794
The Journal of Experimental Biology 214, 794-805
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jeb.051037
RESEARCH ARTICLE
Environmental complexity, seasonality and brain cell proliferation in a weakly electric
fish, Brachyhypopomus gauderio
Kent D. Dunlap1,*, Ana C. Silva2,3 and Michael Chung1
1
Department of Biology, Trinity College, Hartford, CT 06106, USA, 2Unidad Bases Neurales de la Conducta, Instituto de
Investigaciones Biológicas Clemente Estable, Montevideo 11600, Uruguay and 3Laboratorio de Neurociencias, Facultad de
Ciencias, Universidad de la República, Montevideo 11400, Uruguay
*Author for correspondence ([email protected])
Accepted 11 November 2010
SUMMARY
Environmental complexity and season both influence brain cell proliferation in adult vertebrates, but their relative importance and
interaction have not been directly assessed. We examined brain cell proliferation during both the breeding and non-breeding
seasons in adult male electric fish, Brachyhypopomus gauderio, exposed to three environments that differed in complexity: (1) a
complex natural habitat in northern Uruguay, (2) an enriched captive environment where fish were housed socially and (3) a
simple laboratory setting where fish were isolated. We injected fish with BrdU 2.5h before sacrifice to label newborn cells. We
examined the hindbrain and midbrain and quantified the density of BrdU+ cells in whole transverse sections, proliferative zones
and two brain nuclei in the electrocommunication circuitry (the pacemaker nucleus and the electrosensory lateral line lobe).
Season had the largest effect on cell proliferation, with fish during the breeding season having three to seven times more BrdU+
cells than those during the non-breeding season. Although the effect was smaller, fish from a natural environment had greater
rates of cell proliferation than fish in social or isolated captive environments. For most brain regions, fish in social and isolated
captive environments had equivalent levels of cell proliferation. However, for brain regions in the electrocommunication circuitry,
group-housed fish had more cell proliferation than isolated fish, but only during the breeding season (season ⫻ environment
interaction). The regionally and seasonally specific effect of social environment on cell proliferation suggests that addition of new
cells to these nuclei may contribute to seasonal changes in electrocommunication behavior.
Key words: brain cell proliferation, season, environmental enrichment, electrocommunication, electric fish, Brachyhypopomus.
INTRODUCTION
The brains of animals living in complex environments differ
structurally from those of animals living in simplified laboratory
environments. Beginning in the 1960s, researchers showed that
environmental stimuli modify many features of brain structure,
including cortical size, neuronal architecture and synapse formation
(Diamond et al., 1966). More recent studies demonstrate that
environmental complexity also influences cell proliferation and
neurogenesis in the adult brain. For example, rodents housed with
conspecifics and enriched physical environments produce more
neurons in the hippocampus than those living alone in standard
laboratory housing (Kempermann et al., 1997). These studies have
greatly contributed to our understanding of specific stimuli that can
enhance neurogenesis (Fabel et al., 2009; Kozorovitskiy and Gould,
2004; Lu et al., 2003; Stranahan et al., 2006). However, because
they were conducted in laboratory conditions, their conclusions may
not hold true for animals in nature, where environments are much
more complex.
When examined in animals living in natural habitats, brain cell
proliferation or survival is greatly enhanced by the complex physical
and social stimuli in the wild (Amerin et al., 2008; Barnea, 2010;
Barnea and Nottebohm, 1996; Boonstra et al., 2001; DelgadoGonzalez et al., 2008; Hansen and Schmidt, 2004; LaDage et al.,
2010). Most free-living animals face seasonal changes in the
physical and social environment and undergo corresponding drastic
changes in physiology and behavior (e.g. breeding and food
caching). Such natural seasonal changes affect brain cell proliferation
(or survival) in some but not all species (Amerin et al., 2008; Galea
and McEwen, 1999; Hoshooley and Sherry, 2004; Lavenex et al.,
2000; Ormerod and Galea, 2003; Sampedro et al., 2008; Thompson
and Brenowitz, 2005).
Despite many studies showing that enriched captive environments
and complex natural environments can influence brain cell
proliferation, the relative importance of season and environmental
complexity has not been addressed in depth in tetrapods and not at
all in fish. We examined brain cell proliferation in a weakly electric
fish, Brachyhypopomus gauderio, in three environments (natural
habitat, enriched laboratory environment and isolated laboratory
housing) across two seasons to estimate the relative importance of
seasonal, social and physical features of the environment.
Weakly electric fish, such as B. gauderio, are particularly suitable
for addressing this issue because specific brain regions – those used
for electrogenesis and electroreception – are directly linked to the
ways that the fish sense and interact with the physical and social
environment. Fish navigate through the environment by sensing how
nearby objects distort a self-generated electric signal, the electric
organ discharge (EOD). The electric signal originates in a hindbrain
nucleus, the pacemaker nucleus (Pn), which stimulates the discharge
of the electric organ, located primarily in the tail. Electroreceptors
distributed all over the skin of the fish detect spatial and temporal
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Brain cell proliferation in electric fish
variation in electric fields and pass information for primary sensory
processing to the electrosensory lateral line lobe (ELL) in the
hindbrain.
The EOD also is used as the primary means of social
communication. EOD rate and waveform are sexually dimorphic,
hormone sensitive and seasonally variable and thereby convey
information about the sex and reproductive status of the fish
(Salazar and Stoddard, 2009; Silva et al., 2002; Silva et al., 2007;
Silva et al., 2008; Stoddard et al., 2006). Fish can also make rapid
EOD modulations that function in aggression and courtship. Several
EOD modulations are generated by diencephalic nuclei, such as the
prepacemaker nucleus (PPn), that transiently modify the activity of
the Pn (Perrone et al., 2009; Zakon et al., 2002; Zupanc, 2002).
Because these three brain regions – Pn, ELL and PPn – all function
directly in interactions with the environment, we were particularly
interested in their response to environmental change. We examined
whether these specific brain regions or proliferative zones that
contribute cells to these regions showed particularly high levels of
cell proliferation when fish were exposed to environments that varied
in physical and social complexity.
A second advantage of studying environmental influences on
brain cell proliferation in electric fish is that a number of laboratory
studies on one species (Apteronotus leptorhynchus) have previously
detailed patterns of cell proliferation and social influences on cell
addition. In A. leptorhynchus, cell proliferation is abundant
throughout the brain. In a 2h period, approximately 0.2% of all
brain cells undergo mitosis, a rate ~10–100 times that found in
proliferative zones in the adult rodent brain (Zupanc, 2008). Cell
birth is particularly concentrated in proliferation zones in the
cerebellum and the periventricular zone (PVZ) but is also scattered
at lower densities throughout the brain (Zupanc and Horschke, 1995).
