPGi, raphe obscurus,
raphe magnus, locus coeruleus/subcoeruleus, and the PVN. Fewer PRV-labeled cells
were found in the raphe pallidus, central gray, ventral tegmental area, anterior and
lateral hypothalamus and MPOA. No evidence of PRV cell labeling was found in the
lateral preoptic area and BNST (Table 1). The brain regions labeled in pups were
similar to that labeled after LA muscle injections in adults (Table 1).
A comparison between the brain labeling in adults and adolescents is difficult
to assess because of the differences in the size of the spinal cord and brain pathways,
and the time taken to transport the virus. However, the observation that pups had about
twice as many PRV-labeled spinal interneurons compared with the 4 day survival in
adults might suggest that the brain labeling in the pups should reflect the 5 days survival
in adults rather than the 4 days survival time. Fewer PRV-labeled neurons were
observed in the brains of the pups compared to the 5 days survival time. This suggests
that many of the spinal to brain pathways and brain nuclei to brain nuclei pathways are
not fully developed or synaptically connected in the pups.
26
Discussion
The present study demonstrates that PRV injections into the LA muscle labels efferent
spinal and brain pathways that were virtually identical to pathways that have been
reported to innervate the ventral BC muscle in adult male rats (Marson and Carson 3rd
1999; Marson and McKenna 1996; Xu et al. 2006). In addition, feasibility of PRV
labeling in preadolescent pups was verified. These studies also demonstrate that GRP
containing neurons in the medial region of L3-L4 (LSt neurons) are part of the spinal
interneuron network that innervates the BC muscle complex.
This study confirms previous reports that SNB motor neurons innervate the
LA muscle (also termed the dorsal BC muscle) in the male rat (McKenna and Nadelhaft
1986; Ranson et al. 2005; Schroder 1980). In addition, the CNS pathways innervating
the ventral BC muscle (Marson and Carson 3rd 1999; Marson and McKenna 1996; Xu
et al. 2006) and the LA muscle (present study) in adult male rats were similar. In adults
very few SNB neurons were found with a 3 day survival time, while many SNB motor
neurons and spinal interneurons were labeled after 4 and 5 days survival time. In
preadolescent pups SNB motor neurons were consistently labeled with a 3 day survival
time. Contralateral labeling of SNB motor neurons in the pups and with longer survival
times in the adults may occur as a result of synaptic coupling of the ipsilateral and
contralateral SNB neurons (Collins et al. 1991). At 28 days of age the dendritic
arborization of the SNB is twice the length of normal adults, the dendrites then
gradually shrink and by 49 days of age they are the same length of adults (Goldstein et
al. 1990; Sengelaub and Forger 2008). At 28 days of age the dendritic projections
spread medially towards and dorsal to the central canal, as well as to the intermediate
and lateral gray. In addition, the SNB motor neurons project to the ventral horn
(Sengelaub and Forger 2008). This arborization pattern matches the pattern of
PRV-labeled spinal neurons (in the lateral, intermediate and medial gray and ventral
horn) observed in the pups in the present study. In adults the primary dendrites project
towards the medial gray and ventral horn, with sparse innervation of the intermediate
and lateral gray (Sengelaub and Forger 2008). This pattern was also mimicked in the
adults in the present study. In this study, more than twice as many labeled spinal
interneurons in the lateral, intermediate and medial gray of L5-S1 were observed in 30
day old preadolescent pups, compared to the 4 days survival time in adults; this
27
observation may reflect the increased arborization of the SNB neurons. Interestingly,
sexual behavior (intromission, ejaculations, and penile reflexes) in male rats has been
shown to appear around day 49 which matches the neuroanatomical changes seen in the
SNB motor neurons (Sachs and Meisel 1979; Sodersten et al. 1977).
Several studies have documented a group of neurons in the lumbar spinal cord
that exhibit sexual dimorphism in rats; this population of neurons expresses galanin,
cholecystokinin, enkephalin and NK1 receptors (Ju et al. 1987; Newton 1993; Phan and
Newton 1999; Truitt et al. 2003; Xu et al. 2005). These neurons are located within the
third and fourth lumbar segments of the spinal cord in the medial gray (lamina X and
medial lamina VII) and project to the thalamic region of the brain (Ju et al. 1987; Truitt
et al. 2003). These so-called lumbar spinothalamic (LSt) neurons are sexually dimorphic,
with male rats possessing a larger number than females (Ju et al. 1987; Truitt et al.
2003). In the immunohistochemical investigation of GRP, a collection of neurons
expressing GRP in the medial area of the lumbar spinal cord (L3-L4 level) with a clear
male-dominant sexual dimorphism were observed (Sakamoto et al. 2008). These GRP
expressing neurons project to the more caudal segments of the lumbosacral spinal cord
(L5-S1 levels), including the sacral parasympathetic nucleus and SNB, which are both
known for their role in erectile and ejaculatory function (Sakamoto et al. 2010;
Sakamoto et al. 2008). In addition to the parasympathetic nucleus and the SNB, the LSt
neurons project to the sympathetic neurons of the intermediolateral column (IML) in the
thoracic spinal cord, and play a critical role in the emission phase of ejaculation (Coolen
2005).
It has recently been reported that GRPR blockage by the specific antagonist
RC-3095 attenuated bursts of the BC muscles, in response to the dorsal penile nerve
stimuli (Kozyrev et al. 2012). On the other hand, Sakamoto et al. (Sakamoto et al. 2010)
reported the existence of GRP containing afferents to the SNB motoneurons innervating
the ventral BC muscles using high-voltage electron microscopy. It is also previously
reported that the terminals of GRP neurons may form axonal projections with the
dendrites of parasympathetic preganglionic neurons (SPN) (Oti et al. 2012). In the
present study, about a half of the PRV-labeled neurons in the medial gray at the L3-L4
level expressed GRP, after PRV injections into the LA muscle. Interestingly, very few
PRV-labeled spinal interneurons were found in the medial region of L3-L4 in the
preadolescent pups. Taken together these results suggest the existence of direct or
28
indirect synaptic inputs from GRP neurons to SNB motor neurons and autonomic SPN
neurons. GRPR have also been reported in the SPN and SNB of the lumbosacral spinal
cord (Sakamoto et al. 2008).
C-fos positive neurons were found in the L3-L4 medial gray (lamina X and
medial lamina VII) dorsal, lateral and ventro-lateral to the central canal after ejaculation
and. Immunohistochemical staining with galanin demonstrated that 78–80% of the
galanin immuno-positive ejaculatory reflexes in the region that overlaps with the
location of the PRV-labeled neurons in the present study (Marson and Gravitt 2004;
Truitt et al. 2003) neurons were activated with ejaculation and ejaculatory reflexes,
these galanin neurons were primarily located lateral, and ventrolateral to the central
canal (Marson and Gravitt 2004; Truitt et al. 2003). GRP positive neurons in the medial
lumbar spinal cord are more numerous compared to galanin neurons (Sakamoto 2011;
Truitt et al. 2003). The PRV/GRP labeled neurons found in the present study were
located dorsal to that previously reported of the galanin positive neurons. It is possible
that there are 2 or more populations of spinal GRP neurons. One is the smaller soma
population located dorsomedial to the central canal, and the other is the larger soma
population located primarily lateral and ventrolateral to the central canal (Sakamoto et
al. 2008). The smaller galanin negative cells stain for GRP and not galanin, dorsal to the
central canal (Sakamoto et al. 2008), while the larger cells lateral to the central canal
and in the medial part of lamina VII stain for both GRP and galanin (Coolen 2005). In
this study, GRP/PRV positive neurons were primarily found in dorsolateral to the
central canal. These results suggest that the GRP/PRV cells are, at least partly, galanin
negative cells. However, verification of the neurotransmitter content of the LSt neurons
should be examined using molecular biological and gene analysis techniques to verify
the neurotransmitter content since immunohistochemical staining can vary depending
on the antibody sensitivity.
The brain stem regions labeled with PRV after 4 days survival after LA
muscle injections included the ventrolateral medulla, PP region, the LPGi, and the
caudal raphe. Pudendal motoneurons are innervated by serotonergic projections that
arise from these brain regions (Hermann et al. 1998; Holstege and Kuypers 1987;
Johnson 2006; Johnson et al. 2011; Marson and McKenna 1992; Monaghan and
Breedlove 1991; Murphy et al. 1996; Sakamoto et al. 2008). Serotonergic neurons in the
LPGi exert a tonic descending inhibition of ejaculatory-like reflexes, ex copula reflexes
29
and male sexual behavior (Holmes et al. 2002; Johnson 2006; Johnson et al. 2011;
Marson et al. 1992; Marson and McKenna 1990; Yells et al. 1992). Genital sensitive
neurons that respond to pain stimuli are present in the caudal raphe, including the raphe
magnus, and ventrolateral medulla (Hornby and Rose 1976). In addition, electrical
stimulation of the raphe obscurus reduces anorectal activity (Holmes et al. 1997).
Therefore, neuroanatomical and physiological evidence suggests that these brain stem
nuclei play an important role in regulating muscle responses, and possibly nociceptive
changes that occur with sexual function.
Pudendal motoneurons are also densely innervated by noradrenergic fibers
(Kojima et al. 1985; Schroder and Skagerberg 1985). The adrenergic projection most
likely originates in the A5 cell group and the locus coeruleus (Loewy et al. 1986;
Nygren and Olson 1977), which were consistently labeled in the present study. The
noradrenergic input to lumbar spinal motoneuronsmodulates contractility of lower
urinary tract and anal sphincter and may regulate pain and blood flow (Bajic and
Proudfit 1999; Fritschy and Grzanna 1990; Janig and McLachlan 1987; Loewy et al.
1986). Both of these areas were consistently labeled in studies examining the
descending inputs to the pelvic organs.
Consistent labeling was seen in Barrington’s nucleus. This area projects
directly to the parasympathetic preganglionic nucleus of the lumbosacral spinal cord,
the dorsal gray commissure and the pudendal motoneurons (Cano et al. 2000; Ding et al.
1997; Sasaki 2005). Barrington’s nucleus is consistently labeled after injection of PRV
into a variety of pelvic organs (Marson 1997; Marson and Carson 3rd 1999; Marson et
al. 1993; Orr and Marson 1998; Vizzard et al. 1995b). The role of Barrington’s nucleus
in sexual function is still unknown. However, a recent study suggests that it may be
involved in penile erection (Salas et al. 2008). Barrington’s nucleus has also been
shown to be involved in defecation and straining response (Fukuda and Fukai 1986;
Fukuda et al. 1981; Moda et al. 1993; Pavcovich et al. 1998). Therefore Barrington’s
nucleus may be involved in modulating various aspects of pelvic function.
Other regions labeled in this study that project directly to the SNB and
sympathetic
and
parasympathetic
preganglionic
neurons
include
the
lateral
hypothalamus and PVN (Hosoya et al. 1991; Wagner and Clemens 1991). Spinally
projecting PVN neurons have been shown to play a role in androgen dependent steroid
mechanisms (Wagner et al. 1993) and PVN oxytocin mechanism regulate penile
30
erection (Argiolas and Melis 2013).
Longer survival times results in additional brain areas labeled with PRV via
subsequent labeling through passage of the virus from the spinal interneurons and brain
areas that project directly to the spinal cord. These areas include the central gray,
ventral tegmental area, MPOA and BNST; these regions also have a role in modulating
in sexual function (Clement et al. 2012).
In preadolescent pups most of the PRV-labeled cells were found in the
ventrolateral medulla (PP, LPGi and reticular formation), raphe obscurus, raphe magnus,
A5, Barrington’s nucleus and the PVN. The clear synaptic connections of these brain
regions to the BC muscle spinal circuitry in preadolescent rats suggests that these
pathways have an important functional role in excretory processes, sexual function and
nociception that are established early in development. Other brain regions labeled in the
preadolescent pups included the central gray which relays descending informationon
pain, defensive behavior and sexual behavior and the lateral hypothalamus which is
important for food intake. The labeling of the VTA and MPOA pathway suggests the
importance of parenting behavior, motivation and reward in early life that is important
for the establishment of normal sexual behavior.
In summary both the LA and ventral BC muscles are innervated by a common
set of spinal and brain neurons and thus the LA muscle in the male rat may be more
aptly termed the dorsal BC muscle as previously suggested (Holmes et al. 1991; Peshori
et al. 1995; Poortmans and Wyndaele 1998). The motoneurons innervating the dorsal
and ventral BC are intermingled in the SNB of the ventral horn and are innervated by
the pudendal nerve and function to augment penile reflexes and ejaculation (Holmes et
al. 1991; Holmes and Sachs 1994). The present study further demonstrates that spinal
interneurons in the LSt that contain GRP project to the BC muscle complex, further
confirming a role for the LA in male sexual behavior.
31
Table 1
Summary of the distribution of PRV-labeled cells in the brain
The relative distribution is shown. 0 = no cells; + = 1–9 cells; ++ 10–25 cells; +++ 26–49 cells; ++++50–
99; +++++ >101 cells. A5 = A5 noradrenergic cell group; AH = anterior hypothalamus; Bar =
Barrington’s nucleus; BNST = bed nucleus of the stria terminalis; BC = bulbocavernosus; CG = central
gray; Gi/RF = gigantocellular reticular formation of the brain stem; LA = levator ani; LC/SubC = locus
coeruleus/subcoeruleus; LH = lateral hypothalamus; LPGi = lateral paragigantocellularis: LPO = lateral
preoptic area; MPOA = medial preoptic area; PVN = paraventricular nucleus of the hypothalamus; RM =
raphe magnus RO = raphe obscures; PP = parapyramidal region; RP = raphe pallidus; VLM =
ventrolateral medulla; VTA = ventral tegmental area. The table was reproduced from Dobberfuhl et al.
(2014).
32
Figure 1
Diagrammatic representation of a ventral view of the various lobes of the perineal muscles of the adult
male rat. Illustrated by Hannah Darrah. dBC = distal lobe of the bulbocavernosus muscle; IC =
ischiocavernosus muscle; LA = levator ani muscle; vBC = ventral lobe of the bulbocavernosus muscle.
The figure was reproduced from Dobberfuhl et al. (2014).
Figure 2
A schematic diagram of a coronal section of the L6 rat spinal cord. Left side shows location of laminae
I—X [35], and right shows the subdivisions used to count PRV-labeled cells. DH = dorsal horn; L =
lateral gray; I = intermediate gray; M = medial gray; VH = ventral horn. The figure was reproduced from
Dobberfuhl et al. (2014).
33
Figure 3
Coronal sections showing (A) location of SNB motoneurons after PRV injections into the LA muscle in
adults (4 days survival) or (B) ventral BC in 30-day-old pups (3 days survival). Many PRV spinal
interneurons can also be seen in the pups. Scale bars = 250 µm. CC = central canal; SNB = spinal nucleus
of bulbocaverbosus. The figure was reproduced from Dobberfuhl et al. (2014).
Figure 4
The total number and regional distribution of PRV-labeled neurons in the spinal cord 4 days after PRV
injections into the LA muscle of adults. Data represent the mean ± s.e.m. (n = 5). L = lateral gray; I =
intermediate gray; M = medial gray; VH = ventral horn. The figure was reproduced from Dobberfuhl et al.
(2014).
34
Figure 5
Coronal sections of PRVlabeled neurons in the spinal cord after PRV injections to the LA muscle in
adults (A and B) and ventral BC muscle in pups (C and D). Arrows indicate PRV-labeled neurons in the
medial gray of L4 (A and D) and in the lateral gray (parasympathetic preganglionic neurons) and medial
gray (dorsal gray commissure) of L6 (B) and lateral gray (sympathetic preganglionic neurons) and
intermediate gray of L1 (C). Scale bars = 500 µm. The figure was reproduced from Dobberfuhl et al.
(2014).
35
Figure 6
Coronal sections showing presence of PRV-labeled neurons in the autonomic regions of the spinal cord
that are involved in ejaculation. (A) shows the colocalization of PRV and choline acetyl transferase
(ChAT) in sympathetic preganglionic nucleus of L1, 4 days after LA muscle injections. (B) shows the
location of PRV-labeled neurons in the parasympathetic preganglionic nucleus and dorsal gray
commissure of L6 after ventral BC muscle injections in pups. In (A) Red arrow = example of PRV only
labeled cell, green arrow = example of cell labeled with ChAT only, yellow arrows = examples of double
labeled PRV and ChAT neurons. Black arrows in (B) show examples of PRV-labeled cells in the
parasympathetic preganglionic nucleus and dorsal gray commissure. Scale bars = 50 µm in (A) and 500
µm in (B). The figure was reproduced from Dobberfuhl et al. (2014).
36
Figure 7
Double immunofluorescence for gastrin-releasing peptide (GRP) (A) and PRV (B) in the lumbar spinal
cord (L3–L4 level) after PRV injections into the LA muscles. Immunoreactivity against GRP (A; green)
and PRV (B; red) were merged in (C). GRP/PRV-double positive 2 neurons are shown by arrowheads.
(D) Schematic drawing of the lumbar spinal cord (L3–L4 level), indicating the distribution of GRP
neurons by stars. Asterisks (*) indicate the location of the neuronal nuclei expressing GRP and/or PRV.
Scale bar = 20 µm. CC = central canal. The figure was reproduced from Dobberfuhl et al. (2014).
37
Figure 8
Photomicrographs showing PRV-labeled neurons in the brain after PRV injections into the LA muscle.