Cell addition in the PVZ that contributes cells to the PPn is enhanced
by experimental manipulation of social interactions in the laboratory
(Dunlap et al., 2006). Thus, there is good reason to believe that
natural changes in the social environment may affect cell
proliferation, especially in brain regions associated with
electrocommunication.
Among electric fish, B. gauderio is particularly appropriate for
assessing the role of natural stimuli and seasonal change on brain
cell proliferation because of the extensive previous field and
laboratory work documenting seasonal, hormonal and social
influences on their electrocommunication behavior and the key brain
nuclei used in electrocommunication (Quintana et al., 2004; Salazar
and Stoddard, 2009; Silva et al., 2007). Indeed, more is known about
the seasonality, natural behavior and ecology of B. gauderio than
any other South American electric fish. In Uruguay, these fish inhabit
shallow freshwater lakes and streams that are commonly covered
by floating vegetation (Silva et al., 2003). During the breeding season
(October–February), they live in dense concentrations, with fish
usually separated by less than 1m (Miranda et al., 2008). In the
non-breeding season (March–September), fish continue to aggregate,
but their overall density is lower than in the breeding season (A.C.S.,
personal observations).
In our study of B. gauderio, we found that season had a large
effect on cell proliferation rates, with environmental complexity
(natural habitat vs enriched captivity vs isolated captivity) having
a secondary but still significant effect. Fish during the breeding
season and those living in a natural habitat had much greater rates
of cell proliferation throughout the brain than fish during the nonbreeding season and fish in captivity. In addition, fish living in
enriched captive environments that allowed for abundant social
interaction had higher rates of brain cell proliferation than fish in
isolation. This effect was specific to the breeding season and present
only in brain regions that are associated with electrogenesis and
electrosensation. These results suggest that environmentally induced
changes in brain cell proliferation may contribute to changes in social
signaling associated with reproduction.
MATERIALS AND METHODS
We measured brain cell proliferation and plasma 11-ketotestosterone
(11kT) levels in male electric fish, Brachyhypopomus gauderio
(Giora & Balabarba 2009), in three different treatment groups: (1)
fish collected directly from the natural habitat (‘field’); (2) fish
housed in groups held in large, semi-natural outdoor pools (‘group’);
and (3) fish housed in isolation in simple indoor aquaria (‘isolated’).
We conducted this experiment during both the breeding season (midJanuary to February) and the non-breeding season (late May to midJune). The similarities and differences across seasons and
environments are presented in Tables1 and 2. We examined the
brains of six to eight male fish from each subject group. The length
of fish ranged from 14 to 19cm, and all subject groups had equivalent
mean (±s.e.m.) body length (16.1±0.4cm). A previous study has
Table 1. Comparison of environments in different seasons in each treatment group
Group comparisons
Breeding vs non-breeding (field)
Physical environment
Biotic environment
Social environment
Breeding vs non-breeding (group)
Physical environment
Biotic environment
Social environment
Breeding vs non-breeding (isolated)
Physical environment
Similarities
Differences
Size and complexity of physical environment
Water conductivity
Unknown
Living in aggregations
Mean water temperature
Photoperiod
Unknown
Reproductive signaling
Social spacing
Age structure of conspecifics
Size of physical environment
Water conductivity
Presence of retreat sites
Plant cover
Food availability
Sex and age composition of group
Overall density of fish
Mean water temperature
Photoperiod
Size of physical environment
Food availability
Water conductivity
795
None
Reproductive signaling and behavior
Mean water temperature
Photoperiod
THE JOURNAL OF EXPERIMENTAL BIOLOGY
796
K. D. Dunlap, A. C. Silva and M. Chung
Table 2. Comparison of environments in different treatment groups
Group comparisons
Isolated vs group
Physical environment
Biotic environment
Social environment
Group vs field
Physical environment
Biotic environment
Social environment
Similarities
Mean water temperature
Photoperiod
Water conductivity
None
None
Differences
Size of physical environment
Presence of plants
Presence of conspecifics
Size and complexity of habitat
Mean water temperature
Diversity of retreat sites
Photoperiod
Water conductivity
Availability of retreat sites
Presence of plants
Density and diversity of plants, predators, prey and pathogens
Presence of conspecifics in a female-biased sex Density of conspecifics
ratio
Stability of social group
Presence of juveniles
reliably shown that fish greater than 12cm in length are adult animals
(Franchina, 1997) and thus all fish in our study were well within
the size range for adults.
Animals and field site
All fish in the study were collected from a lake, Laguna Lavalle,
in northern Uruguay (Department of Tacuarembo, 31°48⬘S,
55°13⬘W). The ecology and social behavior of this population have
been described previously (Miranda et al., 2008), and the
reproductive cycle and behavior of a neighboring population of B.
gauderio have been studied extensively (Perrone et al., 2009;
Quintana et al., 2004; Silva et al., 2003).
We collected fish in mid-January 2009, during the breeding
season, and in late May 2009, during the non-breeding season. At
the time of sampling, the water temperature was 25–27°C in January
and 17–18°C in May. We located fish using a detector that
transduces the electric signal into an audio signal, and captured fish
with scoop nets. We placed fish individually in inflated plastic bags
partially filled with water and floated them on the lake before
bringing them to the shore. At the shore, approximately one-third
of the fish were injected with bromodeoxyuridine (BrdU) and bled
(see below) to measure cell proliferation and androgen levels in the
field; the remaining fish were packaged for transport to the
laboratory.
Captive conditions
During both the breeding and non-breeding seasons, fish were
housed in captivity under one of two conditions: social groups in
semi-natural outdoor pools or isolated in a controlled environmental
chamber. During each season, we created four pools (250l,
80⫻80⫻40cm length⫻width⫻diameter) on the roof of the
laboratory at Instituto Investigaciones Biologicas Clemente Estable
in Montevideo, Uruguay (Perrone et al., 2009). Each pool housed
12–16 adult fish. The social composition was three to four males
and nine to 12 females per group to maintain a constant sex ratio
of 1:3, which mimicked the adult sex ratio in the natural breeding
population (Miranda et al., 2008). Each pool contained floating
plants (Eichhornia crassipes, Pistia stratiotes) native to the natural
habitat of the fish and six to eight PVC tubes for shelter. During
the breeding season, the mean water temperature of the pools was
27–30°C; during the non-breeding season, it was 16–17°C. In
previous experiments, fish living in these conditions showed
seasonal changes in reproductive physiology and behavior that
parallel the seasonal changes in the wild (Silva et al., 2007;
Quintana et al., 2004).
Isolated fish were housed in individual 10–12l tanks with a shelter
tube and kept at a constant light cycle and temperature that
mimicked field conditions during each season. During the breeding
season, the light cycle was 14h:10h light:dark and the temperature
was a constant 28°C. During the non-breeding season, the light cycle
was 10h:14h light:dark and the temperature was a constant 17°C.
All fish were fed with Tubifex tubifex worms three times per week.
Fish were kept under these conditions for 2–4weeks before they
were terminally anesthetized in late February and mid-June.
BrdU injection, and processing of brains and plasma samples
To label newborn brain cells from fish in the field, we injected fish
with BrdU (Sigma B-9285; 20gg–1 body weight) 15–60min after
capture and held them in plastic boxes filled with lake water (1–2l)
and placed in the shade. Water temperature was 24–27°C during
the period of BrdU incorporation. Approximately 2.5h
(135–165min) following BrdU injection, fish were killed with 2phenoxyethanol (0.075%). Brains were dissected and placed on ice
in buffered paraformaldehyde (4%, 90–120min) followed by a series
of washes in PBS (3⫻30min). Brains were then placed in sucrose
(30%, 4–8h) and a 1:1 mixture of sucrose and mounting medium
for cryoprotection (4–8h), placed in mounting medium alone and
quickly frozen by covering them with pulverized dry ice.
For plasma androgen measurements, fish were bled from the
caudal vein using a heparinized syringe and needle (25G) within
3h after capture and 3min after anesthesia. Blood samples were
placed on ice for 4–8h and then centrifuged. All brain and plasma
samples were stored on dry ice in the field for 1–2d until they were
shipped to the laboratory, where they were stored at –80°C.
Fish were measured for body length and dissected to determine
sex and gonadal state. During the non-breeding season, when the
sex of fish was sometimes ambiguous, gonads were preserved in
paraformaldehyde and later examined under a dissecting scope.
Testes were scored according to three phases in the annual cycle of
this species: 1regressing or recovering gonads, 2maturing gonads
and 3mature gonads (Quintana et al., 2004). Visual inspection did
not allow us to discriminate between regressing and recovering
gonads, so they were pooled in a single group.
For captive fish, the procedures for BrdU injection, brain
processing and plasma sampling were identical to those for the field
fish, except for the following: in the laboratory, fish were injected
with BrdU within 15–20min after capture, water temperature during
the period of BrdU incorporation was 23–24°C and brains were
frozen in 2-methylbutane (–80°C) after sucrose cryoprotection.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Brain cell proliferation in electric fish
BrdU immunohistochemistry
To identify newborn cells, we used BrdU immunohistochemistry
with a protocol identical to that used in a similar study (Dunlap et
al., 2006). In brief, sections were treated with citrate buffer
(5mmoll–1, 10min, 95°C) and pepsin solution (2.5% pepsin in
0.1moll–1 HCl, 3min, 37°C) to expose the BrdU epitope. Sections
were treated sequentially with sheep anti-BrdU (1:200, overnight,
4°C; Capralogics Inc., Hardwick, MA, USA), biotin-conjugated
donkey anti-sheep IgG (1:800, 2h, room temperature; Chemicon
International, Temecula, CA, USA) and streptavidin conjugated to
Alexa 488 (1:800, 1h, room temperature; Molecular Probes, Eugene,
OR, USA).
797
averaged the values of two to five randomly chosen sections in each
axial level.
Measurement of plasma 11-ketotestoterone
We measured 11kT in plasma samples (5–10l) of male fish
(N5–6) using an enzyme linked immunoassay kit (Cayman
Chemical Co., Ann Arbor, MI, USA). The detection limit for this
assay is 6pgml–1, and the cross-reactivity of the 11kT antibody is
<0.05% with other androgens. We assayed plasma samples in
duplicate in a single run of the assay. The intra-assay variation was
3.3%.
Statistical analysis
Quantifying the distribution of BrdU+ cells
In quantifying our observations, we addressed three general
questions: (1) what is the overall, global rate of cell proliferation;
(2) what fraction of newborn cells are generated in specific
proliferation zones; and (3) what is the rate of cell proliferation in
brain regions specifically associated with electrogenesis and
electrosensation? We examined transverse sections of the brain using
a Nikon Eclipse E600 epifluorescence microscope and used the brain
atlas of a closely related electric fish, Apteronotus leptorhynchus
(Maler et al., 1991), as a reference to identify particular brain nuclei.
We estimated the density of BrdU labeling at four axial levels, two
regions of the hindbrain (sections –9 to –7 and sections –7 to –5 of
the Maler atlas) and two regions of the midbrain (sections 14–16
and sections 17–19, which we term the posterior and anterior
midbrain, respectively). Each region spanned ~500m in axial
length.
To estimate the overall cell proliferation, we counted all the
BrdU+ cells in one lateral half of each transverse section and
doubled this value to estimate the number of cells born in the whole
section. To estimate the number of newborn cells in two
proliferative zones, we counted all the BrdU cells in a 100m
band surrounding the ventricle (the PVZ) in the hindbrain and
midbrain and a 100m band along the dorsal border of the
hindbrain cistern, which contains the eminentia granularis pars
medialis (EGm). To estimate the number of cells born outside of
these proliferative zones, the ‘background’ area, we subtracted the
number of BrdU+ cells in the PVZ and EGm from those in the
whole section. To quantify the relative importance of proliferative
zones as sites of cell proliferation, we divided the number of BrdU+
cells in the PVZ and EGm by the total number of cells in each
transverse section. To quantify cell proliferation in the
electrocommunication system, we counted BrdU+ cells unilaterally
in the ELL in atlas sections –9 to –6 and bilaterally in the Pn in
atlas sections –8 to –5. Cell counts in the ELL were doubled to
estimate the total number in the whole section. We did not directly
assess cell proliferation in the PPn. Instead, we used BrdU+ cell
density in anterior midbrain PVZ as an indirect measure of cell
addition to the PPn because, in the closely related electric fish A.
leptorhynchus, the majority of cells added to the PPn during
adulthood arise from the adjacent PVZ in the anterior midbrain
(Zupanc and Zupanc, 1992).
The area of each whole transverse section and brain region was
quantified by capturing an image of the sections and then measuring
the area using NIH Image J software (Bethesda, MD, USA). The
background area was calculated by subtracting the area of the PVZ
and EGm from the area of the whole section. All values were
multiplied by the section thickness (0.030mm) to calculate the
volume of the sample of each region examined, and the density was
expressed as BrdU+ cellsmm–3. For each individual brain, we
We examined variation in BrdU+ cell density and plasma 11kT using
multivariate ANOVA, with region, season and environment as
factors, followed by Bonferroni post hoc tests to identify differences
among groups. In our initial analysis, we found that the two axial
regions of the hindbrain (sections –10 to –8 and –7 to –5) showed
no differences in any subject group (F3.0, P>0.05), and so these
axial regions were combined in subsequent analysis. All data are
presented as means ± s.e.m., and P<0.05 was considered statistically
significant.