(A) 4 days survival time; (B, C, D) 5 days survival time. (A) PRV-labeled cells in the lateral
paragigantocellularis (LPGi), ventrolateral medulla, parapyramidal region (PP) and raphe obscurus (RO).
(B) PRV-labeled cells in Barrington’s nucleus (Bar). (C) PRV-labeled cells in the paraventricular nucleus
of the hypothalamus (PVN) (D) PRV-labeled cells in the medial preoptic area (MPOA) and ventral bed
nucleus of the stria terminalis (BNST). Scale bars = 500 µm. The figure was reproduced from Dobberfuhl
et al. (2014).
38
Figure 9
The total number and regional distribution of PRV-labeled neurons in the spinal cord after injections of
PRV into the ventral bulbocavernosus muscle (BC) of 30 days old pups. Data represent the mean ± s.e.m.
(n = 4). L = lateral gray; I = intermediate gray; M = medial gray; VH = ventral horn. The figure was
reproduced from Dobberfuhl et al. (2014).
39
Chapter 3
The role of androgen receptor in the sexually dimorphic gastrin-releasing peptide
system in the lumbosacral spinal cord during development
40
Summary
Androgens including testosterone, organize the nervous system as well as masculine
external and internal genitalia during the perinatal period. Androgen organization
involves promotion of masculine body features, usually by acting through androgen
receptors (ARs). It is previously demonstrated that the gastrin-releasing peptide (GRP)
system in the lumbar spinal cord also mediates spinal centers promoting penile reflexes
during male sexual behavior in rats. Testosterone may induce sexual differentiation of
this spinal GRP system during development and maintain its activation in adulthood. In
this study, the role of ARs in the nervous system regulating the development of the
sexually dimorphic GRP system was examined. For this purpose, a conditional mouse
line selectively lacking the AR gene in the nervous system was used. AR floxed males
carrying (mutants) or not (controls) the nestin-Cre transgene were castrated in adulthood
and supplemented with physiological amounts of testosterone. Loss of AR expression in
the nervous system resulted in a significant decrease in the number of GRP neurons
compared to control littermates. Consequently, the intensity of GRP axonal projections
onto the lower lumbar and upper sacral spinal cord was greater in control males than in
mutant males. These results suggest that ARs expressed in the nervous system play a
significant role in the development of the GRP system in the male lumbar spinal cord.
The AR-deletion mutation may attenuate sexual behavior and activity of mutant males
via spinal GRP system-mediated neural mechanisms.
41
Introduction
Early in life, androgens induce external and internal genitalia to develop into a
masculine form and also masculinize the developing nervous system. This results in a
permanent organization of neural populations and synaptic connections underlying male
reproductive behavior (Matsuda et al. 2008; Morris et al. 2004; Phoenix et al. 1959). In
adulthood, T acts to activate these neural areas including brain regions (e.g., medial
amygdala, bed nucleus of stria terminalis, and medial preoptic area) and spinal nuclei.
In the spinal cord, androgen organization and activation of neural populations usually
occurs by acting through androgen receptors (ARs) (Matsuda et al. 2008; Morris et al.
2004). However, whether these processes involve the peripheral or central AR remain
unknown. It is previously demonstrated that the gastrin-releasing peptide (GRP) system
in the lumbar spinal cord also mediates spinal centers promoting penile reflexes during
male sexual behavior in rats (Sakamoto 2012; Sakamoto et al. 2008). It is also reported
androgenic effects on the GRP system in the lumbar spinal cord in two animal models,
one involving castration and T replacement in adult male rats and the second using
genetically XY male rats carrying the testicular feminization (Tfm) allele of the AR
gene (Sakamoto et al. 2009b). Both animal models indicate that androgen signaling
plays a pivotal role in the development of the spinal GRP system and in the regulation
of GRP expression in the male lumbar spinal cord. However, in these animal models, it
is difficult to distinguish between central and peripheral effects of androgens, including
behavioral modulations. Therefore, a conditional mouse line selectively lacking the AR
gene in the nervous system (Raskin et al. 2009) was used to elucidate whether central
AR is important for the sex differentiation of the spinal GRP system, which plays a
crucial role in male sexual behavior (Sakamoto et al. 2008). Using this mouse model in
the study of the spinal GRP system is of great interest because this conditional mutation
in mice interferes with the expression of male sexual behavior (Raskin et al. 2009).
42
Materials and Methods
Animals and genotyping
Control males with a floxed AR allele (ARfl/Y) and their mutant littermates (ARfl/Y,
Nes-Cre; ARNesCre) expressing Cre recombinase under the control of the promoter and
the nervous system enhancer of nestin (Nes) gene were obtained as previously described
(Raskin et al. 2009). The ARNesCre mouse line was then backcrossed for at least nine
generations into strain C57BL6J. Control and mutant males obtained in the same litters
were weaned at 24–26 days of age and group housed under a controlled photoperiod (12
h light, 12 h dark cycle; lights on at 07:00 h) and temperature (22 ◦C) and given free
access to food and water. The Cre transgene and floxed or excised AR alleles were
identified by PCR analysis as previously described (De Gendt et al. 2004; Raskin et al.
2009). All studies were performed in accordance with the guidelines for care and use of
laboratory animals [National Institutes of Health (NIH) Guide] and French and
European legal requirements (Decree 87- 848, 86/609/ECC). The experimental
procedures have also been authorized by the Committee for Animal Research, Okayama
University, Japan.
Gonadectomy and T supplementation
Adult males (8–10 weeks of age) of control (ARfl/Y) and ARNesCre were castrated under
general anesthesia (xylazine/ketamine). At the time of castration, all males received 1
cm subcutaneous SILASTIC implants (3.18 mm outer diameter × 1.98 mm inner
diameter; Dow Corning, Midland, MI, USA), either empty or filled with 10 mg T
(Sigma–Aldrich, St.-Quentin Fallavier, France) and sealed at each end with SILASTIC
adhesive. Animals were killed 3–4 weeks later. Circulating levels of T under these
conditions were previously published elsewhere (Marie-Luce et al. 2013; Raskin et al.
2009; Raskin et al. 2012). Circulating T was in the highest physiological range and
similar comparable between controls and mutants treated with T.
Immunohistochemistry and immunofluorescence
Mice were anesthetized and transcardially perfused with physiological saline followed
by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4). Lumbar spinal cords
were quickly removed and immersed in the same fixative for 4 h at room temperature.
43
After immersion in 25% sucrose in 0.1 M PB at 4 C for cryoprotection until they sank,
the preparations were quickly frozen using powdered dry ice and cut into 30 _m-thick
cross or horizontal sections on a cryostat (CM3050 S, Leica, Nussloch, Germany). The
immunohistochemical analysis was performed according to established methods
(Sakamoto et al. 2010; Sakamoto et al. 2008; Sakamoto et al. 2009b). In brief,
endogenous peroxidase activity was eliminated from the sections by incubation in a 1%
H2O2 absolute methanol solution for 30 min followed by three 5-min rinses with
phosphate buffered saline (PBS) (pH 7.4). These processes were omitted for the
immunofluorescence method. After blocking nonspecific binding components with 1%
normal goat serum and 1% BSA in PBS containing 0.3% Triton X-100 for 1 h at room
temperature, sections were incubated with primary rabbit antiserum against GRP
(1:5000) (Phoenix Pharmaceuticals, Burlingame, CA, USA) for 48 h at 4 ◦C.
Immunoreactive (ir) products were detected with a streptavidin-biotin kit (Nichirei,
Tokyo, Japan), followed by diaminobenzidine development according to our previous
method (Sakamoto et al. 2008). The specificity of the antiserum was published
previously (Sakamoto et al. 2008). GRP-ir cells in the spinal cord were localized using
an Olympus Optical (Tokyo, Japan) BH-2 microscope. To determine the effect of
central ARs on the projection site of GRP-ir axons, double-immunofluorescence
staining of GRP and neuronal nitric oxide synthase (nNOS) (A-11; mouse monoclonal
antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:5000 dilution), a marker
protein for neurons in the sacral parasympathetic nucleus (SPN), was performed as
described previously (Sakamoto et al. 2008). Alexa Fluor 546-linked anti-mouse IgG
(Molecular Probes, Eugene, OR, USA) and Alexa Fluor 488-linked anti-rabbit IgG,
both raised in goats (Molecular Probes), were used at a 1:1000 dilution for detection.
Immunostained sections were imaged with a confocal laser scanning microscopy
(Fluoview 1000, Olympus, Tokyo, Japan). For quantitative analysis, GRP-ir cells with
clearly visible round nuclear profiles were counted in the anterior part of the lumbar
spinal cord (L3–L4 level). To determine the optical density (OD) of positive GRPir
fibers in the SPN, dorsal gray commissure (DGC) and dorsal horn (DH), at least six
sections per animal were analyzed using ImageJ software (ImageJ 1.36b) with a set
threshold level. The GRP-ir fiber pixel density was quantified as the average pixel
density in three regions of each animal (SPN, DGC and DH), and were calculated as the
ratio to the density seen in the DH in control males. At least five animals were used for
44
these analyses in each group. The numbers and optical density of the GRP-ir neurons in
the lumbar spinal cord were expressed as the mean ±standard error of the mean (s.e.m.)
in each group, and were analyzed by a Student’s t-test. P < 0.05 was considered
statistically significant.
45
Results
First, using immunohistochemical staining for GRP, the number of GRP-ir neurons
located in the lumbar spinal cord (L3–L4 level; containing somata and dendrites of the
GRP neurons) of control and ARNesCre male mice was studied (Fig. 1). The numbers of
GRP-positive somata in the lumbar spinal cord of mice were 277.3 ±14.1 and 134.0 ±
10.4 in the control and ARNesCre males, respectively (Fig. 1). The number of GRP-ir
neurons was significantly fewer in ARNesCre males than in control males in the upper
lumbar spinal cord (n = 5, t = 8.24, P < 0.05) (Fig. 1). Because all the mice in this study
were castrated and received long-term (3–4 weeks) T supplementation, levels of
circulating T were maintained at a high physiological range, comparable to that between
controls and mutants as previously reported (Marie-Luce et al. 2013; Raskin et al. 2009;
Raskin et al. 2012). Then, the projection of GRP-ir fibers to the SPN in the lower spinal
cord (L5–L6 and S1 level; containing GRP neuronal axon terminals) of male mice was
examined because SPN provides autonomic preganglionic fibers to the genitalia
(Morgan et al. 1981). Double immunofluorescence for GRP and nNOS clearly showed
that GRP-ir fibers projected densely into the SPN (Fig. 2). Consistent with the
difference in the GRP soma area (L3–L4 level), double immunofluorescence for GRP
and nNOS further revealed that the intensity of GRP-ir fibers was greater in control than
in ARNesCre males in the autonomic center SPN whereas changes were observed for
nNOS-ir intensity (Fig. 2). Quantification analysis of GRP-ir clearly confirmed that the
intensity of GRP-ir fibers in the lower lumbar and upper sacral spinal cord (L5–L6 and
S1 level) was greater in control than in ARNesCre males in both the autonomic centers of
the SPN and DGC but not in the somatic sensory layers of the DH (n = 5 each) (Fig. 3).
46
Discussion
The goal of the present study was to determine whether central AR expression directly
regulates the development of the GRP system in the lumbar spinal cord because such a
spinal region is involved in the androgenic modulation of male specific behaviors
(Sakamoto 2012; Sakamoto et al. 2008). There is substantial evidence of androgenic
effects on aggressive and sexual behaviors, although direct effects of the AR signaling
on the GRP system in the lumbar spinal cord have not been fully investigated.
Therefore, the present study focused on the effects of central AR deletion on the spinal
GRP system, which controls male copulatory behavior (Sakamoto et al. 2010;
Sakamoto et al. 2008; Sakamoto et al. 2009b) by using genetically modified mutant
mice lacking AR in the nervous system (Marie-Luce et al. 2013; Raskin et al. 2009;
Raskin et al. 2012). The male urogenital tract of ARNesCre mutant develops normally and
mice are able to produce offspring, although with reduced fertility (Raskin et al. 2009;
Raskin et al. 2012). Regardless of attenuated sexual motivation and performance,
ARNesCre males exhibited a sexual behavior against females (Raskin et al. 2009).
ARNesCre males also exhibit a very low aggressive behavior compared to their control
littermates (Marie-Luce et al. 2013; Raskin et al. 2009). During mating, the mean length
of grooming erection is also significantly reduced in mutant males (Raskin et al. 2009;
Raskin et al. 2012). In the lumbar spinal cord of ARNesCre males, the number of GRP
neurons was significantly decreased compared to control littermates, despite
normalization of circulating levels of T between the two genotypes. In addition, GRP-ir
fibers were much more prominent in control males than in ARNesCre males in both the
SPN and DGC surrounding the central canal. Both the SPN and DGC provide
autonomic preganglionic fibers to genitalia (Morgan et al. 1981). Fig. 2 showed many
overlaps between GRP-ir fibers and SPN neuron somata with the proximal dendrites.
Using double immunoelectron microscopy, it is previously demonstrated that
GRP-containing presynaptic boutons innervate nNOS-positive dendrites in the SPN in
rats (Oti et al. 2012; Sakamoto et al. 2008). Therefore, these overlaps might be possible
sites of synaptic contacts also in mice. On the other hand, orchiectomy of adult male
rats significantly reduced the expression of GRP in the lumbar spinal cord after 1 month
and this reduction was prevented by long-term androgen replacement treatment
(Sakamoto et al. 2008; Sakamoto et al. 2009b). Conversely, long-term T treatment of
47
castrated adult female rats did not fully masculinize GRP expression (Sakamoto et al.
2008), suggesting that, at least in rats, androgens exert organizational effects on GRP
neuronal cell number only during the perinatal critical period (Sakamoto et al. 2009b).
These GRP-expressing neurons in the lumbar spinal cord of males also express AR but
do not express estrogen receptor a in rats (Sakamoto et al. 2008) and mice (Tamura et
al., my personal communication). Collectively, a male-dominant sexual dimorphism in
the spinal GRP system may be developed through AR-mediated mechanisms by the
androgen surge during the critical period. This male specific GRP neuronal cell number
is perhaps maintained with considerably low plasticity in adulthood. However, it is
suggested that not only the number of GRP neurons but also ir of GRP in the GRP
neurons might be decreased in ARNesCre males in adulthood.
The Tfm rodent is a unique model for examining the role of ARs in the
central nervous system because a point mutation in the AR gene renders the protein
dysfunctional (Zuloaga et al. 2008b). Because, these dysfunctional ARs in Tfm models
are expressed in the whole body including the brain, spinal cord, skeletal muscles,
reproductive organs and so on (Bardin et al. 1971; Beach and Buehler 1977), it is
difficult to distinguish the tissue-specific AR functions. Additionally, rat and mouse
Tfm models differ in terms of circulating T levels (Zuloaga et al. 2008b). Tfm male
mice have significantly less endogenous T compared to their wild-type littermates
(Jones et al. 2003; Zuloaga et al. 2008a), while Tfm male rats and ARNesCre males have
circulating T levels in the high male range (Raskin et al. 2009; Raskin et al. 2012;
Roselli et al. 1987; Sakamoto et al. 2009b). It is previously demonstrated that the spinal
cord of Tfm male rats is hyperfeminine in terms of the number of GRP-expressing
neurons in the upper lumbar region (L3–L4 level), although circulating T levels tended
to be high (Sakamoto et al. 2008; Sakamoto et al. 2009b). In the present study, a similar
feminization in the spinal GRP system of ARNesCre male mice were observed. Taken
together, the present results from ARNesCre mice further imply that ARs expressed in the
peripheral muscles and/or reproductive organs could not directly oppose the
organizational effects on the sexually dimorphic GRP system in the lumbar spinal cord.
In rodents, the spinal nucleus of the bulbocavernosus (SNB) is located in the lower
lumbar and upper sacral spinal cord. It is also a sexually dimorphic nucleus that
innervates the perineal muscles involved in male copulatory behavior (Breedlove and
Arnold 1980; Sengelaub and Forger 2008). Male rats have more and larger SNB
48
motoneurons than do females, a dimorphism that results from differences in perinatal
androgen signaling through an AR-mediated mechanism (Breedlove and Arnold 1980;
Sengelaub and Forger 2008). The sex-related difference in the number of SNB motor
neurons develops perinatally in rats and mice. Prior to birth, the number of motor
neurons in the SNB increases and reaches a maximum in both sexes; at this point,
functional neuromuscular junctions have been established in the SNB system at this
time. However, in females, these components (both motor neurons and muscles) die
near the time of birth unless the animals are exposed to testosterone during the critical
period (androgen surge) (Nordeen et al. 1985). If an androgen surge occurs, it results in
higher expression of AR in the perineal muscles and spinal motor neurons. Interestingly,
the number of SNB motor neurons is unrelated to ARNesCre mutation status and adult
changes in T levels (Raskin et al. 2012), although it is likely that the ARNesCre mutation
caused a significant reduction in the number of the spinal GRP neurons in adult males.
In the SNB, the central AR participates in the developmental regulation of soma size
and dendritic length but not the survival of SNB motoneurons. The AR
immunohistochemistry in the lumbosacral spinal cord also demonstrated the expression
of AR in the cellular nuclei of SNB motoneurons in controls but not in ARNesCre males
(Raskin et al. 2012). It is suggested that AR could be differently involved in the
organization of cell populations in the spinal cord. It is reported that the high-voltage
electron microscopy (HVEM) analysis provides a clear three-dimensional visualization
of multiple synaptic contacts from the GRP system in the lumbar spinal cord onto the
dendrites of SNB motor neurons in rats (Sakamoto et al. 2010). Further investigations
of the AR dependent male specific organizational effects on these GRP and SNB
systems located both in the lower spinal cord and the physiological approaches for their
functional correlations are needed to draw firm conclusions.
Conclusions
It is demonstrated, for the first time in mice, that ARs expressed in the nervous system
play a significant role in the development of the GRP system in the male lumbar spinal
cord. Thus, the central but not peripheral ARs are critical for developmental regulation
and maintenance of the male dominant cell number and dendritic arborizations in the
spinal GRP system in rodents. Alteration by the ARNesCre mutation may attenuate sexual
behavior and activity of mutant males via spinal GRP system-mediated neural
49
mechanisms.
50
Figure 1
Immunohistochemical analysis for gastrin-releasing peptide (GRP) in the upper lumbar spinal cord (L3–
L4 level) of adult male mice. GRP-immunoreactive neurons were found in the upper lumbar spinal cord
of control males (A) and mutant males, selectively lacking the androgen receptor (AR) gene in the
nervous system (ARNesCre) (B). The number of GRP-immunoreactive neurons was fewer in ARNesCre males
than in control males in the upper lumbar spinal cord. Arrows indicate possible GRP-positive cell bodies.
Dashed lines indicate the level of the mid-line. Scale bar = 50 µm. The figure was reproduced from
Sakamoto et al. (2014).
51
Figure 2
Distribution of GRP-immunoreactivity in the lower lumbar and upper sacral spinal cord (L5–L6 and S1
level) of adult male mice. In the sacral parasympathetic nucleus (SPN) located in the lower lumbar and
upper sacral spinal cord, GRP-immunoreactive fibers (green) were observed both in control (A) and
ARNesCre (B) males. ARNesCre males present decreased GRP-immunoreactive fibers compared with control
littermates in this region. The neuronal nitric oxide synthase (nNOS) serves as a marker for SPN neurons,
and the immunoreactivity for nNOS in the SPN neurons (magenta) showed no difference between control
(C) and ARNesCre (D) males. Double immunofluorescence for GRP and nNOS revealed closely
appositions of GRP-containing fibers of control (E) and ARNesCre (F) males with the cell bodies and
proximal dendrites of nNOS-positive neurons in the SPN. Scale bar = 50 µm. The figure was reproduced
from Sakamoto et al. (2014).
52
Figure 3
Quantification analysis for the intensity of GRP-immunoreactive fibers in the lower lumbar and upper
sacral spinal cord (L5-L6 and S1 level) in control and ARNesCre males. (A) A schematic drawing of the
lower lumbar and upper sacral spinal cord, indicating the intra-spinal location of the SPN (A1), dorsal
gray commissure (DGC) (A2) and the dorsal horn (DH) (A3) in this spinal cord level. The blocked areas
are enlarged in (B). These higher magnification images show the distribution of GRP-immunoreactive
fibers in the SPN (B1), DGC (B2) and DH (B3), respectively. The lower panels in (B) show the
black-and-white images with a set threshold level corresponding to each upper panel. The optical density
of GRP fibers in the DGC, SPN and DH of control males versus ARNesCre males were analyzed in (C).
The intensity of GRP-immunoreactive fibers in the lower lumbar and upper sacral spinal cord was greater
in control than in ARNesCre males both in the autonomic centers of the DGC and SPN but not in the
somatic sensory layers of the DH. The optical density of the GRP-ir in the lumbar spinal cord was
expressed as the mean ±standard error of the mean (s.e.m.) in each group. CC; central canal. **P < 0.01
compared with control males (n = 5 mice in each group). Scale bars = 100 µm. The figure was
reproduced from Sakamoto et al. (2014).
53
Chapter 4
Postnatal development of the gastrin-releasing peptide system in the lumbosacral
spinal cord
54
Summary
A sexually dimorphic spinal gastrin-releasing peptide (GRP) system in the lumbosacral
spinal cord, which projects to the lower spinal centers, controls erection and ejaculation
in rats. However, little is known about the postnatal development of this system. In this
study, therefore the postnatal development of the male-dominant spinal GRP system
and its sexual differentiation in rats were examined using immunohistochemistry. Our
results show that male-dominant expression of GRP is prominent from the onset of
puberty and that sexually dimorphism persists into adulthood. These results suggest that
androgen surge during male puberty plays an important role in the development and
maintenance of the male-specific GRP function in the rat spinal cord.
55
Introduction
During perinatal life in mammals, androgens such as testosterone (T) induce external
and internal genitalia to develop into a masculine form and also masculinize the
developing brain and spinal cord. The spinal nucleus of the bulbocavernosus (SNB) is a
male-specific, sexually dimorphic nucleus in the lumbosacral spinal cord (L5–S1 level)
that innervates perineal striated muscles attached to the base of the penis (Breedlove and
Arnold 1983a, b; Sengelaub and Forger 2008). The sexual dimorphism is caused by sex
differences in perinatal androgen exposure (Breedlove and Arnold 1983a, b; Sengelaub
and Forger 2008). Recent research has demonstrated that pubertal sex hormones also
organize sex differentiation in brain functions for sexual behavior in adulthood (Sisk
and Zehr 2005). These results in a permanent masculinization of neural populations and
synaptic connections underlying male sexual behavior (Matsuda et al. 2008; Morris et al.
2004; Phoenix et al. 1959). In adulthood, T activates these neural areas including
several brain areas and spinal nuclei. Thus, T influences the brain and spinal cord via
organizational (perinatal) and activational (adulthood) effects (Sakamoto et al. 2012). In
the SNB neuromuscular system, sexual dimorphism in the spinal cord is organized
through an androgen receptor-mediating mechanism (Morris et al. 2004). Neurons
located dorsolateral to the central canal in lamina X within the third and fourth lumbar
spinal cord project to the thalamic region of the brain (Ju et al. 1987; Truitt et al. 2003).
These so-called lumbar spinothalamic neurons are sexually dimorphic, with males
possessing greater number than females (Ju et al. 1987; Truitt et al. 2003).
Truitt and Coolen (2002) demonstrated that a population of the lumbar
spinothalamic neurons acts as a “spinal ejaculation generator” because specific toxin
treatments that selectively lesion these galanin-positive neurons completely eliminate
ejaculation in rats. Independently, Sakamoto et al. (2008) demonstrated that the
gastrin-releasing peptide (GRP) system in the lumbosacral segments controls spinal
centers promoting penile erection and ejaculation during copulatory behavior in male
rats. It has also been demonstrated that GRP neurons in the lumbar spinal region (L3–
L4 level) are more numerous in males than in females, revealing a novel sexual
dimorphism in the spinal cord (Sakamoto et al. 2008). GRP neurons project axons into
the more caudal lumbosacral spinal cord (L5–S1 level), forming synaptic contacts on
the sacral parasympathetic nucleus (SPN) and SNB (Dobberfuhl et al. 2014; Oti et al.
56
2012; Sakamoto et al. 2010). These nuclei are also known to control erection and
ejaculation in an androgen-dependent manner in adulthood (Kozyrev et al. 2012;
Sakamoto 2014; Truitt and Coolen 2002). Considering these findings, it appears likely
that a population of the lumbar spinothalamic neurons, at least partly, expresses both
galanin and GRP (Sakamoto et al. 2008). Moreover, GRP is a possible candidate for
neurotransmitters involved in the modulation of lumbosacral spinal activity, generating
ejaculation at the moment of male orgasm (Sakamoto et al. 2008).
Little is known about the postnatal development of this sexually dimorphic
spinal GRP system in relation to puberty. In this study, using immunohistochemistry,
the postnatal development of the male-dominant GRP system in the lumbosacral spinal
cord controlling male reproductive function and its sexual differentiation around
puberty in rats were examined.
57
Materials and Methods
Animals
Male and female rats of the Wistar strain (Charles River Japan, Yokohama, Japan) were
used on postnatal day (PND) 16, 23, 30, 37, 44, or 70 (n = 5, of each sex, respectively).
All rats were maintained on a 12-h light/dark cycle and provided with unlimited access
to water and rodent chow. All experimental procedures have been authorized by the
Committee for Animal Research, Okayama University, Japan.
Tissue preparation
Rats were overdosed with sodium pentobarbital (100 mg/kg body weight) and perfused
via the left ventricle with 10–100 ml of physiological saline followed by 20–200 ml of
4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The amount of fixative and
its duration were determined in proportion to body size. Subsequently, spinal cords
were immediately removed and post-fixed in the same fixative for 3 h at room
temperature.
Immunohistochemistry
Immunohistochemistry for GRP was performed as previously described (Sakamoto et al.
2008; Sakamoto et al. 2009b; Takanami et al. 2014). In brief, cryosections (30 µm in
thickness) were prepared using a cryostat (CM3050 S, Leica, Nussloch, Germany).
After blocking nonspecific binding, the sections were incubated with the primary rabbit
antiserum against GRP (1:2,000 dilution) (11081; AssayPro, St. Charles, MO, USA).
Immunoreactive products were detected with a streptavidin-biotin kit (Nichirei, Tokyo,
Japan), followed by 3,3’-diaminobenzidine (Dojindo, Kumamoto, Japan) development.
To determine the intensity of GRP-immunoreactivity in fibers in the lower lumbosacral
spinal cord (L5–S1 level), at least 10 cross sections (30-µm thick) per animal were
randomly selected, and the digital images of three regions [the SPN, dorsal gray
commissure (DGC), and dorsal horn (DH)] were prepared (magnification, ×200 per
section). The unit area (343 × 469 µm2) was analyzed to localize the nuclei at the center
of each area. The optical density of GRP staining was analyzed using ImageJ software
(ImageJ 1.44p; National Institutes of Health, Bethesda, MD, USA) (see Fig. 1)
according to our established methods (Sakamoto et al. 2008; Sakamoto et al. 2014;
58
Sakamoto et al. 2009b). Briefly, each threshold optical density was determined by
normalizing the data to those of the (preabsorbed negative) control sections. The
GRP-positive-fiber pixel density was quantitated as the average pixel density in the
SPN, DGC, and DH of each animal, and the data were expressed as the ratio of each to
the density of the DH in males on PND 70. All micrographs were coded and evaluated
without the knowledge of sexes and PNDs, and the code was not broken until the
analysis was complete.
Immunofluorescence staining for GRP in the upper lumbosacral spinal cord
(L3–L4 level) was performed as described above using horizontal sections (at least n =
5 on each sex and PND) to count the number of GRP-positive neurons per animal as
described previously (Sakamoto et al. 2008; Sakamoto et al. 2014; Sakamoto et al.
2009b). In brief, digital microphotographs were processed with Adobe PhotoShop
computer software (Adobe Systems, San Jose, CA, USA) at 300 dots per inch resolution.
The number of GRP-positive cell bodies was counted at ×200 magnification in all
sections and was analyzed a 600-µm2 area (see Fig. 1). GRP neurons were identified by
their following characteristics: densely immunostained, anatomical localization (mainly
dorsal, dorsolateral, or both to the central canal in lamina X of lumbar segments III–IV),
relatively large cell bodies (diameters approximately 20–30 µm), and clear round nuclei
(diameters approximately 10–15 µm). To avoid overestimating cell number, only
GRP-positive neurons that contained a round, transected nucleus were counted. Because
the mean diameter of the nuclei in the GRP neurons is much smaller than the 30-µm
thick sections, this analysis reduced the overestimation of the number of neurons. All
micrographs were coded and evaluated without the knowledge of sexes and PNDs, and
the code was not broken until the analysis was complete. Alexa Fluor 488-linked
anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) was used for detection at a
1:1,000 dilution. Immunostained sections were imaged with a confocal laser scanning
microscope (FluoView 1000, Olympus, Tokyo, Japan).
Statistics
The number of GRP-positive neurons in the lumbar spinal cord was reported as the
mean ± s.e.m. in each group of animals. The differences between sexes and PNDs were
assessed statistically using a two-way analysis of variance (ANOVA) (expressed as
mean ± s.e.m.). Statistical analyses of the number of GRP-positive neurons and the
59
optical density of GRP-positive fibers (expressed as mean ± s.e.m.) were performed
using a one-way ANOVA. When significant main effects were found using an ANOVA,
post hoc Tukey’s tests were performed.
60
Results
Figure 1 shows the analytical protocol for the morphological quantification used in this
study. First, using immunohistochemistry for GRP, developmental changes in the
number of GRP-positive neurons in the upper lumbar spinal cord (L3–L4 level) were
examined at different ages, especially before and around puberty (PND 16, 23, 30, 37,
44, and 70) (Fig. 2). Most GRP-positive somata and arborizations were greater in males
relative to females in all experimental periods examined (Fig. 2). Interestingly, on PND
30 and 37, GRP-positive neurons were hardly detected in females, and very few were
detected in females on PND 44 (Fig. 2).
Quantification analysis showed that, on PND 16, 30, 37, 44, and 70, the
number of GRP-positive neurons was significantly greater in males than in females
[F(1,48) = 793.3, P < 0.001] (Fig. 3). In contrast, on PND 23, no significant difference
was found in the number of GRP-positive neurons between sexes (Fig. 3). In males, the
number of GRP-positive neurons was much greater on PND 70 than on PND 16,
although the number of GRP-positive neurons was fewer on PND 30 than on PND 16
[F(5,48) = 38.19, P < 0.05] (Fig. 3). In females, the number of GRP-positive neurons
was greater on PND 23 than on PND 16 (P < 0.01) (Fig. 3).
In
the
lumbosacral
spinal
cord
(L5–S1
level)
of
males,
the
immunohistochemistry for GRP demonstrated that the intensities of GRP-positive fibers
both in the SPN [F(5,24) = 24.55] and DGC [F(5,24) = 17.42] were significantly higher
on PND 44 and 70 than on PND 16 (P < 0.05) (Fig. 4A). However, in the somatic
sensory layers of the DH, a slightly lower intensity of GRP-positive fibers was observed
on PND 37 relative to PND 16 [F(5,24) = 3.770, P < 0.05] (Fig. 4A). In females, the
intensities of GRP-positive fibers in the SPN were greater on PND 23, 44, and 70 than
on PND 16 [F(5,24) = 7.921, P < 0.05] (Fig. 4B). In the DGC, the intensity of
GRP-positive fibers was greater on PND 23, 44, and 70 than on PND 16 [F(5,24) =
15.62, P < 0.05] (Fig. 4B). In the DH, no significant difference was detected in the
intensity of GRP-positive fibers in females at any time point [F(5,24) = 2.484] (Fig. 4B).
Collectively, our findings showed that expression levels of GRP were lower in females
than in males at all time-points in the SPN and DCG, but were comparable in the DH
(Fig. 4). These expression patterns and the intensity of GRP-positive fibers in the SPN,
DGC, and DH are in keeping with previous studies in adult male and female rats
61
(Sakamoto et al. 2008; Sakamoto et al. 2012).
62
Discussion
While it is previously demonstrated the male-dominant sexual dimorphism of the spinal
GRP system which plays an important role in the male reproductive function in adult
rats (Sakamoto et al. 2008), little is known about the developmental changes in the
spinal center during ontogeny. In this study, this system at several stages from
pre-puberty to post-puberty (sexual maturation) was investigated to assess the effects of
the escalated circulation sex steroids during puberty, and compared the development of
the spinal GRP system between sexes. Only on PND 23, which is considered to be
pre-puberty, a sex difference was not observed in the number of GRP neurons . These
results suggest that male-dominant expression of GRP is prominent from the onset of
puberty, and that this sexual dimorphism persists into adulthood (PND 70). The PND 60
male rats (post-puberty) displayed significantly higher levels of plasma T than PND 30
male rats (pre-puberty) (Viau et al. 2005), suggesting that androgens begin to rise
during puberty. Thus, androgens appear to be a likely contributor to changes in the
expression of GRP during puberty.
The GRP system in the spinal cord shows sexual dimorphism, even in PND
16, suggesting that the GRP system is organized by sex steroids during the perinatal
critical period, similar to the SNB neuromuscular system (Breedlove and Arnold 1983a,
b; Sengelaub and Forger 2008). The number of GRP-positive neurons in females
increased from PND 16 to 23, and no difference in the number of GRP neurons between
sexes was observed at PND 23. Levels then rose again by PND 70 after having dropped
to non-detectable levels in females. It appears as though the number of
GRP-immunoreactive neurons decreased in males compared with PND 16, whereas it
increased in females. These results suggest two different possibilities: the change in
numbers was due to changes in number of cells or in the expression of GRP in the cells.
Further, the number of GRP neurons in the lumbosacral spinal cord markedly increased
from PND 30 onward. Because sex-related endocrine changes take place during puberty
in rats (Viau et al. 2005), PND 30 might be the approximate age of puberty onset in
male rats. Circulating T levels also significantly increase in post-pubertal (~PND 60)
compared with those in pre-pubertal (~PND 30) male rats (Viau et al. 2005). It is
demonstrated, for the first time, that the spinal GRP system shows a remarkable
male-dominant sexual dimorphism during puberty at the level of the spinal neural
63
circuit, when plasma T levels are dramatically increased. This suggests that the T
increase during puberty establishes the male-specific spinal GRP system by
upregulating the expression of GRP in cells in the lumbosacral spinal cord.
From PND 30 onward in females, few GRP-positive cell bodies were detected
and that GRP staining was not intense. Vaginal opening occurs around PND 33 in rats
(Franssen et al. 2014), suggesting that this is the age of puberty onset in females.