RESULTS
Our quantitative analysis of BrdU labeling allowed us to estimate
the overall rates of cell proliferation across the hindbrain and
midbrain, the relative importance of proliferation zones as sites of
cell birth and the density of newborn cells in specific brain nuclei
associated with electrogenesis (Pn) and electrosensation (ELL). In
addition, our experimental design allowed us to partition variance
in BrdU+ cell density and plasma 11kT levels to season (breeding
vs non-breeding), environment (isolated vs group housed vs field)
and their interaction.
General patterns of cell proliferation
Averaged over all brain regions, environments and seasons,
transverse sections had a mean of 135±32BrdU+ cells, a mean
volume of 0.124±0.002mm3 and a mean density of 1088±293BrdU+
cellsmm–3. In all subject groups, BrdU+ cell density was greater in
the hindbrain than in the midbrain (F6.9, P<0.005; for field active
animals, see Fig.3). The overall distribution of BrdU+ cells was
similar to that reported for A. leptorhynchus (Zupanc and Horschke,
1995), with the exception that the cerebellum of B. gauderio
appeared to have relatively fewer BrdU+ cells than that of A.
leptorhynchus.
Hindbrain
In the hindbrain, the EGm was the primary site of cell proliferation,
containing ~20–55% of the newborn cells in the transverse section
and a BrdU+ cell density three to five times greater than that of the
background area. Although newborn cells were found surrounding
the entire cistern, the vast majority were located in the dorsal border
of the cistern in the EGm (Fig.1). The PVZ of the hindbrain was
also a proliferative zone, but was quantitatively less important than
the EGm. The PVZ contained ~5–10% of all the newborn cells in
the transverse section and a BrdU+ cell density two to three times
greater than that of the background area.
Midbrain
The anterior and posterior midbrain differed according to season
and environment (F32.2, P<0.0001), and thus these regions were
kept separate in our analysis. Cell proliferation was concentrated in
THE JOURNAL OF EXPERIMENTAL BIOLOGY
798
K. D. Dunlap, A. C. Silva and M. Chung
Fig.1. Seasonal variation in brain cell proliferation in the eminentia
granularis pars medialis, the major proliferative zone of the hindbrain, of
adult Brachyhypopomus gauderio. (A)Breeding season; (B) non-breeding
season. The cavity in the lower right of each photograph is the cerebromedullary cistern. Scale bar, 100m.
the PVZ (Fig.2A), which contained 15–40% of all labeled cells in
the transverse section and a BrdU+ cell density three to six times
greater than background. BrdU+ cells were scattered throughout the
torus semicircularis and along the medial edge of optic tectum and
were in particularly high density in the ventral hypothalamus.
Pn and ELL
In the Pn and ELL, BrdU+ cells were found at approximately the
same density as the background areas, and thus these nuclei cannot
be considered areas of concentrated mitosis. Within the Pn (Fig.2B),
labeled cells were located at all axial regions and tended to be more
abundant in the ventral half of the Pn. In field fish during the
breeding season (the subject group with the highest Pn cell
proliferation), each 30m section of the Pn contained a mean of
4.1±1.1BrdU+ cells per section. Given that the Pn is ~500m in
axial length (Quintana et al., 2010), we estimate that the entire
nucleus would generate 60–70 new cells in a 2.5h period. This is
a rate approximately half that reported from the Pn in Apteronotus
(Zupanc and Horschke, 1995). Labeled cells were found throughout
the ELL, although they were in much greater density in the
pyramidal and granule layers than in the molecular layers (Fig.2C).
Similar to another electric fish, Gymnotus (Castello et al., 2008),
BrdU+ cells also tended to be more concentrated in the lateral
segment than the central segments, although this difference was not
quantified. This apparent concentration of newborn cells in the lateral
segment deserves future research because, in other electric fish
species, this is the subsection of the ELL that is specifically involved
with processing social electric signals (Metzner and Juranek, 1997;
Marsat et al., 2009).
Effect of season
There was a strong overall effect of season on BrdU+ cell density
for all regions of the brain (Table3, Figs1, 3), with season
accounting for ~30–60% of the overall variation in BrdU+ cell
Fig.2. BrdU labeling in the brain of adult Brachyhypopomus gauderio. In all
photographs, dorsal is towards the top of the figure. (A)Periventricular zone
of the anterior midbrain. The cavity at the center is the ventricle. Scale bar,
100m. (B)Pacemaker nucleus. Arrows point to BrdU+ cells. The large
cells are relay cells, which only show background labeling. Scale bar,
50m. (C)Electrosensory lateral line lobe (ELL). Figure shows one lateral
half of the ELL, with lateral toward the left. Scale bar, 50m.
density. Across all treatment groups and brain regions, fish during
the breeding season had BrdU+ cell densities three to seven times
greater than fish during the non-breeding season.
Effect of environmental complexity
For all brain regions, there was a significant effect of environment
(isolation vs group housing vs field) on cell proliferation (Table3,
Fig.4), with environment accounting for ~10–30% of the overall
variation in BrdU+ cell density. In both seasons and in all brain
regions, field active fish had greater (approximately two to five
times) BrdU+ cell density than the two captive groups (isolation
and group housed). One of the only methodological differences in
sampling field and captive fish was that, for logistical reasons, fish
sampled in the field spent approximately 30min longer in holding
boxes before they were injected with BrdU than captive fish. Such
capture stress inhibits cell proliferation in some species (Amerin et
al., 2008), so it is likely that our report underestimates the true
difference between field and captive fish.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Brain cell proliferation in electric fish
799
Table 3. Results of ANOVA showing the effects of season, environment and their interaction on BrdU+ cell density in three axial regions of
the brain
Season
Brain region
Hindbrain
Whole
Background
PVZ
EGm
Pn
ELL
Posterior midbrain
Whole
Background
PVZ
Anterior midbrain
Whole
Background
PVZ
Season ⫻ Environment
Environment
% Var
F
P
% Var
F
P
% Var
F
P
58.7
56.7
33.7
60.4
28.6
48.1
53.3
49.5
20.7
66.0
24.2
176.5
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
7.3
12.0
10.1
8.1
25.3
30.5
3.3
5.2
3.4
4.4
10.7
55.0
<0.05
<0.05
<0.05
<0.05
<0.0005
<0.0001
1.8
3.3
6.6
4.6
11.1
20.4
0.8
1.5
2.0
2.5
4.6
37.1
>0.05
>0.05
>0.05
>0.05
<0.05
<0.0001
43.8
56.0
45.1
36.8
77.9
38.3
<0.0001
<0.0001
<0.0001
12.9
17.6
12.6
5.7
12.2
5.3
<0.01
<0.0005
<0.01
6.2
9.4
3.2
2.8
3.5
1.3
>0.05
>0.05
>0.05
57.1
56.9
48.5
82.2
77.4
84.1
<0.0001
<0.0001
<0.0001
18.6
21.9
18.9
13.3
14.9
16.3
<0.0001
<0.0001
<0.0001
6.8
8.3
16.1
2.9
2.7
13.9
>0.05
>0.05
<0.0001
% Var, the percentage of the variation in BrdU+ cell density that is attributable to each factor, as determined by ANOVA.