Circulating estrogen levels markedly increase around the time of vaginal opening in
female rats (Viau et al. 2005). Interestingly, in females, GRP-positive neurons are
barely detected on PND 30 and 37 when circulating estrogen levels are high. While
circulating T levels during male puberty significantly increased the GRP expression in
the lumbosacral spinal cord, in females, the higher circulating estrogen levels during
puberty might attenuate GRP expression. Thus, estrogens may play an important role in
the feminization of the male-specific spinal GRP system, possibly by inhibiting the
expression of GRP in the lumbosacral spinal cord during female puberty. On the other
hand, there is evidence that circulating estrogen levels are very high in both male and
female rats (and do not differ) up until about 6 weeks of age (Dohler et al. 1984), so the
observed decrease in females and increase in males during the same period are unlikely
to be estrogen-mediated. Notwithstanding such a discrepancy, GRP neurons in the
spinal cord of female rats can again be detected in adulthood, although the number is
still lower than that in males. Therefore, it is suggested that the initiation of the estrous
cycle in females might influence the expression of GRP in the rat spinal cord. Further
studies examining the effects of estrogens (e.g., estradiol-17β) are needed to draw a firm
conclusion.
The expression of GRP in the spinal DH is considered to be involved in the
transmission of the itch sensation (Sun and Chen 2007; Sun et al. 2009; Takanami and
Sakamoto 2014; Takanami et al. 2014). In this study, the expression of GRP in the DH
did not significantly change during ontogeny. Further, the two GRP systems in the
spinal cord, sexual and somatosensory, differ in their sensitivity to androgens, the
former being sensitive to androgens and the latter being insensitive. A possible
explanation may be the presence of androgen receptors in the GRP neurons of the
sexual system with an absence of such receptors in the somatosensory system.
64
Figure 1
Quantification analysis of the number of gastrin-releasing peptide (GRP)-positive neurons and the
intensity of GRP-immunoreactivity in fibers in the spinal cord. (A) A representative image of
immunofluorescence for GRP (left panel). Using the ImageJ software package, the image was inverted to
a bright field (inversion) image (right panel). (B) A schematic drawing of the lower lumbar spinal cord,
indicating the intraspinal location of the sacral parasympathetic nucleus (SPN) (B1), dorsal gray
commissure (DGC) (B2), and the dorsal horn (DH) (B3) at the lumbar spinal cord level (L6). The black
squares (B1–3) are enlarged in (C). These higher magnification images show the distribution of
GRP-immunoreactive fibers in the SPN (C1), DGC (C2), and DH (C3). The lower panels in (C) show the
black-and-white images with a set threshold level corresponding to each upper panel. Scale bars = 100
µm (applies to all). *Asterisk marks the central canal. The figure was reproduced from Katayama et al.
(2016).
65
Figure 2
Representative photomicrographs of developmental changes and sex differences in gastrin-releasing
peptide (GRP)-immunoreactivity analysis in the upper lumbosacral spinal cord (L3-L4 level) on postnatal
day (PND) 16, 23, 30, 37, 44, and 70. Scale bar = 50 µm (applies to all). The figure was reproduced from
Katayama et al. (2016).
66
Figure 3
Quantitative analyses of developmental changes in the number of gastrin-releasing peptide (GRP)
neurons in the upper lumbosacral spinal cord (L3-L4 level) of male and female rats. *Asterisks indicate
the sex differences in each postnatal day (PND) (P < 0.001). Letters denote differences among means (P
< 0.05); means with the same letter (capital letters in males; small letters in females) are not significantly
different. N.D.: not detectable. The figure was reproduced from Katayama et al. (2016).
Figure 4
Quantitative analyses of developmental changes in the intensity of gastrin-releasing peptide
(GRP)-positive fibers in the sacral parasympathetic nucleus (SPN), dorsal gray commissure (DGC), and
dorsal horn (DH) in the lower lumbar and upper sacral spinal cord (L5–S1 level) of male (A) and female
(B) rats. *P < 0.05, between postnatal days (PNDs); †P < 0.05, vs. PND 16; ‡P < 0.05, vs. PND 37; §P <
0.05, vs. PND 44. The figure was reproduced from Katayama et al. (2016).
67
Chapter 5
Perinatal development of the sexually dimorphic gastrin-releasing peptide system
in the lumbosacral spinal cord that mediates male reproductive function
68
Summary
In rats, a sexually dimorphic spinal gastrin-releasing peptide (GRP) system in the
lumbosacral spinal cord projects to spinal centers that control erection and ejaculation.
This system controls the sexual function of adult males in an androgen-dependent
manner. In this study, the influence of androgen exposure on the spinal GRP system
was assessed during a critical period of the development of sexual dimorphism.
Immunohistochemistry was used to determine if the development of the spinal GRP
system is regulated by the perinatal androgen surge. First, the responses of neonates
administered the anti-androgen flutamide were studied. To remove endogenous
androgens, rats were castrated at birth. Further, neonatal females were administered
androgens during a critical period to evaluate the development of the male-specific
spinal GRP system. Treatment of neonates with flutamide on postnatal days 0 and 1
attenuated the spinal GRP system during adulthood. Castrating male rats at birth
resulted in a decrease in the number of GRP neurons and the intensity of neuronal GRP
in the spinal cord during adulthood despite testosterone supplementation during puberty.
This effect was prevented if the rats were treated with testosterone propionate
immediately after castration. Moreover, treating female rats with androgens on the day
of birth and the next day masculinized the spinal GRP system during adulthood, which
resembled the masculinized phenotype of adult males and induced a hypermasculine
appearance. The perinatal androgen surge plays a key role in masculinization of the
spinal GRP system that controls male sexual behavior. Further, the present study
provides potentially new approaches to treat sexual disorders of males.
69
Introduction
Androgens such as testosterone (T) and 5α-DHT play a key role in the sexual
differentiation of the central nervous system through a mechanism that is mediated by
signaling through the nuclear androgen receptor (AR) during the perinatal life
(Breedlove and Arnold 1983a, b; Matsuda et al. 2008; Morris et al. 2004). In males, a
considerable amount of T secreted transiently from the testes represents the “androgen
surge” that masculinizes the developing brain as well as the external and internal
genitalia (Matsuda et al. 2008; Morris et al. 2004; Phoenix et al. 1959). Sex differences
caused by the androgen surge are restricted to a limited period called the “sensitive” or
“critical” period (Morris et al. 2004; Phoenix et al. 1959). For example, a critical period
in rats occurs 18–27 days after fertilization when masculinization of the brain and spinal
cord typically depends on transiently higher levels of circulating androgens (Matsuda et
al. 2008; Morris et al. 2004).
The spinal nucleus of the bulbocavernosus (SNB), which is located in the
lumbosacral spinal cord (L5–S1 level), is homologous to Onuf’s nucleus in humans
(Onufrowicz 1899) and innervates perineal striated muscles attached to the base of the
penis; the bulbocavernosus (BC) muscles in mammals such as rats (Breedlove and
Arnold 1980; Sengelaub and Forger 2008), mice (Wee and Clemens 1987), and
monkeys (Ueyama et al. 1985). The SNB-BC neuromuscular system is male-dominant
in adults, and this sexual dimorphism is caused by sex differences in perinatal androgen
exposure (Breedlove and Arnold 1980; Sengelaub and Forger 2008). Although
androgens appear to play an important role in the establishment of SNB motoneuron
number (Breedlove 1985; Freeman et al. 1996; Nordeen et al. 1985; Sengelaub and
Arnold 1986), soma size (Goldstein et al. 1990; Verhovshek et al. 2010), and
neuromuscular synapse elimination (Jordan et al. 1989), estrogens might also be
critically involved in SNB dendritic development (Burke et al. 1997). Taken together,
these results suggest that, in the developing SNB-BC neuromuscular system, somal and
dendritic growth may occur through separate developmental mechanisms, and that
androgens and estrogens act synergistically to support normal masculine SNB dendritic
development (Burke et al. 1997; Sengelaub and Forger 2008).
Gastrin-releasing peptide (GRP), a brain-gut peptide, is expressed and
distributed widely in the central nervous system as well as in the gastrointestinal tract of
70
mammals (Panula et al. 1988). GRP regulates many physiological processes, including
food intake (Ladenheim et al. 1996), circadian rhythms (Shinohara et al. 1993), anxiety
(Merali et al. 2006), and the sensation of itch (Sun and Chen 2007; Takanami et al.
2014). Further, it is demonstrated that neurons that express GRP (GRP neurons) in the
lumbosacral spinal cord mediate the function of spinal centers that promotes penile
reflexes during the sexual behavior of rats (Sakamoto and Kawata 2009; Sakamoto et al.
2008). Further, the number of GRP neurons in the lumbar spinal region (L3–L4 level) is
greater in males than that in females (Sakamoto and Kawata 2009; Sakamoto et al.
2008). These GRP neurons project axons into the more caudal lumbosacral spinal cord
(L5–S1 level), forming synaptic contacts with neurons in the sacral parasympathetic
nucleus (SPN) and motor neurons in the SNB (Dobberfuhl et al. 2014; Oti et al. 2012;
Sakamoto et al. 2010; Sakamoto and Kawata 2009; Sakamoto et al. 2008; Sakamoto et
al. 2009a). These nuclei control erection and ejaculation in an androgen-dependent
manner during adulthood (Kozyrev et al. 2012; Sakamoto et al. 2008; Truitt and Coolen
2002). However, little is known about the effects of the androgen surge during perinatal
development of this sexually dimorphic spinal GRP system.
To study the mechanisms underlying the development of the sexually
dimorphic spinal GRP system in rats, the effects of administering the anti-androgen
flutamide to neonatal male rats were determined. Further, newborn male rats were
castrated to prevent the production of endogenous androgens. In addition, androgen
treatments to neonatal female rats were performed to evaluate their effect on the
development of the male-specific spinal GRP system during a critical period.
71
Materials and Methods
Animals
Male and female Wistar rats (Charles River Japan, Yokohama, Japan) were housed
under a 12-h light/dark cycle and were provided unlimited access to water and rodent
chow. The Committee for Animal Research, Okayama University, Japan authorized the
experimental procedures.
Flutamide treatment of male neonates
To determine if the development of the sexually dimorphic spinal GRP system is
regulated by the androgen surge, flutamide (Zhang et al. 2008; Zhang et al. 2010)
(Sigma-Aldrich, St. Louis, MO, USA) or sesame oil (Nacalai, Kyoto, Japan) (control)
was administered to male neonates under deep, cool anesthesia. Male pups from
multiple dams were randomly divided into the groups as follows: Male pups were
injected subcutaneously (s.c.) with 0.25 mg flutamide in 100 µl of sesame oil on the day
of birth (PND 0) and 24 h later (PND 1) (designated the demasculinization group, n = 5).
This treatment resulted in a demasculinization of behavior in adult rats (Zhang et al.
2008; Zhang et al. 2010). Male pups (n = 5) from the same cohort were injected s.c.
with 100 µl of sesame oil (designated the control group).
Orchiectomy (ORX) and testosterone propionate (TP) treatment of male neonates
To examine the effects of the androgen surge on GRP expression in the lumbosacral
spinal cord, neonates under deep, cool anesthesia were castrated and administered TP or
sesame oil. Male pups from multiple dams were randomly divided into three groups. On
PND 0, males (n = 3) underwent bilateral ORX to ablate androgen production and were
then injected s.c. with 1.0 mg of TP (Nacalai) in 100 µl sesame oil and again on PND 1
for the purpose of androgen replacement (masculinization group) (Hagiwara et al. 2007;
Holman and Hutchison 1991). Other males (n = 4) underwent bilateral ORX on PND 0
and were injected s.c. with 100 µl of sesame oil. Control males (n = 6) were
sham-operated and injected s.c. with 100 µl of sesame oil on PNDs 0 and 1. On PND 30,
which is approximately the onset of puberty, ORX or ORX + TP males were deeply
anesthetized with 2% isoflurane and implanted s.c. with 50-mm Silastic capsules (inner
diameter = 1.59 mm, outer diameter = 3.18 mm; Companie de Saint-Gobain,
72
Courbevoie, France) containing crystalline T (Tokyo Chemical, Tokyo, Japan) to
maintain T levels similar to those of males at puberty for 50 days (Sakamoto et al. 2008;
Sakamoto et al. 2009b). Intact control males were implanted s.c. with empty 50-mm
Silastic capsules.
Ovariectomy (OVX) and TP or 5α-DHT treatments of female neonates
To assess the effect of androgen administration on the development of the male-specific
spinal GRP system during a critical period in females, neonatal females under deep,
cool anesthesia were administered androgens or sesame oil. Female pups from multiple
dams were randomly assigned to five treatment groups. On PNDs 0 and 1, females (n =
5) were injected s.c. with 0.1 or 1.0 mg of TP in 100 µl of sesame oil, and other females
(n ≥ 3) were injected s.c. with 0.1 or 1.0 mg with the non-aromatizable androgen
5α-DHT (Sigma-Aldrich) in 100 µl of sesame oil on PNDs 0 and 1 (all groups were
designated masculinization groups). These doses of TP and 5α-DHT approximate the
physiological levels present in female neonates (Goldstein and Sengelaub 1992;
Hagiwara et al. 2007; Sengelaub et al. 1989). Females (n = 6) from the same cohort
were injected with 100 µl of sesame oil on PNDs 0 and 1 and served as controls. On
PND 30, all females under deep isoflurane anesthesia underwent bilateral OVX to
remove circulating estrogens, and 50-mm Silastic capsules containing T were
immediately implanted s.c. into their backs where they remained for 54 days to maintain
the levels of T similar to those of the males described above.
Tissue preparation
After receiving the treatments described above, 11–12-week-old male and female rats
were administered an overdose of sodium pentobarbital (100 mg/kg body weight) and
perfused through the left ventricle with 100 ml of physiological saline followed by 200
ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Their spinal cords
were immediately removed and fixed in the fixative described above for 3 h at room
temperature.
Immunohistochemistry (IHC) and immunofluorescence
The fixed lumbrosacral spinal cords were cryoprotected by immersing them in 25%
sucrose in 0.1 M PB for 48 h at 4°C, quickly frozen using powdered dry ice, and cut
73
into 30 µm-thick cross or horizontal sections using a cryostat (CM3050 S, Leica,
Nussloch, Germany). Endogenous peroxidase activity was eliminated by incubating the
sections in absolute methanol containing 1% H2O2 for 30 min and then rinsed three
times with phosphate buffered saline (PBS, pH 7.4) for 5 min. This procedure was
omitted for samples subjected to immunofluorescence analysis. After blocking
nonspecific binding sites with 1% normal goat serum and 1% bovine serum albumin in
PBS containing 0.3% Triton X-100 for 1 h at room temperature, the sections were
incubated with a primary rabbit antiserum against GRP (1:2,000 dilution) (11081;
AssayPro, St. Charles, MO, USA) (Takanami et al. 2014). The specificity of the
anti-GRP serum for detected GRP in the spinal cord was demonstrated previously
(Takanami et al. 2014). Immunoreactive (ir) products were detected using a
streptavidin-biotin kit (Nichirei, Tokyo, Japan) and diaminobenzidine (Dojindo,
Kumamoto, Japan) (Sakamoto et al. 2008; Sakamoto et al. 2014; Takanami et al. 2014).
To
determine
the
sites
of
projection
of
GRP-ir
axons,
the
immunofluorescence analysis of the expression of GRP and neuronal nitric oxide
synthase (nNOS) was performed. The latter serves as a marker for neurons in the SPN.
To detect nNOS, a mouse monoclonal antibody (A-11, 1:5,000 dilution; Santa Cruz
Biotechnology, Santa Cruz, CA, USA) was used. Both primary antibodies were
combined to probe the sections. Alexa Fluor 546-conjugated anti-mouse IgG (Molecular
Probes, Eugene, OR, USA) and Alexa Fluor 488-conjugated anti-rabbit IgG (Molecular
Probes) were used for detection (each diluted 1:1,000). Immunostained sections were
imaged using a confocal laser scanning microscope (FluoView 1000, Olympus, Tokyo,
Japan).
Morphological analysis
First, GRP neurons in the lumbosacral spinal cord were counted. Immunofluorescence
analysis of GRP expression (GRP-ir) in neurons of the anterior lumbar spinal cord (L3–
L4 level) was performed as described above using horizontal sections (approximately
eighteen–twenty-two 30-µm-thick sections per animal) (Sakamoto et al. 2008;
Sakamoto et al. 2009a; Sakamoto et al. 2009b). Briefly, the number of GRP-ir cell
bodies was counted at ×200 magnification in all sections and was analyzed a 600-µm2
area localized to the midline at the center. Five–15 micrographs per section were
acquired, the number of which depended on the distribution of the GRP-ir neurons.
74
These digital micrographs were selected and processed using Adobe PhotoShop (Adobe
Systems, San Jose, CA, USA) and printed at 300 dots per inch on photographic paper.
GRP neurons were identified by their following characteristics: densely immunostained,
anatomical localization (mainly dorsal, dorsolateral, or both to the central canal in
lamina X of lumbar segments III–IV), relatively large cell bodies (diameters
approximately 20–30 µm), and clear round nuclei (diameters approximately 10–15 µm).
To avoid overestimating cell number, only GRP-ir neurons that contained a round,
transected nucleus were counted. Because the mean diameter of the nuclei in the GRP
neurons is much smaller than the 30-µm thick sections, this analysis reduced the
overestimation of the number of neurons. All micrographs were coded and evaluated
without the knowledge of the experimental group designation, and the code was not
broken until the analysis was complete.
Next, a semi-quantitative analysis of GRP expression was performed. To
determine the density of GRP-ir fibers in the lumbosacral spinal cord (L5–S1 level), at
least 10 cross sections (30-µm thick) per animal were randomly selected, and the digital
images of three regions [the SPN, dorsal gray commissure (DGC), and dorsal horn
(DH)] were prepared (magnification, ×200 per section). The unit area (343 × 469 µm2)
was analyzed to localize the nuclei at the center of each area. The optical density of
GRP staining was determined using black-and-white images that were converted from
micrographs using ImageJ software (ImageJ 1.44p; National Institutes of Health,
Bethesda, MD) (see Figure 1) according to our established methods (Sakamoto et al.
2008; Sakamoto et al. 2009a; Sakamoto et al. 2014). Briefly, the optical density of
background labeling was estimated by comparisons with similar areas of the control
sections reacted with the anti-GRP antiserum that was incubated first with an excess of
peptide antigen (50 µg/mL). GRP expression was undetectable in these sections. Each
threshold optical density was determined by normalizing the data to those of the
preabsorbed sections. The GRP-ir-fiber pixel density was semi-quantitated as the
average pixel density in the SPN, DGC, and DH of each animal, and the data were
expressed as the ratio of each to the density of the DH, which was used as the standard
in each analysis. It was demonstrated that this procedure eliminates the variability in
background staining among sections, animals, or both (Sakamoto et al. 2008; Sakamoto
et al. 2009a; Sakamoto et al. 2014). Moreover, this analysis substantially reflects
mirrored changes in the levels of GRP mRNA and protein after androgen treatment of
75
adult rats as the previous study (Sakamoto et al. 2009b). Micrographs were coded and
evaluated without the knowledge of the experimental group designation, and the code
was not broken until the analysis was complete.
Enzyme immunoassay
Before perfusing the rats with formaldehyde, circulating blood was collected from the
cardiac left ventricle, and the plasma was stored at −80°C. To measure the T
concentration, each plasma sample was assayed using a T enzyme immunoassay kit
(Cayman Chemical, Ann Arbor, MI, USA) (Sakamoto et al. 2009b).
Statistics
The number of GRP-ir neurons in the lumbar spinal cord of each group of animals was
presented as the mean ± standard error of the mean (s.e.m.). The sex differences
between males treated with flutamide and controls were assessed using the Student’s
t-test (mean ± s.e.m.). Statistical analyses of the number of GRP-ir neurons, the optical
density of GRP-ir fibers, and plasma T concentrations (mean ± s.e.m.) were performed
using one-way analysis of variance (ANOVA). When significant main effects were
found using ANOVA, the post-hoc Tukey test or the Steel-Dwass test was performed.
76
Results
Administration of flutamide demasculinizes the spinal GRP system of neonatal male
rats
To determine if the development of the sexually dimorphic spinal GRP system was
regulated by the androgen surge, the specific AR antagonist flutamide was administered
to neonatal male rats. Immunohistochemical analysis of GRP expression in the upper
lumbar spinal cord (L3–L4 level) revealed slightly fewer GRP-ir neurons in neonatal
flutamide-treated males than in controls (Figure 2, left panels). Further, flutamide
treatment decreased the intensity of GRP-ir dendrites in adult males (Figure 2, left
panels). Immunofluorescence analysis of GRP and nNOS expression revealed that the
intensity of GRP-ir fibers was greater in control males than that in flutamide-treated
males in the SPN, although the intensity of nNOS-ir staining was unchanged in the
lumbosacral spinal cord (L5–S1 level) (Figure 2, right panels). The number of
GRP-positive neurons in the upper lumbar spinal cord (L3–L4 level) was significantly
fewer in flutamide-treated males than that in controls males [t(8) = 2.480, *P < 0.05]
(Figure 3A). The intensity of GRP-ir fibers in the lumbosacral spinal cord (L5–S1 level)
was lower in flutamide-treated males than that in control males in the SPN [t(8) = 2.832,
*P < 0.05] and DGC [t(8) = 5.718, *P < 0.05], but not in the DH [t(8) = 1.173] (Figure
3B). In adults, the plasma concentrations of T were 4.34 ± 0.56 and 6.18 ± 1.25 ng/ml in
oil-treated controls (n = 5) and the neonatal flutamide-treated group (n = 5), respectively.
The adult level of plasma T after neonatal flutamide administration was not
significantly different, which is consistent with the findings of a previous study
(Mikkila et al. 2006).
Effects of the androgen surge on neonatal development of the spinal GRP system in
males
The testes of neonates were removed to assess the effects of the androgen surge on GRP
expression in the lumbosacral spinal cord. ORX males were treated with T starting at
PND 30. Immunohistochemical analysis of GRP expression in the upper lumbar spinal
cord (L3–L4 level) of adults revealed fewer GRP-positive neurons and lower intensity
of GRP-ir somata in ORX males than in control males, which were not significantly
different in neonates (ORX + TP males) administered TP independent of T
77
supplementation during adulthood (Figure 4, left panels). Immunofluorescence analysis
of GRP and nNOS expression in the SPN showed that the intensity of the GRP-ir fibers
was lower in neonatal ORX males than in control and ORX + TP males (Figure 4, right
panels) independent of T supplementation during adulthood. The number of
GRP-positive neurons in the upper lumbar spinal cord (L3–L4 level) was significantly
decreased in ORX males compared with sham-operated controls [F(2, 10) = 12.37, *P <
0.01] (Figure 5A). The intensity of GRP-ir fibers in the lumbosacral spinal cord (L5–S1
level) was greater in sham-operated controls than that in ORX males in the SPN [F(2,
10) = 24.91, *P < 0.01] and DGC [F(2, 10) = 18.67, *P < 0.01] but not in the DH [F(2,
10) = 0.109] (Figure 5B). Because the number of GRP-positive neurons in ORX +
TP-treated males was not significantly different compared with sham-operated male
controls, neonatal T replacement restored the number of GRP-positive neurons in adults
(Figure 5A). Moreover, castrates displayed a significantly lower GRP expression than
sham-operated groups and the ORX + TP-treated group in the SPN (†P < 0.01) and
DGC (†P < 0.01), but the expression was constant in the DH (Figure 5B). Because all
castrates were implanted with T-capsules on PND 30, plasma concentrations of T in
sham-operated controls, ORX, and ORX + TP-treated males ranged from 2.16 ± 0.29
ng/ml (ORX and ORX + TP) to 3.45 ± 0.61 ng/ml (sham-operated control), suggesting
that all the values were in the physiological ranges of those of the adult males.
Neonatal administration of androgens masculinizes the spinal GRP system of females
Androgens were administered to females to assess the development of the male-specific
spinal GRP system during a critical period (Figure 6). Immunohistochemical analysis of
GRP expression in the upper lumbar spinal cord (L3–L4 level) in neonatal females
administered 5α-DHT (1.0 mg) significantly increased the intensity of the GRP signal
as well as the number of GRP-ir neurons compared with controls (Figure 6, left panels).
Immunofluorescence analysis of GRP and nNOS expression in the lumbosacral spinal
cord (L5–S1 level) showed that the intensity of GRP-ir fibers in the SPN was greater in
5α-DHT-treated females than that in controls (Figure 6, right panels). Moreover, there
was no significant difference between the number of GRP neurons or the intensity of
GRP-ir in the lumbosacral spinal cord of 5α-DHT-treated females compared with adult
males (Figures 6). To our knowledge, the present study is the first to show such an
expression of male-specific GRP in the lumbosacral spinal cord of females. Moreover,
78
the number of GRP-positive neurons in the upper lumbar spinal cord (L3–L4 level) was
significantly greater in 5α-DHT-treated (0.1 and 1.0 mg) females than that in controls
[F(4, 19) = 15.35, *P < 0.05] (Figure 7A). Further, neonatal females administered TP
(0.1 and 1.0 mg) significantly increased the intensity of GRP-ir fibers in the SPN
compared with controls [F(4, 19) = 15.68, *P < 0.05] and DGC [F(4, 19) = 66.60, *P <
0.05] (Figure 7B), although there was no significant difference in the number of
GRP-positive neurons (Figure 7A). Further, the administration of 5α-DHT (0.1 and 1.0
mg) to neonatal females significantly increased the intensities of GRP-ir fibers relative
to controls (*P < 0.05) in the SPN and DGC (Figure 7B), and the expression levels were
similar to those of adult males (Figure 3B), mirroring the number of GRP cells (Figure
7A and B). Significant increases in the levels of GRP-ir in the DGC were detected in the
5α-DHT-treated group (0.1 and 1.0 mg) and in the group treated with 1.0 mg of TP
compared with the group treated with 0.1 mg of TP (†P < 0.05) (Figure 7B). In the DH,
small but significant differences in the intensity of GRP-ir fibers were observed among
groups (*P < 0.05, vs. Control) (Figure 7B). All the females in this study were OVX
and treated with T from PND 30. In adults, plasma T concentrations were slightly but
statistically lower in 0.1 mg 5α-DHT-treated females than in oil-treated control only
[F(4, 19) = 4.029, P < 0.05]. Nevertheless, plasma concentrations of T in oil-treated
controls, TP-, and 5α-DHT-treated females ranged from 1.95 ± 0.15 (0.1 mg 5α-DHT)
to 3.66 ± 0.44 ng/ml (oil-treated control), suggesting that all the values were in the
physiological ranges of those of the adult males.
79
Discussion
In the present study, the mechanisms of sex-specific hormonal regulation, particularly
androgen signaling, that mediate the development of the sexually dimorphic spinal GRP
system were examined in rats. It is demonstrated that androgen treatment of neonates
altered the spinal GRP system in adulthood. Interestingly, neonatal androgen treatment
induced complete masculinization of the spinal GRP system in XX females. This is the
first demonstration that the spinal cords of XX females were completely masculinized
with respect to the spinal GRP system and that this male-specific sexual dimorphism
persisted into adulthood.
Androgens masculinize the spinal GRP system in XX females
In this study, 5α-DHT treatment of female neonates increased the number and fiber
density of GRP neurons in the lumbosacral spinal cord. This upregulation of the spinal
GRP system in females approximated that of the adult males and led to a
hypermasculine appearance. The intensities of GRP-fibers at the SPN and DGC were
increased by treating neonates with TP, and the effect was dependent on the
concentration of TP, although there were no significant changes in the number of GRP
neurons in TP-treated groups. Thus, treating females with TP had no significant effect
on the number of GRP-positive neurons, although TP increased the optical density of
GRP-positive fibers. These findings indicate the possibility that administration of TP to
neonates did not affect the number of GRP neurons but increased the level of ARs in the
GRP neurons or altered androgen sensitivity. The higher levels of exogenous androgens,
which are present in pubescent males, might increase the density of GRP-positive fibers
during adulthood.
Together, these results indicate that 5α-DHT treatment of neonates induced
masculinization of the spinal GRP system in contrast to the partial affect of TP. Because
5α-DHT binds with a higher affinity to the AR than the T, these differences in androgen
actions suggest that the doses of TP used here were lower than the threshold level
required to induce an effect in the developing spinal cord. Comparing these results with
the SNB neuromuscular system in rats, the combination of prenatal and postnatal TP
treatments can induce a complete masculinization of the SNB system in females
(Sengelaub and Arnold 1986; Sengelaub et al. 1989). Goldstein and Sengelaub (1992)
80
found that the treatment of female perinatal rats with 5α-DHT propionate treatment
increases the motoneuron number, somal size, and dendritic arborization in the SNB to
the levels of an adult male. Our present results are consistent with these studies,
suggesting that the androgen surge during perinatal life plays a significant role in
masculinization of the spinal GRP system as well as that of the SNB-BC neuromuscular
system. The synergistic effects of androgens on these spinal systems may play an
important role in normal sexual differentiation in the rat spinal cord.
Effects of androgens on sexual differentiation of the spinal GRP system during a critical
period
Neonatal blockade of the androgen surge using the anti-androgen flutamide decreases
the number and density of GRP neurons in the adult male spinal cord, although the
endogenous T level is maintained or increased in adulthood (McCormick and Mahoney
1999; Mikkila et al. 2006). Moreover, in this study, neonatal ORX decreased the
number and density of GRP neurons in adulthood despite T supplementation
administered around puberty (PND 30). Further, neonatal administration of TP to ORX
pups, which might mimic the androgen surge, prevented the loss of the number or
density of GRP neurons in the spinal cord during adulthood. Therefore, it is suggested
that the mechanisms involved in the neonatal effects of androgens on the spinal GRP
system are related to the anti-apoptotic or neurotrophic effects of androgens in males
during a critical period or are associated with the effects of epigenetic modifications.
It is previously demonstrated an entirely feminine pattern or a hyperfeminine
appearance of the spinal GRP system in genetic males using two AR-deficient models
as follows: (i) genetically male (XY) rats carrying a testicular feminization mutation of
AR genes express a defective AR protein (Sakamoto et al. 2008; Sakamoto et al. 2009b)
and (ii) a mouse line specifically lacking AR genes in the nervous system (Sakamoto et
al. 2014). Nonetheless, analysis of these mutants did not address the involvement of AR
in the development of the sexually dimorphic spinal GRP system during neonatal life
because these two models lack AR function.
In contrast, a large body of literature shows that the androgen surge during a
critical period also plays a pivotal role in the sexual differentiation of the rat brain
(Matsuda et al. 2008; Morris et al. 2004). However, the masculinization of the brain
appears to depend on estrogens converted from T by the enzymatic activities of the
81
cytochrome P450 aromatase expressed in the brain (Matsuda et al. 2008; McCarthy
2008; Morris et al. 2004). The sexually dimorphic nucleus of the preoptic area
(SDN-POA) is one of the most important in the brain that is involved in the regulation
of male sexual behavior in rats (Matsuda et al. 2008; Morris et al. 2004; Sakamoto
2012). Many studies have shown that the volume of the SDN-POA is several times
larger in adult male rats than that in adult female rats (Dohler et al. 1984; McCarthy
2008; Sakamoto 2012). Treating female rats with testosterone as well as a synthetic
estrogen, diethylstilbestrol immediately before and after birth enlarges the size of the
SDN-POA of adults to that of normal males (Dohler et al. 1984), whereas the
SDN-POA is smaller and feminized in adult male rats castrated at birth (Matsuda et al.
2008; Morris et al. 2004). Thus, it is suggested that estrogens are exclusively
responsible for the establishment of this sexual dimorphism in the brain (Matsuda et al.
2008; McCarthy 2008; Morris et al. 2004). Similarly to the brain, it is likely that
estrogens might play a role in a normal masculinization of the spinal GRP system. On
the other hand, in this study, disrupting androgen signaling on PNDs 0 and 1
significantly attenuated the spinal GRP system, suggesting that these two days (PNDs 0
and 1) might be sufficient for masculinization of the spinal GRP system in rats. These
findings also suggested that the incomplete demasculinization of the spinal GRP system
in male rats may be attributed to the unpreventable effects of neonatal surgery or
treatment on the androgen surge during embryonic life, the level of secreted T before
castration on the day of birth, or both.
Somatosensory GRP system in the spinal cord
Evidence indicates that the expression of GRP in the spinal DH is involved in the
transmission of the itch sensation (Sun and Chen 2007; Takanami et al. 2014).
Significant effects on the expression of GRP were not observed following neonatal
administration of androgens or their antagonists on the expression of GRP in the adult
spinal DH. Long-term ORX (28 days) or ORX combined with TP treatment of adult rats
did not change the density of GRP-fibers in the DH of either sex (Sakamoto et al. 2008;
Sakamoto et al. 2009b). These results are consistent with present data, suggesting that
androgens do not affect the expression of GRP in the spinal somatosensory system.
These and the present findings show that the differences between the sexual and
somatosensory systems in the spinal cord are not mediated by different modes of
82
androgen signaling and that only one system is androgen sensitive.
Conclusions
It is demonstrated here that the androgen surge during a critical period in rats plays a
key role in the development of the sexually dimorphic GRP system in the lumbosacral
spinal cord. Further understanding of the mechanisms of sexual differentiation in the
spinal cord may provide new avenues for treating male sexual disorders.
83
Figure 1
Semi-quantitative analysis of the expression of gastrin-releasing peptide (GRP) in the lumbosacral spinal
cord (L5–S1 level). (A) Depiction of the lower lumbar and upper sacral spinal cord (L5–S1 level),
indicating the intraspinal location of the sacral parasympathetic nucleus (SPN, A1), dorsal gray
commissure (DGC, A2), and dorsal horn (DH, A3). The areas enclosed in boxes are enlarged in (B).
These magnified images show the distribution of GRP-immunoreactive fibers in the SPN (B1), DGC (B2),
and DH (B3). The lower panels in (B) show the black-and-white images with a set threshold level
corresponding to each upper panel. Scale bars = 100 µm. The figure was reproduced from Oti et al.
(2016).
84
Figure 2
Flutamide administered to neonatal male rats demasculinizes the spinal GRP system. Flutamide decreased
the number and intensity of GRP-immunoreactive neurons (left panels). Immunohistochemical analysis
of the expression of GRP (green) and neuronal nitric oxide synthase (magenta) shows a decrease in the
intensity of GRP-immunoreactive fibers in the SPN. Arrowheads indicate possible GRP-positive cell
bodies. Scale bars = 100 µm, left panel; 50 µm, right panel. The figure was reproduced from Oti et al.
(2016).