Season ⫻ environment interaction
Relative importance of proliferation zones as a function of
season and environment
For almost all comparisons, season and environment had no
significant effect on the fraction of BrdU+ cells located in
proliferative zones (PVZ and EGm). Thus, overall, it appears that
environmental stimuli affect cell proliferation uniformly in
proliferative and background regions. There is one exception to this
generality. In the hindbrain, the relative importance of the PVZ as
a site of new cell birth was affected by season: the percentage of
BrdU+ cells in the PVZ compared with the whole transverse section
at the same axial level was significantly lower during the breeding
season (5.3±0.7%) than during the non-breeding season (12.8±1.3%)
(F15.9; P<0.001). The hindbrain PVZ appears less responsive to
seasonal change than other hindbrain regions: the PVZ only showed
an approximately threefold increase in BrdU+ cell density from the
non-breeding to the breeding season whereas the whole brain section,
background, EGm, ELL and Pn showed an approximately five- to
seven-fold increase.
Testis maturity and plasma 11-ketotestosterone levels
In our January sampling, most fish captured from the field (five of
eight) and those in group housing (six of nine) had fully mature
testes (gonadal score3) and the remaining fish had testes of
intermediate maturity (gonadal score2). The mean gonadal maturity
8000
Anterior midbrain
4000
0
8000
Posterior midbrain
Cell proliferation (BrdU+ cells mm3)
The effect of environment varied by season in some brain regions
(Table3, Fig. 5). During the breeding season, group-housed fish had
greater BrdU+ cell density in the Pn, ELL and the anterior midbrain
PVZ than isolated fish, but lower BrdU+ cell density than field active
fish. However, during the non-breeding season, group-housed and
isolated fish had equivalent BrdU+ cell densities (P>0.05; Fig.5).
Thus group housing elevated cell proliferation in these regions to
an intermediate level during the breeding season, but had no effect
during the non-breeding season. There were no significant
environment ⫻ season interactive effects on BrdU cell density in
the whole transverse sections and background regions in any axial
region, the hindbrain PVZ and EGm, or the posterior midbrain PVZ
(Table3). Thus these regions were enhanced by field conditions but
not group housing during both seasons.
Breeding season
Non-breeding season
4000
0
Whole Backsection ground
16,000
PVZ
Hindbrain
12,000
8000
4000
0
Whole Backsection ground
PVZ
EGm
ELL
Pn
Fig.3. Effect of season on cell proliferation in different brain regions of field
active Brachyhypopomus gauderio. The anterior midbrain comprises
sections 17–19 and the posterior midbrain comprises sections 14–16,
according the Maler brain atlas (Maler et al., 1991). EGm, eminentia
granularis pars medialis; ELL, electrosensory lateral line lobe; Pn,
pacemaker; PVZ, periventricular zone. Whole section is the density of
BrdU+ cells in the whole transverse section. Background is the density of
BrdU+ cells in the transverse section outside of proliferative zones (PVZ
and EGm). Cell proliferation was significantly higher (P<0.0001) during the
breeding season than during the non-breeding season in all brain regions.
N6–8 fish per sample.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
800
K. D. Dunlap, A. C. Silva and M. Chung
Hindbrain
4000
*
2000
2000
0
0
4000
Cell proliferation (BrdU+ cells mm3)
Whole
section
Midbrain
4000
Isolated
Whole
section
Group
*
*
4000
Background
2000
2000
*
0
0
Background
Field
Fig.4. Effect of environmental complexity on cell proliferation
in whole brain, background regions and proliferation zones
[eminentia granularis pars medialis (EGm) and
periventricular zone (PVZ)] of Brachyhypopomus gauderio.
Data are presented only from the breeding season. Note the
difference in y-axis scale. *, significant difference from both
isolated and group fish; **, significant difference from
isolated fish and field fish. N6–8 fish per sample.
*
8000
PVZ
8000
*
*
*
4000
4000
0
0
Posterior
16,000
EGm
*
PVZ
**
Anterior
*
8000
0
scores were 2.63±0.24 and 2.66±0.21 for the field and group fish,
respectively. All isolated fish during the breeding season and all
fish during the non-breeding season had fully regressed gonads
(gonadal score1).
Plasma 11kT concentrations varied significant by season (F7.58,
P<0.01), environment (F3.64, P<0.05) and their interaction
(F2.82, P<0.05). Concentrations of 11kT were higher during the
breeding season than during the non-breeding season (Fig.4).
During the breeding season, field fish had significantly higher 11kT
levels than fish in either captive group, and group-housed fish had
significantly higher levels than isolated fish. During the nonbreeding season, fish in all environment groups had equivalent
values (P>0.05). Fish were held in holding boxes for ~3h following
capture (during the period of BrdU incorporation), and because such
acute stress commonly inhibits androgen secretion, the levels for
11kT that we report may be less than those experienced by
undisturbed fish. Nevertheless, our values are well within the range
of those reported in other studies of B. gauderio, in which fish were
bled immediately after capture (Gavassa et al., 2010; Salazar and
Stoddard, 2009). More importantly, fish were treated equivalently
across subject groups, allowing us to make comparisons of androgen
concentration among groups.
captive fish in all the brain regions we examined. This result
corroborates the findings of other studies (Amerin et al., 2008;
Barnea, 2010; Boonstra et al., 2001; LaDage et al., 2010), indicating
that examining animals in captivity tends to underestimate the
proliferative capacity of the adult vertebrate brain. We also found
that group-housed captive fish had greater rates of cell proliferation
than isolated fish, but only in brain regions associated with
electrosensation and electrogenesis. Thus the degree of
environmental complexity is correlated with the specificity of the
effect: the highly complex natural environment had a broad, global
effect on the brain whereas the limited enhancement of the captive
environment had a more regionalized, specific effect.
Although the effect of environmental complexity was significant,
it was quantitatively surpassed by the effect of season. The overall
effect of season on the brain was likely attributable to seasonal
changes in temperature or photoperiod whereas regionally specific
changes were likely due to changes in reproductive physiology and
behavior. Thus the role of external stimuli in promoting brain
plasticity must be understood in the context of annual cycles of the
environment and reproduction.