85
Figure 3
Semi-quantitative analyses of the effects on the spinal GRP system of the administration of flutamide to
neonates. The number of GRP neurons (A) and the intensity of GRP-immunoreactive fibers (B) in the
lumbosacral spinal cord. *P < 0.05 vs. Control. The figure was reproduced from Oti et al. (2016).
86
Figure 4
Neonatal castration demasculinizes the spinal GRP system. Effects of the androgen surge on neonatal
development of the spinal GRP system in males (left panels). Immunohistochemical analysis of GRP
(green) and nNOS (magenta) expression showed that the intensity of GRP-immunoreactive fibers in the
SPN was decreased by neonatal orchiectomy (ORX) but was prevented by administering testosterone
propionate (TP) (ORX + TP) to neonates. Arrowheads indicate possible GRP-positive cell bodies. Scale
bars = 100 µm, left panel; 50 µm, right panel. The figure was reproduced from Oti et al. (2016).
87
Figure 5
Semi-quantitative analysis of the effects of the androgen surge on the developing spinal GRP system of
males. Castrating rats at birth (ORX) decreased the number and intensity of GRP-immunoreactive
neurons in the spinal cord during adulthood. These effects were prevented if the castrates were treated
with testosterone propionate (ORX + TP) immediately after castration. Number of GRP neurons (A) and
intensity of GRP-immunoreactive fibers (B) in the lumbosacral spinal cord. *P < 0.01 vs. Control; †P <
0.01 vs. ORX. The figure was reproduced from Oti et al. (2016).
88
Figure 6
Masculinization of the spinal GRP system in female neonates exposed to androgens. Representative
photomicrographs of female neonates treated with 5 α -dihydrotestosterone (5α-DHT). Neonatal
administration of 5α-DHT increased the number and intensity of GRP-immunoreactive neurons (left
panels). Immunohistochemical analysis of GRP (green) and nNOS (magenta) showing significantly
increased intensity of GRP-immunoreactive fibers in the SPN induced by neonatal administration of
5α-DHT. Arrowheads indicate possible GRP-positive cell bodies. Scale bars = 100 µm, left panel; 50 µm,
right panel. The figure was reproduced from Oti et al. (2016).
89
Figure 7
Semi-quantitative analyses of the effects of the androgen surge on the developing spinal GRP system in
females. TP and 5α-DHT (0.1 mg or 1.0 mg each) were injected into female neonates on PNDs 0 and 1.
The number of GRP neurons (A) and the intensity of GRP-immunoreactive fibers (B) in the lumbosacral
spinal cord. *P < 0.05 vs. Control; †P < 0.05 vs. 0.1 mg TP; §P < 0.05 vs. 1.0 mg TP. The figure was
reproduced from Oti et al. (2016).
90
General Discussion
Neural circuits underlying male sexual function are composed of several nuclei located
in the brain and spinal cord (Sakamoto 2012, 2014). Previously, it has been
demonstrated that the sexually dimorphic spinal GRP system mediated the male sexual
function such as erection and ejaculation (Sakamoto 2011, 2014; Sakamoto et al. 2010;
Sakamoto and Kawata 2009; Sakamoto et al. 2008; Sakamoto et al. 2009a; Sakamoto et
al. 2014). GRP neurons project axons into the lower lumbar and upper sacral spinal cord
(L5–S1 level), including the SPN and SNB, controlling penile erection and ejaculation
(Sakamoto 2011, 2012; Sakamoto et al. 2010; Sakamoto and Kawata 2009; Sakamoto et
al. 2008). However, little information is available for the local neural circuitry in the
lumbosacral spinal cord regulating the male reproductive function. In Chapter 1, to
reveal the synaptic contacts from GRP neurons onto SPN neurons, a dual electron
microscopic technique and HVEM tomography assisted by light microscopy to a study
of the 3-D chemical neuroanatomy of the rat lower spinal cord were used. This study
demonstrated that the terminals of GRP neurons formed 3-D axonal projections with the
dendrites of SPN neurons, suggesting that these structures may be possible sites of
synaptic contacts. This is the first demonstration that 3-D axonal projections were
revealed using HVEM tomography applied a dual immunoelectron microscopic
technique. In Chapter 2, to reveal the local neural circuit from GRP neurons to SNB
motoneurons innervating perineal striated muscles attached to the base of the penis; the
bulbocavernosus muscle and levator ani muscle, PRV, a viral trans-synaptic retrograde
tracing techniques, was used. In this result, approximately half of PRV-positive neurons
in the medial gray at the L3-L4 level expressed GRP after PRV injection into the levator
ani muscle, suggesting that the existence of neural circuits from the levator ani muscle
to GRP neurons. It has been reported that GRPRs are expressed both in the SPN and
SNB (Sakamoto et al. 2008; Takanami and Sakamoto 2014). Therefore, the spinal
GRP/GRPR system could generate an ejaculatory response by activating autonomic and
somatic centers e.g., SPN and SNB, in the lumbosacral spinal cord (Takanami and
Sakamoto 2014). These findings support the hypothesis that the GRP/GRPR system
may regulate male sexual behavior via afferents to both SPN and SNB neurons and
organize autonomic and somatic functions, in response to penile reflexes during male
sexual behavior (Sakamoto and Oti 2015; Takanami and Sakamoto 2014). Taken
91
together, these results suggests that the GRP/GRPR system may regulate male sexual
behavior via synaptic inputs from GRP neurons onto both SPN and SNB neurons and
organize autonomic and somatic functions, in response to penile reflexes during male
sexual behavior (Sakamoto 2014; Sakamoto and Oti 2015).
The GRP somata express AR and the number of GRP neurons is greater in
males than that in females (Sakamoto 2014; Sakamoto et al. 2008). Sakamoto et al.
(2008; 2009b) demonstrated a hyperfeminine appearance of the spinal GRP system in
genetic males using genetically male (XY) rats carrying a testicular feminization
mutation of AR genes express a defective AR protein. It is suggested that ARs play a
key role in the masculinization of the spinal GRP system. However, these dysfunctional
ARs in Tfm models are expressed in the whole body including the brain, spinal cord,
skeletal muscles, reproductive organs and so on (Bardin et al. 1971; Beach and Buehler
1977). So, it is difficult to distinguish the tissue-specific AR functions. In Chapter 3,
therefore, using a mouse line specifically lacking AR genes in the nervous system
(ARNesCre mice), the mechanism underlying sexual differenciation of the spinal GRP
system was examined. The number of GRP neurons was significantly fewer in ARNesCre
males than in control males in the upper lumbar spinal cord. These results show that AR
expression in the nervous system plays an important role in the masclinization of the
spinal GRP system. However, these mutants never addressed the involvement of AR in
establishment of the sexually dimorphic spinal GRP system during the developmental
period, because these two models lack AR function all through life. Hence, in Chapter 4,
the spinal GRP system at several stages from pre-puberty to post-puberty (sexual
maturation) were investigated to assess the effects of the escalated circulation androgens
during puberty, and compared the development of the spinal GRP system between sexes.
Only on PND 23, which is considered to be pre-puberty, a sex difference was not
observed in the number of GRP neurons. Male-dominant expression of GRP had
already been established before PND 16. Further, male-dominant expression of GRP
was prominent from the onset of puberty (PND 30), and that this sexual dimorphism
persisted into adulthood (PND 70). These results suggest that the androgen surge from
the testes after puberty contribute to the establishment of the male-specific GRP
expression in adulthood. Interestingly, in females, GRP-positive neurons were barely
detected on PND 30 and 37. In addition, a transgenic rat expressing a yellow fluorescent
protein (Venus) under control of the GRP promoter was recently generated. Preliminary
92
results show that more Venus-positive neurons were observed in the upper lumber
spinal cord of OVX mutants compared with sham operated females. In females, plasma
estrogen and progesterone levels are higher in postpuberty (~PND60) than in prepuberty
(~PND 30) (Viau et al. 2005). These results indicate that in females, the higher
circulating estrogen and progesterone levels after puberty attenuate the GRP expression.
Taken together, these results suggest that the androgen surge after puberty contributed
to the establishment of the male-specific GRP expression in adulthood and estrogen and
progesterone surge after puberty might play a role in the feminization of the spinal GRP
system.
In males, a considerable amount of T secreted transiently from the testes
represents the perinatal “androgen surge” that masculinizes the developing brain as well
as the external and internal genitalia (Matsuda et al. 2008; Morris et al. 2004). Sex
differences caused by the androgen surge are restricted to a limited period called the
“critical period” (Morris et al. 2004; Phoenix et al. 1959). In Chapter 4, the
male-dominat sexually dimorphism of the spinal GRP system perhaps appears on PND
16. These results indicate that the androgen surge during a critical period might
masculinize the spinal GRP system. In Chapter 5, to identify the mechanisms of
sex-specific hormonal regulation during a neonatal life, the androgen surge was
manipulated by anti-androgen treatment or ORX to rat neonates. Neonatal blockade of
the androgen surge using the anti-androgen flutamide decreased the number and density
of GRP neurons in the adult male spinal cord. Moreover, the neonatal ORX also
decreased the number and density of GRP neurons in adulthood despite T
supplementation
administered
around
puberty
(PND
30).
Further,
neonatal
administration of TP to ORX pups, which might mimic the androgen surge, prevents the
loss of the number or density of GRP neurons in the spinal cord during adulthood.
These results indicate that the androgen surge during neonatal life is essential for a
normal development of the spinal GRP system. In addition, 5α-DHT treatment of
female neonates significantly increased the number and fiber density of GRP neurons in
the adult lumbosacral spinal cord. This upregulation of the spinal GRP system in
females approximated that of the adult males and led to a hypermasculine appearance.
This is the first demonstration that the spinal cords of XX females were completely
masculinized with respect to the spinal GRP system and that this male-specific sexual
dimorphism persisted into adulthood. Previously, Goldstein and Sengelaub (1992)
93
reported that the treatment of female perinatal rats with 5α-DHT propionate treatment
increased the motoneuron number, somal size, and dendritic arborization in the SNB to
the levels of an adult male. Taken together, these results show that the androgen surge
during the critical period plays a significant role in masculinization of the spinal GRP
system as well as that of the SNB-BC neuromuscular system.
In conclusion, the GRP/GRPR system may regulate male sexual behavior via
synaptic inputs from GRP neurons onto both SPN and SNB neurons and organize
autonomic and somatic functions, in response to penile reflexes during male sexual
behavior. Further, the androgen surge during the critical period play a key role in
development of the sexually dimorphic GRP system in the lumbosacral spinal cord, and
the androgen surge from the testes after puberty also contribute to the establishment of
the male-specific GRP expression in adulthood. Further understanding of the spinal
GRP system will help us to consider the neural circuits underlying male sexual function
and the mechanisms of sexual differentiation in the spinal cord.
94
Acknowledgments
I am grateful to my supervisor Associate Prof. Hirotaka Sakamoto for his valuable
comments and disscussions through this work. I also thank Prof. Sumio Takahashi for
their valuable comments and encouragement on this work. I also thank Prof. Tatsuya
Sakamoto, Assistant Prof. Yasuhisa Kobayasi, Naoaki Tsutsui and Dr. Keiko Takanami
for their valuable comments and encouragement on this work. I would like to thank Prof.
Mitsuhiro Kawata, Associate Prof. Ken Ichi Matsuda and Kazuyoshi Murata for
valuable comments and suggestions on this work. The discussion and cooperation with
all colleagues have contributed substantially to this work. I would like to acknowledge
the Japan Society for the Promotion of Science (JSPS) for supporting me a Reseach
Fellowship for Young Scientists.
95
References
Allard J, Truitt WA, McKenna KE, Coolen LM (2005) Spinal cord control of
ejaculation. World J Urol 23 (2):119-126. doi:10.1007/s00345-004-0494-9
Argiolas A, Melis MR (2013) Neuropeptides and central control of sexual behaviour
from the past to the present: a review. Prog Neurobiol 108:80-107.
doi:10.1016/j.pneurobio.2013.06.006
Bajic D, Proudfit HK (1999) Projections of neurons in the periaqueductal gray to
pontine and medullary catecholamine cell groups involved in the modulation of
nociception. The Journal of comparative neurology 405 (3):359-379
Bardin CW, Bullock L, Blackburn WR, Sherins RJ, Vanha-Perttula T (1971)
Testosterone metabolism in the androgen-insensitive rat: a model for testicular
feminization. Birth Defects Orig Artic Ser 7 (6):185-192
Beach FA, Buehler MG (1977) Male rats with inherited insensitivity to androgen show
reduced
sexual
behavior.
Endocrinology
100
(1):197-200.
doi:10.1210/endo-100-1-197
Belichenko PV, Dahlstrom A (1995) Studies on the 3-dimensional architecture of
dendritic spines and varicosities in human cortex by confocal laser scanning
microscopy and Lucifer yellow microinjections. J Neurosci Methods 57
(1):55-61
Breedlove SM (1985) Hormonal control of the anatomical specificity of
motoneuron-to-muscle innervation in rats. Science 227 (4692):1357-1359
Breedlove SM, Arnold AP (1980) Hormone accumulation in a sexually dimorphic
motor nucleus of the rat spinal cord. Science 210 (4469):564-566
Breedlove SM, Arnold AP (1983a) Hormonal control of a developing neuromuscular
system. I. Complete Demasculinization of the male rat spinal nucleus of the
bulbocavernosus
using
the
anti-androgen
flutamide.
The
Journal
of
neuroscience : the official journal of the Society for Neuroscience 3 (2):417-423
Breedlove SM, Arnold AP (1983b) Hormonal control of a developing neuromuscular
system. II. Sensitive periods for the androgen-induced masculinization of the rat
spinal nucleus of the bulbocavernosus. The Journal of neuroscience : the official
journal of the Society for Neuroscience 3 (2):424-432
Bremer RE, Barber MD, Coates KW, Dolber PC, Thor KB (2003) Innervation of the
96
levator ani and coccygeus muscles of the female rat. Anat Rec A Discov Mol
Cell Evol Biol 275 (1):1031-1041. doi:10.1002/ar.a.10116
Brown DJ, Hill ST, Baker HW (2006) Male fertility and sexual function after spinal
cord injury. Prog Brain Res 152:427-439. doi:10.1016/S0079-6123(05)52029-6
Burke KA, Widows MR, Sengelaub DR (1997) Synergistic effects of testosterone
metabolites on the development of motoneuron morphology in a sexually
dimorphic rat spinal nucleus. J Neurobiol 33 (1):1-10
Cano G, Card JP, Rinaman L, Sved AF (2000) Connections of Barrington's nucleus to
the sympathetic nervous system in rats. J Auton Nerv Syst 79 (2-3):117-128
Card JP, Rinaman L, Lynn RB, Lee BH, Meade RP, Miselis RR, Enquist LW (1993)
Pseudorabies virus infection of the rat central nervous system: ultrastructural
characterization of viral replication, transport, and pathogenesis. The Journal of
neuroscience : the official journal of the Society for Neuroscience 13
(6):2515-2539
Card JP, Rinaman L, Schwaber JS, Miselis RR, Whealy ME, Robbins AK, Enquist LW
(1990) Neurotropic properties of pseudorabies virus: uptake and transneuronal
passage in the rat central nervous system. The Journal of neuroscience : the
official journal of the Society for Neuroscience 10 (6):1974-1994
Clement P, Laurin M, Compagnie S, Facchinetti P, Bernabe J, Alexandre L, Giuliano F
(2012) Effect of dapoxetine on ejaculatory performance and related brain
neuronal activity in rapid ejaculator rats. The journal of sexual medicine 9
(10):2562-2573. doi:10.1111/j.1743-6109.2012.02884.x
Collins WF, 3rd, Erichsen JT, Rose RD (1991) Pudendal motor and premotor neurons
in the male rat: a WGA transneuronal study. The Journal of comparative
neurology 308 (1):28-41. doi:10.1002/cne.903080104
Coolen LM (2005) Neural control of ejaculation. The Journal of comparative neurology
493 (1):39-45. doi:10.1002/cne.20784
De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin M, Devos A, Tan K,
Atanassova N, Claessens F, Lecureuil C, Heyns W, Carmeliet P, Guillou F,
Sharpe RM, Verhoeven G (2004) A Sertoli cell-selective knockout of the
androgen receptor causes spermatogenic arrest in meiosis. Proceedings of the
National Academy of Sciences of the United States of America 101
(5):1327-1332. doi:10.1073/pnas.0308114100
97
Denk W, Svoboda K (1997) Photon upmanship: why multiphoton imaging is more than
a gimmick. Neuron 18 (3):351-357
Ding YQ, Zheng HX, Gong LW, Lu Y, Zhao H, Qin BZ (1997) Direct projections from
the lumbosacral spinal cord to Barrington's nucleus in the rat: a special reference
to micturition reflex. The Journal of comparative neurology 389 (1):149-160
Dobberfuhl AD, Oti T, Sakamoto H, Marson L (2014) Identification of CNS neurons
innervating the levator ani and ventral bulbospongiosus muscles in male rats.