DISCUSSION
Across all brain regions and treatment groups, fish produced
approximately three to seven times more new cells during the
breeding season than during the non-breeding season. Seasonality
in brain cell proliferation has been documented in other vertebrate
We found that environmental complexity enhanced the proliferation
of brain cells in the weakly electric fish, B. gauderio. Fish in the
natural environment had greater rates of cell proliferation than
Seasonality in cell proliferation, reproductive state and
physical environment
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Brain cell proliferation in electric fish
Breeding season
8000
Midbrain PVZ
Non-breeding season
*
*
Midbrain PVZ
2000
*
*
Cell proliferation (BrdU+ cells mm3)
4000
*
*
1000
0
0
Posterior
4000
*
** *
ELL
Anterior
*
Posterior
1000
Anterior
ELL
801
Fig.5. Season ⫻ environment interactive effect on
brain cell proliferation in brain regions of
Brachyhypopomus gauderio associated with
electrogenesis and electrosensation. Note the
difference in y-axis scale. The pacemaker nucleus
(Pn) initiates the production of the electric organ
discharge, the electrosensory lateral line lobe
(ELL) is the primary electrosensory processing
region and the anterior midbrain periventricular
zone (PVZ) likely contributes cells to the
prepacemaker nucleus, which regulates the
production of social electric signals. *, significant
difference from both isolated and group fish; **,
significant different from isolated fish and field fish.
N6–8 fish per sample.
Isolated
2000
*
500
**
Group
Field
0
4000
0
1000
Pn
Pn
*
2000
0
**
*
500
0
classes, such as cyclostomes (Vidal Pizarro et al., 2004), amphibians
(Dawley et al., 2000), reptiles (Delgado-Gonzalez et al., 2008;
Ramirez et al., 1997), birds (Barnea and Nottebohm, 1994; Goldman,
2008) and mammals (Amerin et al., 2008; Galea and McEwen, 1999;
Lavenex et al., 2000), but our study is the first report of seasonality
in brain cell proliferation in a teleost fish. Such seasonal variation
in cell proliferation could be attributable to physiological and/or
behavioral changes in the annual reproductive cycle of the animal
or to changes in physical environment, such as temperature and
photoperiod. In considering both possibilities, we conclude that,
although reproductive physiology and behavior may influence
seasonal changes in brain cell proliferation, annual cycles of
temperature or photoperiod explain most of the seasonal variation
in cell proliferation.
To assess the reproductive state of fish during each season, we
examined testis maturity and plasma levels of the primary teleost
androgen, 11kT. In our breeding season sample, most field active
and group-housed fish had fully mature testes, but some had testes
of intermediate maturity. Plasma levels of 11kT were also elevated
in field active and group-housed fish, although group-housed fish
had significantly lower 11kT levels than field active fish. The
presence of some fish with partially regressed testes and low 11kT
levels combined with other data from this same population (Gavassa
et al., 2010) indicate that our sampling period was toward the end
of the breeding season.
In many vertebrates, brain cell proliferation peaks during or just
prior to the breeding season (Chetverukhin and Polenov, 1993;
Dawley et al., 2000; Delgado-Gonzalez et al., 2008; Vidal Pizarro
et al., 2004), and some of our results are consistent with the idea
that high levels of cell proliferation are causally related to seasonal
changes in reproductive physiology and behavior. For example,
isolation during the breeding season caused the regression of testes,
inhibition of plasma 11kT levels and a corresponding decrease in
cell proliferation. Conversely, 11kT levels were highest in breeding
fish from the field, which also had the highest levels of cell
proliferation.
However, a closer look at our results indicates that cell
proliferation is only partially tied to reproductive physiology and
behavior. The fact that season affected cell proliferation rates even
in isolated fish – which had regressed gonads, low 11kT levels
and no social interaction during both seasons – demonstrates that
seasonal changes in reproductive physiology and social signaling
are not the sole factors driving the seasonal change in cell
proliferation. Similarly, the fact that, during the non-breeding
season, field active fish had higher rates of cell proliferation than
captive fish yet all fish had equivalently low levels of 11kT
indicates that cell proliferation is not strictly attributable to
seasonal changes in androgens. Finally, during the breeding
season, field active fish had higher rates of cell proliferation than
group-housed fish even though their testes were at equivalent
stages of maturity, and thus proliferation rates are not strictly
correlated to testes maturity. Thus, some factor beyond
reproductive state must play a large role in seasonal changes in
cell proliferation rate.
For temperate species, the breeding season typically has high
temperatures and a long photoperiod. These features of the physical
environment commonly stimulate changes in reproductive
physiology and behavior. Indeed, for B. gauderio, seasonal changes
in temperature are likely the crucial zeitgeber stimulating
reproductive activity (Quintana et al., 2004). However, changes in
the thermal and photic environment can also have direct effects on
cellular processes. For example, mitotic rate increases dramatically
with temperature (Rieder and Cole, 2002) and, for many ectotherms,
mean body temperature is higher during the breeding season. Thus,
THE JOURNAL OF EXPERIMENTAL BIOLOGY
K. D. Dunlap, A. C. Silva and M. Chung
the seasonal changes in brain cell proliferation we found are likely
influenced directly by temperature.
When using BrdU to assess cell proliferation, temperature could
influence the basal mitotic rates and/or the transport and metabolism
of BrdU during the period following BrdU injection. In our
sampling, the temperature of the water that fish inhabited varied
from approximately 27°C during the breeding season to 17°C during
the non-breeding season. We kept water temperature during the
period of BrdU incorporation within a narrower range, but it
nonetheless varied from 23 to 27°C. To estimate the effect of this
temperature variation during the period of BrdU incorporation, we
conducted an experiment on A. leptorhynchus in which all fish were
housed at the same temperature (27°C) but were then shifted to 23
or 27°C for 2.5h during the period of BrdU incorporation. We found
no effect of temperature on BrdU+ cell density in the midbrain PVZ
(4858±1367 BrdU+ cellsmm–3 at 23°C vs 4539±1359 at 27°C;
t0.16, P>0.05). Thus it is likely that any effect of temperature on
BrdU labeling in our B. gauderio study is related to mitotic rate in
the environment where they were living, rather than to the dynamics
of BrdU uptake and metabolism.
The effect of the thermal environment and photoperiod on brain
cell proliferation has received little attention, but several studies
indicate they have a potent influence. Radmilovich et al. examined
cell proliferation in the brains of turtles acclimated to warm and
cold temperatures (Radmilovich et al., 2003). Turtles held at 20°C
produced approximately six times more cells in brain proliferative
zones than those held at 11°C. Similarly, Peñafiel et al. found that
lizards (Psammodromus algirus) maintained at 20°C had
approximately three to 10 times more BrdU-labeled cells than lizards
maintained at 10°C (Peñafiel et al., 2001). Ramirez et al. found no
effect of temperature (10 vs 25°C) on brain cell proliferation in
another lizard species (Podarcis hispanica), but long daylength (16
vs 8h) increased brain cell proliferation by approximately threefold
(Ramirez et al., 1997). Our results from isolated laboratory
conditions showed that fish during the breeding season housed at
28°C with long daylengths (14h) produced approximately three to
eight times more newborn cells than those held at 17°C at short
daylengths (10h) during the non-breeding season. For most brain
regions (those except Pn, ELL and anterior midbrain PVZ), fish in
all three environments had similar magnitudes of seasonal change
(i.e. no season ⫻ environment interaction). Because temperature
and photoperiod were the only common features shared by these
environments, we believe that seasonal changes in temperature and
photoperiod are sufficient to explain seasonal differences in cell
proliferation in most brain regions. However, as discussed below,
season likely affects cell proliferation in the Pn, ELL and anterior
midbrain PVZ through seasonal changes in reproductive physiology
and/or associated changes in electrocommunication behavior.
Cell proliferation and environmental complexity
Simple vs enriched captive environment
Compared with isolated fish, group-housed fish had both abundant
opportunity for social interaction and larger, more complex physical
environments (Table2). During the breeding season, they were also
in a more advanced reproductive state, as evidenced by testis
maturity and androgen levels (Fig.6). Yet, for most brain regions,
cell proliferation rates were equivalent between these two captive
groups. This indicates that, for most of the brain, the size and
complexity of the physical environment, the presence of plants and
conspecifics, and the reproductive status of the fish do not affect
brain cell proliferation. However, for the Pn, ELL and anterior
midbrain PVZ, group housing enhanced cell proliferation. This effect
Plasma 11kT (pg ml–1)
802
*
1000
Isolated
Group
Field
500
**
0
Breeding
season
Non-breeding
season
Fig.6. Plasma 11-ketotesterone (11kT) levels in Brachyhypopomus
gauderio according to season and environment. *, significant difference
from both isolated and group fish; **, significant different from isolated fish
and field fish. N5–6 fish per sample.
only occurred during the breeding season, when the reproductive
system is active and most of the social signaling occurs (Fig. 5).
The structure of the physical environment did not change from the
breeding to the non-breeding seasons (Table1) and cannot account
for this season ⫻ environment interaction. Thus, the higher rates
of cell proliferation in group-housed fish than in isolated fish during
the breeding season is most closely associated with differences in
social environment and/or reproductive physiology.
Our study does not enable us to determine whether elevated rates
of cell proliferation are the cause or effect of socially induced
changes in behavior and reproductive physiology. Moreover, our
study only examined the birth of cells, and we do not know the
long-term fate of these cells. While recognizing these limitations,
we hypothesize that social stimuli and/or activation of the
reproductive system enhance the production of cells in brain regions
involved with electrocommunication. Addition of cells to these
nuclei and their differentiation into neurons may then contribute to
socially induced changes in electrocommunication behavior.
The Pn drives the production of electric signals for
electrocommunication and reproductive signaling and, in B.
gauderio, its activity is modulated by social interactions and
breeding conditions (Pouso et al., 2010; Quintana et al., 2010; Silva
et al., 2007). For example, the nocturnal EOD rate of males is greater
during the breeding season than during the non-breeding season and
is further elevated when males are housed with females (Silva et
al., 2007). The Pn expresses androgen receptors, which likely play
a role in reproductive and social changes in Pn activity (Pouso et
al., 2010). One possibility is that the cells born under the influence
of social interactions and breeding conditions differentiate into
neurons and incorporate into the Pn circuitry. These new neurons
could then modify Pn activity either by providing sites for novel
inputs to the Pn or by directly changing its intrinsic firing rate. Such
incorporation of new neurons could be mediated by androgen
binding to receptors on these new cells or on adjacent cells of the
Pn.
The ELL also shows elevated cell proliferation in response to
group housing and is crucial for electrocommunication. However,
less is known about how the ELL changes functionally in response
to social interaction and reproductive state. In other electric fish
(Sternopygus), electroreceptors in the periphery, which send inputs
directly to the ELL, change their tuning properties in response to
androgens (Keller et al., 1986). In addition, the ELL of Gymnotus
shows accelerated cell proliferation as fish enter adulthood (Castello
et al., 2008). These observations indicate that examining
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Brain cell proliferation in electric fish
reproductive influences on ELL function and cell proliferation may
be a fruitful area for future research.
Group housing also enhances cell proliferation in the anterior
midbrain PVZ. The fate of cells born in this proliferation zone is
unknown in B. gauderio. In the closely related fish, Eigenmannia
sp., some of these cells migrate laterally and become neurons in the
PPn, which controls an important electrocommunication signal
termed chirping (Zupanc and Zupanc, 1992). In another electric fish,
A. leptorhynchus, approximately two-thirds of the cells born in this
region differentiate into neurons (Dunlap et al., 2008). Cell addition
to the PVZ adjacent to the PPn increases in response to social
housing and correlates with socially induced changes in chirping
behavior (Dunlap et al., 2002; Dunlap et al., 2006). Male B. gauderio
also exhibit chirping behavior (Perrone et al., 2009), and in other
members of the genus, chirping is controlled by the PPn (Kawasaki
and Heiligenberg, 1989). If the anterior midbrain PVZ also
contributes cells to the PPn in B. gauderio, then social enhancement
of cell addition to the PPn may also participate in socially induced
changes in chirping behavior. However, it is important to recognize
that the anterior midbrain PVZ may also contribute cells to other
nearby regions of the brain (e.g. the hypothalamus), so, without
further study, it is difficult to assess whether the social effect on
the PVZ is restricted to the electrocommunication system.
The regionally specific effect of social interaction on brain cell
proliferation in B. gauderio and A. leptorhynchus is also found
in a frog, Hyla cinerea. In this frog, exposure to conspecific
acoustic stimuli elevates cell proliferation rates in brain regions
involved with calling behavior, but not in control regions that are
unrelated to calling behavior (Almli and Wilczynski, 2009).
Together, these results indicate that cell proliferation in brain
regions involved with communication behavior is particularly
responsive to social stimuli and may contribute to adaptive
changes in social behavior.
Enriched captive environment vs complex natural environment
Fish from the field had higher rates of cell proliferation in all brain
regions and during both seasons than captive fish. Even in brain
regions that are stimulated by group housing (ELL, Pn and anterior
midbrain PVZ), cell proliferation is further enhanced by field
conditions during the breeding season.
There are many physical, social and biotic differences between
the enriched laboratory environment and the natural environment
that could contribute to differences in brain cell proliferation
(Table2). Compared with group-housed fish, field active fish
experienced a more complex structural environment. Enhancing the
structural complexity of the environment stimulates cell proliferation
in the telencephalon of zebrafish (von Krogh et al., 2010), and the
greater number and diversity of objects in the natural habitat of B.
gauderio (compared with the pools in captivity) may broadly
stimulate brain activity and promote brain cell proliferation in B.
gauderio. In addition, the natural environment is simply larger than
the artificial pools of group-housed fish. At Laguna Lavalle, males
during the reproductive season move a span of ~4m over a 3–4d
period (Miranda et al., 2008). This is approximately three times
farther than the maximum dimension in the pools in our study.