The journal of sexual medicine 11 (3):664-677. doi:10.1111/jsm.12418
Dohler KD, Coquelin A, Davis F, Hines M, Shryne JE, Gorski RA (1984) Pre- and
postnatal influence of testosterone propionate and diethylstilbestrol on
differentiation of the sexually dimorphic nucleus of the preoptic area in male
and female rats. Brain Res 302 (2):291-295
Fishman RB, Breedlove SM (1992) Local perineal implants of anti-androgen block
masculinization of the spinal nucleus of the bulbocavernosus. Brain Res Dev
Brain Res 70 (2):283-286
Forger NG, Breedlove SM (1986) Sexual dimorphism in human and canine spinal cord:
role of early androgen. Proceedings of the National Academy of Sciences of the
United States of America 83 (19):7527-7531
Franssen D, Ioannou YS, Alvarez-real A, Gerard A, Mueller JK, Heger S, Bourguignon
JP, Parent AS (2014) Pubertal timing after neonatal diethylstilbestrol exposure
in female rats: neuroendocrine vs peripheral effects and additive role of prenatal
food restriction. Reprod Toxicol 44:63-72. doi:10.1016/j.reprotox.2013.10.006
Freeman LM, Watson NV, Breedlove SM (1996) Androgen spares androgen-insensitive
motoneurons from apoptosis in the spinal nucleus of the bulbocavernosus in rats.
Hormones and behavior 30 (4):424-433. doi:10.1006/hbeh.1996.0047
Fritschy JM, Grzanna R (1990) Demonstration of two separate descending
noradrenergic pathways to the rat spinal cord: evidence for an intragriseal
trajectory of locus coeruleus axons in the superficial layers of the dorsal horn.
The
Journal
of
comparative
neurology
291
(4):553-582.
doi:10.1002/cne.902910406
Fukuda H, Fukai K (1986) Postural change and straining induced by distension of the
rectum, vagina and urinary bladder of decerebrate dogs. Brain Res 380
(2):276-286
98
Fukuda H, Fukai K, Yamane M, Okada H (1981) Pontine reticular unit responses to
pelvic nerve and colonic mechanical stimulation in the dog. Brain Res 207
(1):59-71
Gerstenberg TC, Levin RJ, Wagner G (1990) Erection and ejaculation in man.
Assessment of the electromyographic activity of the bulbocavernosus and
ischiocavernosus muscles. Br J Urol 65 (4):395-402
Giuliano F (2011) Neurophysiology of erection and ejaculation. The journal of sexual
medicine 8 Suppl 4:310-315. doi:10.1111/j.1743-6109.2011.02450.x
Giuliano F, Rampin O (2000) Central neural regulation of penile erection. Neurosci
Biobehav Rev 24 (5):517-533
Goldstein LA, Kurz EM, Sengelaub DR (1990) Androgen regulation of dendritic
growth and retraction in the development of a sexually dimorphic spinal nucleus.
J Neurosci 10 (3):935-946
Goldstein LA, Sengelaub DR (1992) Timing and duration of dihydrotestosterone
treatment affect the development of motoneuron number and morphology in a
sexually dimorphic rat spinal nucleus. The Journal of comparative neurology
326 (1):147-157. doi:10.1002/cne.903260113
Guan KP, Yuan ZW, Wang WL, Gao F, Su PJ (2007) [Quantitative analysis of sensory
neurons innervating the muscle levator ani in rats with anorectal malformation].
Zhonghua Yi Xue Za Zhi 87 (2):131-133
Hagiwara H, Funabashi T, Mitsushima D, Kimura F (2007) Effects of neonatal
testosterone treatment on sex differences in formalin-induced nociceptive
behavior
in
rats.
Neurosci
Lett
412
(3):264-267.
doi:10.1016/j.neulet.2006.11.020
Hama K, Kosaka T (1981) Neurobiological applications of high voltage electron
microscopy. Trends Neuroscience 4:193-196
Hart BL, Melese-D'Hospital PY (1983) Penile mechanisms and the role of the striated
penile muscles in penile reflexes. Physiol Behav 31 (6):807-813
Hell SW (2007) Far-field optical nanoscopy. Science 316 (5828):1153-1158.
doi:10.1126/science.1137395
Hermann GE, Bresnahan JC, Holmes GM, Rogers RC, Beattie MS (1998) Descending
projections from the nucleus raphe obscurus to pudendal motoneurons in the
male rat. The Journal of comparative neurology 397 (4):458-474
99
Holman SD, Hutchison JB (1991) Lateralized action of androgen on development of
behavior and brain sex differences. Brain research bulletin 27 (2):261-265
Holmes GM, Chapple WD, Leipheimer RE, Sachs BD (1991) Electromyographic
analysis of male rat perineal muscles during copulation and reflexive erections.
Physiol Behav 49 (6):1235-1246
Holmes GM, Hermann GE, Rogers RC, Bresnahan JC, Beattie MS (2002) Dissociation
of the effects of nucleus raphe obscurus or rostral ventrolateral medulla lesions
on eliminatory and sexual reflexes. Physiol Behav 75 (1-2):49-55
Holmes GM, Martau JM, Hermann GE, Rogers RC, Bresnahan JC, Beattie MS (1997)
Nucleus raphe obscurus (nRO) regulation of anorectal motility in rats. Brain Res
759 (2):197-204
Holmes GM, Sachs BD (1994) Physiology and mechanics of rat levator ani muscle:
evidence for a sexual function. Physiol Behav 55 (2):255-266
Holstege JC, Kuypers HG (1987) Brainstem projections to lumbar motoneurons in rat--I.
An ultrastructural study using autoradiography and the combination of
autoradiography and horseradish peroxidase histochemistry. Neuroscience 21
(2):345-367
Hornby JB, Rose JD (1976) Responses of caudal brain stem neurons to vaginal and
somatosensory stimulation in the rat and evidence of genital-nociceptive
interactions. Exp Neurol 51 (2):363-376
Hosoya Y, Sugiura Y, Okado N, Loewy AD, Kohno K (1991) Descending input from
the hypothalamic paraventricular nucleus to sympathetic preganglionic neurons
in the rat. Exp Brain Res 85 (1):10-20
Hull EM, Dominguez JM (2007) Sexual behavior in male rodents. Hormones and
behavior 52 (1):45-55. doi:10.1016/j.yhbeh.2007.03.030
Janig W, McLachlan EM (1987) Organization of lumbar spinal outflow to distal colon
and pelvic organs. Physiological reviews 67 (4):1332-1404
Johansen JA, Breedlove SM, Jordan CL (2007) Androgen receptor expression in the
levator ani muscle of male mice. Journal of neuroendocrinology 19 (10):823-826.
doi:10.1111/j.1365-2826.2007.01592.x
Johnson RD (2006) Descending pathways modulating the spinal circuitry for
ejaculation: effects of chronic spinal cord injury. Prog Brain Res 152:415-426.
doi:10.1016/S0079-6123(05)52028-4
100
Johnson RD, Chadha HK, Dugan VP, Gupta DS, Ferrero SL, Hubscher CH (2011)
Bilateral bulbospinal projections to pudendal motoneuron circuitry after chronic
spinal cord hemisection injury as revealed by transsynaptic tracing with
pseudorabies virus. J Neurotrauma 28 (4):595-605. doi:10.1089/neu.2009.1180
Jones RD, Pugh PJ, Hall J, Channer KS, Jones TH (2003) Altered circulating hormone
levels, endothelial function and vascular reactivity in the testicular feminised
mouse. Eur J Endocrinol 148 (1):111-120
Jordan CL, Letinsky MS, Arnold AP (1989) The role of gonadal hormones in
neuromuscular synapse elimination in rats. I. Androgen delays the loss of
multiple innervation in the levator ani muscle. The Journal of neuroscience : the
official journal of the Society for Neuroscience 9 (1):229-238
Ju G, Melander T, Ceccatelli S, Hokfelt T, Frey P (1987) Immunohistochemical
evidence for a spinothalamic pathway co-containing cholecystokinin- and
galanin-like immunoreactivities in the rat. Neuroscience 20 (2):439-456
Katayama N, Oti T, Takanami K, Sakamoto T, Sakamoto H (2016) Postnatal
development of the gastrin-releasing peptide system in the lumbosacral spinal
cord controlling male reproductive function in rats. Proceedings of the Japan
Academy, Series B, Physical and Biological Sciences
Kojima M, Matsuura T, Tanaka A, Amagai T, Imanishi J, Sano Y (1985) Characteristic
distribution of noradrenergic terminals on the anterior horn motoneurons
innervating
the
perineal
striated
muscles
in
the
rat.
An
immuno-electromicroscopic study. Anat Embryol (Berl) 171 (3):267-273
Kozyrev N, Lehman MN, Coolen LM (2012) Activation of gastrin-releasing peptide
receptors in the lumbosacral spinal cord is required for ejaculation in male rats.
The
journal
of
sexual
medicine
9
(5):1303-1318.
doi:10.1111/j.1743-6109.2012.02688.x
Ladenheim EE, Taylor JE, Coy DH, Moore KA, Moran TH (1996) Hindbrain GRP
receptor blockade antagonizes feeding suppression by peripherally administered
GRP. The American journal of physiology 271 (1 Pt 2):R180-184
Loewy AD, Marson L, Parkinson D, Perry MA, Sawyer WB (1986) Descending
noradrenergic pathways involved in the A5 depressor response. Brain Res 386
(1-2):313-324
Marie-Luce C, Raskin K, Bolborea M, Monin M, Picot M, Mhaouty-Kodja S (2013)
101
Effects of neural androgen receptor disruption on aggressive behavior, arginine
vasopressin and galanin systems in the bed nucleus of stria terminalis and lateral
septum. Gen Comp Endocrinol 188:218-225. doi:10.1016/j.ygcen.2013.03.031
Marson L (1997) Identification of central nervous system neurons that innervate the
bladder body, bladder base, or external urethral sphincter of female rats: a
transneuronal tracing study using pseudorabies virus. The Journal of
comparative neurology 389 (4):584-602
Marson L, Carson 3rd CC (1999) Central Nervous System Innervation of the Penis,
Prostate, and Perineal Muscles: A Transneuronal Tracing Study. Mol Urol 3
(2):43-50
Marson L, Gravitt K (2004) Spinal neurons activated with the urethrogenital reflex in
the male rat. Brain Res 1026 (1):108-115. doi:10.1016/j.brainres.2004.08.019
Marson L, List MS, McKenna KE (1992) Lesions of the nucleus paragigantocellularis
alter ex copula penile reflexes. Brain Res 592 (1-2):187-192
Marson L, McKenna KE (1990) The identification of a brainstem site controlling spinal
sexual reflexes in male rats. Brain Res 515 (1-2):303-308
Marson L, McKenna KE (1992) A role for 5-hydroxytryptamine in descending
inhibition of spinal sexual reflexes. Exp Brain Res 88 (2):313-320
Marson L, McKenna KE (1996) CNS cell groups involved in the control of the
ischiocavernosus and bulbospongiosus muscles: a transneuronal tracing study
using pseudorabies virus. The Journal of comparative neurology 374
(2):161-179.
doi:10.1002/(SICI)1096-9861(19961014)374:2&lt;161::AID-CNE1&gt;3.0.CO;
2-0
Marson L, Platt KB, McKenna KE (1993) Central nervous system innervation of the
penis as revealed by the transneuronal transport of pseudorabies virus.
Neuroscience 55 (1):263-280
Matsuda K, Sakamoto H, Kawata M (2008) Androgen action in the brain and spinal
cord for the regulation of male sexual behaviors. Current opinion in
pharmacology 8 (6):747-751. doi:10.1016/j.coph.2008.08.010
McCarthy MM (2008) Estradiol and the developing brain. Physiological reviews 88
(1):91-124. doi:10.1152/physrev.00010.2007
McCormick CM, Mahoney E (1999) Persistent effects of prenatal, neonatal, or adult
102
treatment with flutamide on the hypothalamic-pituitary-adrenal stress response
of
adult
male
rats.
Hormones
and
behavior
35
(1):90-101.
doi:10.1006/hbeh.1998.1500
McIntyre BS, Barlow NJ, Foster PM (2001) Androgen-mediated development in male
rat offspring exposed to flutamide in utero: permanence and correlation of early
postnatal changes in anogenital distance and nipple retention with malformations
in androgen-dependent tissues. Toxicological sciences : an official journal of the
Society of Toxicology 62 (2):236-249
McKenna KE, Chung SK, McVary KT (1991) A model for the study of sexual function
in anesthetized male and female rats. The American journal of physiology 261
(5 Pt 2):R1276-1285
McKenna KE, Nadelhaft I (1986) The organization of the pudendal nerve in the male
and female rat. The Journal of comparative neurology 248 (4):532-549.
doi:10.1002/cne.902480406
Merali Z, Bedard T, Andrews N, Davis B, McKnight AT, Gonzalez MI, Pritchard M,
Kent P, Anisman H (2006) Bombesin receptors as a novel anti-anxiety
therapeutic target: BB1 receptor actions on anxiety through alterations of
serotonin activity. The Journal of neuroscience : the official journal of the
Society
for
Neuroscience
26
(41):10387-10396.
doi:10.1523/JNEUROSCI.1219-06.2006
Meston CM, Frohlich PF (2000) The neurobiology of sexual function. Arch Gen
Psychiatry 57 (11):1012-1030
Mikkila TF, Toppari J, Paranko J (2006) Effects of neonatal exposure to
4-tert-octylphenol, diethylstilbestrol, and flutamide on steroidogenesis in
infantile rat testis. Toxicological sciences : an official journal of the Society of
Toxicology 91 (2):456-466. doi:10.1093/toxsci/kfj156
Miyawaki A (2003) Fluorescence imaging of physiological activity in complex systems
using GFP-based probes. Curr Opin Neurobiol 13 (5):591-596
Moda Y, Yamane M, Fukuda H, Okada H (1993) Excitation and inhibition of neuronal
activity in the pontine micturition center by pelvic rectal and pudendal anal
afferents in dogs. J Auton Nerv Syst 43 (1):59-68
Molander C, Xu Q, Grant G (1984) The cytoarchitectonic organization of the spinal
cord in the rat. I. The lower thoracic and lumbosacral cord. The Journal of
103
comparative neurology 230 (1):133-141. doi:10.1002/cne.902300112
Monaghan EP, Breedlove SM (1991) Brain sites projecting to the spinal nucleus of the
bulbocavernosus. The Journal of comparative neurology 307 (3):370-374.
doi:10.1002/cne.903070303
Morgan C, Nadelhaft I, de Groat WC (1981) The distribution of visceral primary
afferents from the pelvic nerve to Lissauer's tract and the spinal gray matter and
its relationship to the sacral parasympathetic nucleus. The Journal of
comparative neurology 201 (3):415-440. doi:10.1002/cne.902010308
Morris JA, Jordan CL, Breedlove SM (2004) Sexual differentiation of the vertebrate
nervous system. Nature neuroscience 7 (10):1034-1039. doi:10.1038/nn1325
Murphy AZ, Shipley MT, Vanderhorst VG, Holstege G (1996) Descending projections
from the caudal ventrolateral medulla of the male rat selectively innervate
discrete motoneuronal pools within the lumbosacral cord: Possible role in
reproductive behavior. Society for Neuroscience Abstract 22
Nadelhaft I, Booth AM (1984) The location and morphology of preganglionic neurons
and the distribution of visceral afferents from the rat pelvic nerve: a horseradish
peroxidase study. The Journal of comparative neurology 226 (2):238-245.
doi:10.1002/cne.902260207
Newton BW (1993) Galanin immunoreactivity in rat spinal lamina IX: emphasis on
sexually dimorphic regions. Peptides 14 (5):955-969
Nishi M, Kawata M (2006) Brain corticosteroid receptor dynamics and trafficking:
Implications
from
live
cell
imaging.
Neuroscientist
12
(2):119-133.
doi:10.1177/1073858405279691
Nordeen EJ, Nordeen KW, Sengelaub DR, Arnold AP (1985) Androgens prevent
normally occurring cell death in a sexually dimorphic spinal nucleus. Science
229 (4714):671-673
Nygren LG, Olson L (1977) A new major projection from locus coeruleus: the main
source of noradrenergic nerve terminals in the ventral and dorsal columns of the
spinal cord. Brain Res 132 (1):85-93
Onufrowicz B (1899) Notes on the arrangement and function of the cell groups in the
sacral region of the spinal cord. J Nerv Ment Dis 26:498-504
Orr R, Marson L (1998) Identification of CNS neurons innervating the rat prostate: a
transneuronal tracing study using pseudorabies virus. J Auton Nerv Syst 72
104
(1):4-15
Oti T, Satoh K, Saito K, Murata K, Kawata M, Sakamoto T, Sakamoto H (2012)
Three-dimensional evaluation of the spinal local neural network revealed by the
high-voltage electron microscopy: a double immunohistochemical study.
Histochemistry
and
cell
biology
138
(4):693-697.
doi:10.1007/s00418-012-0976-6
Oti T, Takanami K, Katayama N, Edey T, Satoh K, Sakamoto T, Sakamoto H (2016)
Perinatal testosterone exposure is critical for the development of the
male-specific sexually dimorphic gastrin-releasing peptide system in the
lumbosacral spinal cord that mediates erection and ejaculation. Biol Sex Differ
7:4. doi:10.1186/s13293-016-0058-x
Owens W, Zeiger E, Walker M, Ashby J, Onyon L, Gray LE, Jr. (2006) The OECD
program to validate the rat Hershberger bioassay to screen compounds for in
vivo androgen and antiandrogen responses. Phase 1: use of a potent agonist and
a potent antagonist to test the standardized protocol. Environ Health Perspect
114 (8):1259-1265
Pacheco P, Martinez-Gomez M, Whipple B, Beyer C, Komisaruk BR (1989)
Somato-motor components of the pelvic and pudendal nerves of the female rat.
Brain Res 490 (1):85-94
Panula P, Nieminen O, Falkenberg M, Auvinen S (1988) Localization and development
of bombesin/GRP-like immunoreactivity in the rat central nervous system.
Annals of the New York Academy of Sciences 547:54-69
Pavcovich LA, Yang M, Miselis RR, Valentino RJ (1998) Novel role for the pontine
micturition center, Barrington's nucleus: evidence for coordination of colonic
and forebrain activity. Brain Res 784 (1-2):355-361
Peshori KR, Erichsen JT, Collins WF, 3rd (1995) Differences in the connectivity of rat
pudendal motor nuclei as revealed by retrograde transneuronal transport of
wheat germ agglutinin. The Journal of comparative neurology 353 (1):119-128.
doi:10.1002/cne.903530111
Phan DC, Newton BW (1999) Cholecystokinin-8-like immunoreactivity is sexually
dimorphic in a midline population of rat lumbar neurons. Neurosci Lett 276
(3):165-168
Phoenix CH, Goy RW, Gerall AA, Young WC (1959) Organizing action of prenatally
105
administered testosterone propionate on the tissues mediating mating behavior
in
the
female
guinea
pig.
Endocrinology
65:369-382.
doi:10.1210/endo-65-3-369
Pierce LM, Reyes M, Thor KB, Dolber PC, Bremer RE, Kuehl TJ, Coates KW (2005)
Immunohistochemical evidence for the interaction between levator ani and
pudendal motor neurons in the coordination of pelvic floor and visceral activity
in the squirrel monkey. Am J Obstet Gynecol 192 (5):1506-1515.
doi:10.1016/j.ajog.2004.10.607
Poortmans A, Wyndaele JJ (1998) M. levator ani in the rat: does it really lift the anus?
Anat Rec 251 (1):20-27
Ranson RN, Dowling P, Santer RM, Watson AH (2007) The effects of ageing on the
distribution of vesicular acetylcholine transporter immunoreactive inputs to
pelvic motoneurons of male Wistar rats. Neuroscience 144 (2):636-644.
doi:10.1016/j.neuroscience.2006.09.036
Ranson RN, Santer RM, Watson AH (2005) Biogenic amine and neuropeptide inputs to
identified pelvic floor motoneurons that also express SRC-1. Neurosci Lett 382
(3):248-253. doi:10.1016/j.neulet.2005.03.040
Raskin K, de Gendt K, Duittoz A, Liere P, Verhoeven G, Tronche F, Mhaouty-Kodja S
(2009) Conditional inactivation of androgen receptor gene in the nervous
system: effects on male behavioral and neuroendocrine responses. The Journal
of neuroscience : the official journal of the Society for Neuroscience 29
(14):4461-4470. doi:10.1523/JNEUROSCI.0296-09.2009
Raskin K, Marie-Luce C, Picot M, Bernard V, Mailly P, Hardin-Pouzet H, Tronche F,
Mhaouty-Kodja S (2012) Characterization of the spinal nucleus of the
bulbocavernosus neuromuscular system in male mice lacking androgen receptor
in
the
nervous
system.
Endocrinology
153
(7):3376-3385.
doi:10.1210/en.2012-1001
Roppolo JR, Nadelhaft I, de Groat WC (1985) The organization of pudendal
motoneurons and primary afferent projections in the spinal cord of the rhesus
monkey revealed by horseradish peroxidase. The Journal of comparative
neurology 234 (4):475-488. doi:10.1002/cne.902340406
Roselli CE, Salisbury RL, Resko JA (1987) Genetic evidence for androgen-dependent
and independent control of aromatase activity in the rat brain. Endocrinology
106
121 (6):2205-2210. doi:10.1210/endo-121-6-2205
Sachs B (1983) Potency and fertelity. Hormonal and mechanical causes and effects of
penile actions in rats. In: Balthazart J, Prove E, Gilles R (eds) Hormones and
behavior in higher vertabrates. Springer-Verlag, Berlin, pp 87-110
Sachs BD, Meisel RL (1979) Pubertal development of penile reflexes and copulation in
male rats. Psychoneuroendocrinology 4 (4):287-296
Sakamoto H (2011) Gastrin-releasing peptide system in the spinal cord mediates
masculine
sexual
function.
Anat
Sci
Int
86
(1):19-29.
doi:10.1007/s12565-010-0097-z
Sakamoto H (2012) Brain-spinal cord neural circuits controlling male sexual function
and behavior. Neurosci Res 72 (2):103-116. doi:10.1016/j.neures.2011.11.002
Sakamoto H (2014) Sexually dimorphic nuclei in the spinal cord control male sexual
functions. Front Neurosci 8:184. doi:10.3389/fnins.2014.00184
Sakamoto H, Arii T, Kawata M (2010) High-voltage electron microscopy reveals direct
synaptic inputs from a spinal gastrin-releasing peptide system to neurons of the
spinal
nucleus
of
bulbocavernosus.
Endocrinology
151
(1):417-421.
doi:10.1210/en.2009-0485
Sakamoto H, Kawata M (2009) Gastrin-releasing peptide system in the spinal cord
controls male sexual behaviour. Journal of neuroendocrinology 21 (4):432-435.
doi:10.1111/j.1365-2826.2009.01847.x
Sakamoto H, Matsuda K, Zuloaga DG, Hongu H, Wada E, Wada K, Jordan CL,
Breedlove SM, Kawata M (2008) Sexually dimorphic gastrin releasing peptide
system in the spinal cord controls male reproductive functions. Nature
neuroscience 11 (6):634-636. doi:10.1038/nn.2126
Sakamoto H, Matsuda K, Zuloaga DG, Nishiura N, Takanami K, Jordan CL, Breedlove
SM, Kawata M (2009a) Stress affects a gastrin-releasing peptide system in the
spinal cord that mediates sexual function: implications for psychogenic erectile
dysfunction. PloS one 4 (1):e4276. doi:10.1371/journal.pone.0004276
Sakamoto H, Oti T (2015) Oxytocin and the gastrin-releasing peptide system in the
spinal cord: Implications for male sexual problems. Interdisciplinary
Information Sciences 21 (3):235-242. doi:10.4036/iis.2015.B.08
Sakamoto H, Saito K, Marie-Luce C, Raskin K, Oti T, Satoh K, Tamura K, Sakamoto T,
Mhaouty-Kodja S (2014) Androgen regulates development of the sexually
107
dimorphic gastrin-releasing peptide neuron system in the lumbar spinal cord:
evidence from a mouse line lacking androgen receptor in the nervous system.
Neurosci Lett 558:109-114. doi:10.1016/j.neulet.2013.10.068
Sakamoto H, Takahashi H, Matsuda K, Nishi M, Takanami K, Ogoshi M, Sakamoto T,
Kawata M (2012) Rapid signaling of steroid hormones in the vertebrate nervous
system. Front Biosci (Landmark Ed) 17:996-1019
Sakamoto H, Takanami K, Zuloaga DG, Matsuda K, Jordan CL, Breedlove SM, Kawata
M (2009b) Androgen regulates the sexually dimorphic gastrin-releasing peptide
system in the lumbar spinal cord that mediates male sexual function.
Endocrinology 150 (8):3672-3679. doi:10.1210/en.2008-1791
Salas JC, Iwasaki H, Jodo E, Schmidt MH, Kawauchi A, Miki T, Kayama Y, Otsuki M,
Koyama Y (2008) Penile erection and micturition events triggered by electrical
stimulation of the mesopontine tegmental area. Am J Physiol Regul Integr Comp
Physiol 294 (1):R102-111. doi:10.1152/ajpregu.00226.2007
Sasaki M (2005) Role of Barrington's nucleus in micturition. The Journal of
comparative neurology 493 (1):21-26. doi:10.1002/cne.20719
Schmidt MH, Schmidt HS (1993) The ischiocavernosus and bulbospongiosus muscles
in mammalian penile rigidity. Sleep 16 (2):171-183
Schroder HD (1980) Organization of the motoneurons innervating the pelvic muscles of
the male rat. The Journal of comparative neurology 192 (3):567-587.
doi:10.1002/cne.901920313
Schroder HD (1981) Onuf's nucleus X: a morphological study of a human spinal
nucleus. Anat Embryol (Berl) 162 (4):443-453
Schroder HD (1985) Anatomical and pathoanatomical studies on the spinal efferent
systems innervating pelvic structures. 1. Organization of spinal nuclei in animals.
2. The nucleus X-pelvic motor system in man. J Auton Nerv Syst 14 (1):23-48
Schroder HD, Skagerberg G (1985) Catecholamine innervation of the caudal spinal cord
in the rat. The Journal of comparative neurology 242 (3):358-368.
doi:10.1002/cne.902420305
Sengelaub DR, Arnold AP (1986) Development and loss of early projections in a
sexually dimorphic rat spinal nucleus. The Journal of neuroscience : the official
journal of the Society for Neuroscience 6 (6):1613-1620
Sengelaub DR, Forger NG (2008) The spinal nucleus of the bulbocavernosus: firsts in
108
androgen-dependent neural sex differences. Hormones and behavior 53
(5):596-612. doi:10.1016/j.yhbeh.2007.11.008
Sengelaub DR, Nordeen EJ, Nordeen KW, Arnold AP (1989) Hormonal control of
neuron number in sexually dimorphic spinal nuclei of the rat: III. Differential
effects of the androgen dihydrotestosterone. The Journal of comparative
neurology 280 (4):637-644. doi:10.1002/cne.902800413
Shinohara K, Tominaga K, Isobe Y, Inouye ST (1993) Photic regulation of peptides
located in the ventrolateral subdivision of the suprachiasmatic nucleus of the rat:
daily variations of vasoactive intestinal polypeptide, gastrin-releasing peptide,
and neuropeptide Y. The Journal of neuroscience : the official journal of the
Society for Neuroscience 13 (2):793-800
Sipski ML (1998) Sexual functioning in the spinal cord injured. Int J Impot Res 10
Suppl 2:S128-130; discussion S138-140
Sisk CL, Zehr JL (2005) Pubertal hormones organize the adolescent brain and behavior.
Front Neuroendocrinol 26 (3-4):163-174. doi:10.1016/j.yfrne.2005.10.003
Sodersten P, Damassa DA, Smith ER (1977) Sexual behavior in developing male rats.
Hormones and behavior 8 (3):320-341
Studeny S, Vizzard MA (2005) Corticotropin-releasing factor (CRF) expression in
postnatal and adult rat sacral parasympathetic nucleus (SPN). Cell Tissue Res
322 (3):339-352. doi:10.1007/s00441-005-0014-2
Sun YG, Chen ZF (2007) A gastrin-releasing peptide receptor mediates the itch
sensation
in
the
spinal
cord.
Nature
448
(7154):700-703.
doi:10.1038/nature06029
Sun YG, Zhao ZQ, Meng XL, Yin J, Liu XY, Chen ZF (2009) Cellular basis of itch
sensation. Science 325 (5947):1531-1534. doi:10.1126/science.1174868
Takanami K, Sakamoto H (2014) The Gastrin-Releasing Peptide Receptor (GRPR) in
the Spinal Cord as a Novel Pharmacological Target. Curr Neuropharmacol 12
(5):434-443. doi:10.2174/1570159X12666140923201432
Takanami K, Sakamoto H, Matsuda KI, Satoh K, Tanida T, Yamada S, Inoue K, Oti T,
Sakamoto T, Kawata M (2014) Distribution of gastrin-releasing peptide in the
rat trigeminal and spinal somatosensory systems. The Journal of comparative
neurology 522 (8):1858-1873. doi:10.1002/cne.23506
Thor KB, de Groat WC (2010) Neural control of the female urethral and anal
109
rhabdosphincters and pelvic floor muscles. Am J Physiol Regul Integr Comp
Physiol 299 (2):R416-438. doi:10.1152/ajpregu.00111.2010
Thor KB, Morgan C, Nadelhaft I, Houston M, De Groat WC (1989) Organization of
afferent and efferent pathways in the pudendal nerve of the female cat. The
Journal of comparative neurology 288 (2):263-279. doi:10.1002/cne.902880206
Truitt WA, Coolen LM (2002) Identification of a potential ejaculation generator in the
spinal cord. Science 297 (5586):1566-1569. doi:10.1126/science.1073885
Truitt WA, Shipley MT, Veening JG, Coolen LM (2003) Activation of a subset of
lumbar spinothalamic neurons after copulatory behavior in male but not female
rats. The Journal of neuroscience : the official journal of the Society for
Neuroscience 23 (1):325-331
Ueyama T, Mizuno N, Takahashi O, Nomura S, Arakawa H, Matsushima R (1985)
Central distribution of efferent and afferent components of the pudendal nerve in
macaque monkeys. The Journal of comparative neurology 232 (4):548-556.
doi:10.1002/cne.902320411
Verhovshek T, Buckley KE, Sergent MA, Sengelaub DR (2010) Testosterone
metabolites differentially maintain adult morphology in a sexually dimorphic
neuromuscular
system.
Developmental
neurobiology
70
(4):206-221.
doi:10.1002/dneu.20780
Viau V, Bingham B, Davis J, Lee P, Wong M (2005) Gender and puberty interact on
the stress-induced activation of parvocellular neurosecretory neurons and
corticotropin-releasing hormone messenger ribonucleic acid expression in the rat.
Endocrinology 146 (1):137-146. doi:10.1210/en.2004-0846
Vizzard MA, Erdman SL, de Groat WC (1995a) Increased expression of neuronal nitric
oxide synthase (NOS) in visceral neurons after nerve injury. The Journal of
neuroscience : the official journal of the Society for Neuroscience 15 (5 Pt
2):4033-4045
Vizzard MA, Erickson VL, Card JP, Roppolo JR, de Groat WC (1995b) Transneuronal
labeling of neurons in the adult rat brainstem and spinal cord after injection of
pseudorabies virus into the urethra. The Journal of comparative neurology 355
(4):629-640. doi:10.1002/cne.903550411
Wagner CK, Clemens LG (1991) Projections of the paraventricular nucleus of the
hypothalamus to the sexually dimorphic lumbosacral region of the spinal cord.
110
Brain Res 539 (2):254-262
Wagner CK, Sisk CL, Clemens LG (1993) Neurons in the paraventricular nucleus of the
hypothalamus that project to the sexually dimorphic lower lumbar spinal cord
concentrate 3H-estradiol in the male rat. Journal of neuroendocrinology 5
(5):545-551
Wallner C, Dabhoiwala NF, DeRuiter MC, Lamers WH (2009) The anatomical
components
of
urinary
continence.
Eur
Urol
55
(4):932-943.
doi:10.1016/j.eururo.2008.08.032
Wee BE, Clemens LG (1987) Characteristics of the spinal nucleus of the
bulbocavernosus are influenced by genotype in the house mouse. Brain Res 424
(2):305-310
Xu C, Giuliano F, Yaici ED, Conrath M, Trassard O, Benoit G, Verge D (2006)
Identification of lumbar spinal neurons controlling simultaneously the prostate
and the bulbospongiosus muscles in the rat. Neuroscience 138 (2):561-573.
doi:10.1016/j.neuroscience.2005.11.016
Xu C, Yaici ED, Conrath M, Blanchard P, Leclerc P, Benoit G, Verge D, Giuliano F
(2005) Galanin and neurokinin-1 receptor immunoreactive [corrected] spinal
neurons controlling the prostate and the bulbospongiosus muscle identified by
transsynaptic
labeling
in
the
rat.
Neuroscience
134
(4):1325-1341.
doi:10.1016/j.neuroscience.2005.06.002
Yarbrough WG, Quarmby VE, Simental JA, Joseph DR, Sar M, Lubahn DB, Olsen KL,
French FS, Wilson EM (1990) A single base mutation in the androgen receptor
gene causes androgen insensitivity in the testicular feminized rat. J Biol Chem
265 (15):8893-8900
Yells DP, Hendricks SE, Prendergast MA (1992) Lesions of the nucleus
paragigantocellularis: effects on mating behavior in male rats. Brain Res 596
(1-2):73-79
Zhang JM, Konkle AT, Zup SL, McCarthy MM (2008) Impact of sex and hormones on
new cells in the developing rat hippocampus: a novel source of sex dimorphism?
The
European
journal
of
neuroscience
27
(4):791-800.
doi:10.1111/j.1460-9568.2008.06073.x
Zhang JM, Tonelli L, Regenold WT, McCarthy MM (2010) Effects of neonatal
flutamide treatment on hippocampal neurogenesis and synaptogenesis correlate
111
with depression-like behaviors in preadolescent male rats. Neuroscience 169
(1):544-554. doi:10.1016/j.neuroscience.2010.03.029
Zuloaga DG, Morris JA, Jordan CL, Breedlove SM (2008a) Mice with the testicular
feminization mutation demonstrate a role for androgen receptors in the
regulation of anxiety-related behaviors and the hypothalamic-pituitary-adrenal
axis. Hormones and behavior 54 (5):758-766. doi:10.1016/j.yhbeh.2008.08.004
Zuloaga DG, Morris JA, Monks DA, Breedlove SM, Jordan CL (2007)
Androgen-sensitivity of somata and dendrites of spinal nucleus of the
bulbocavernosus (SNB) motoneurons in male C57BL6J mice. Hormones and
behavior 51 (2):207-212. doi:10.1016/j.yhbeh.2006.10.003
Zuloaga DG, Puts DA, Jordan CL, Breedlove SM (2008b) The role of androgen
receptors in the masculinization of brain and behavior: what we've learned from
the testicular feminization mutation. Hormones and behavior 53 (5):613-626.
doi:10.1016/j.yhbeh.2008.01.013
112