Considering that locomotor behavior stimulates brain cell
proliferation in some vertebrates (van Praag, 2008), it is possible
that field active fish have higher rates of cell proliferation because
they swim more than group-housed fish. However, the only test of
this idea in fish indicates that, in laboratory environments that
promote swimming, fish actually have lower rates of brain cell
proliferation (von Krogh et al., 2010).
803
The social environment was also more complex in the field than
in the group laboratory housing. Certain features of the natural social
environment, such as the presence of fish of both sexes in a realistic
sex ratio, were preserved in group housing, but the composition of
the social environment was fixed and limited to only adults. The
more dynamic and age-diverse social stimuli fish receive in the wild
might contribute to higher rates of proliferation throughout the brain
and/or the particular enhancement of proliferation in the Pn, ELL
and anterior midbrain PVZ. In zebra finches, social complexity
significantly influences cell recruitment into brain regions subserving
vocal communication (Lipkind et al., 2002) and, in B. gauderio,
the history and complexity of social interactions affect
electrocommunication (Salazar and Stoddard, 2009). Thus, it seems
plausible that the greater social complexity of the natural
environment contributes to the elevated rates of cell proliferation
in the electrocommunication circuity of B. gauderio.
Finally, fish in the field were exposed to more biotic complexity,
with greater numbers and diversity of plants, prey, predators and
pathogens. Sensory cues from predators inhibit brain cell
proliferation in rats (Tanapat et al., 2001) but, otherwise, nothing
is known about how the biotic environment affects brain cell
dynamics, and it is difficult to evaluate its influence on B. gauderio.
Relative importance of proliferation zones as sites of cell
proliferation
In many vertebrate species, most cells added to the adult brain
originate from discrete proliferation zones (Chapouton et al., 2007).
Although fish clearly possess such proliferation zones, some of
which are likely homologous to those of other vertebrates (Zupanc,
2008; Zupanc and Horschke, 1995), they also generate a substantial
number of new cells scattered throughout the brain, a phenomenon
we term ‘background proliferation’. In B. gauderio, we found that
~15–55% of new cells were born in proliferation zones. This
proportion was greater in the hindbrain than in the midbrain.
However, in almost all cases, the relative importance of the
proliferative zones as sites of cell birth was independent of season
and environment. Thus field conditions and breeding season
uniformly increase cell proliferation in proliferative and background
zones.
The only exception to this generality was that the hindbrain PVZ
was relatively less important as a site of cell proliferation during
the breeding season compared with the non-breeding season. The
overall quantitative effect of this seasonal difference is small,
because only ~10% of all newborns cells in the hindbrain originate
in the PVZ. Nevertheless, this result shows that, in some cases, the
environment can influence the distribution of newborn cells in and
out of proliferative zones.
CONCLUSIONS
We found that the natural environment and the breeding season had
a large positive effect on the overall proliferative activity of the
brain. We are uncertain about the precise features that stimulate cell
proliferation, but we suggest that the structural and social complexity
of the natural environment and the warm temperatures and long
daylengths of the breeding season globally enhance brain cell
proliferation.
Our study also highlights the importance of regional and seasonal
specificity in the effect of the environment of cell proliferation.
Experimental manipulation of the social environment only affects
specific brain regions – the Pn, ELL and anterior midbrain PVZ –
and only during the breeding season. These brain regions are
important components of the electrocommunication circuitry that
THE JOURNAL OF EXPERIMENTAL BIOLOGY
804
K. D. Dunlap, A. C. Silva and M. Chung
contribute to seasonal changes in social signaling. This correlation
between regionally and seasonally specific brain cell proliferation
in the electrocommunication nuclei and changes in
electrocommunication behavior support the idea that selective
addition of new cells may be a mechanism contributing to behavioral
responses to environmental change.
LIST OF ABBREVIATIONS
11kT
BrdU
EGm
ELL
EOD
Pn
PPn
11-ketotestosterone
bromodeoxyuridine
eminentia granularis pars medialis
electrosensory lateral line lobe
electric organ discharge
pacemaker nucleus
prepacemaker nucleus
ACKNOWLEDGEMENTS
We thank R. Perrone, G. Batista, L. Zubizarreta, L. Quintana and T. de los
Campos for their generous and invaluable assistance in the field; T. Williams for
comments on the manuscript; and O. Macadar for inspiration. This work was
supported by fellowships from the US Fulbright Scholar program and the Charles
Dana Foundation to K.D.D. M.C. was supported by grants to K.D.D. from the NIH
(R15H080731-01) and Trinity College Faculty Research Committee. Deposited in
PMC for release after 12 months.
REFERENCES
Almli, L. M. and Wilczynski, W. (2009). Sex-specific modulation of cell proliferation by
socially relevant stimuli in the adult green treefrog brain (Hyla cinerea). Brain Behav.
Evol. 74, 143-154.
Amerin, I., Lipp, H. P., Boonstra, R. and Wojtowicz, J. (2008). Adult hippocampal
neurogenesis in natural populations. In Adult Neurogenesis (ed. F. H. Gage, G.
Kempermann and H. Song), pp. 645-660. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press.
Barnea, A. (2010). Wild neurogenesis. Brain Behav. Evol. 75, 86-87.
Barnea, A. and Nottebohm, F. (1994). Seasonal recruitment of hippocampal neurons
in adult free-ranging black-capped chickadees. Proc. Natl. Acad. Sci. USA 91,
11217-11221.
Barnea, A. and Nottebohm, F. (1996). Recruitment and replacement of hippocampal
neurons in young and adult chickadees: an addition to the theory of hippocampal
learning. Proc. Natl. Acad. Sci. USA 93, 714-718.
Boonstra, R., Galea, L., Matthews, S. and Wojtowicz, J. M. (2001). Adult
neurogenesis in natural populations. Can. J. Physiol. Pharmacol. 79, 297-302.
Castello, M. E., Iribarne, L. and Caputi, A. A. (2008). Postnatal brain development of
Gymnotus omari. Abstr. Soc. Neurosci. 38.
Chapouton, P., Jagasia, R. and Bally-Cuif, L. (2007). Adult neurogenesis in nonmammalian vertebrates. BioEssays 29, 745-757.
Chetverukhin, V. K. and Polenov, A. L. (1993). Ultrastructural radioautographic
analysis of neurogenesis in the hypothalamus of the adult frog, Rana temporaria,
with special reference to physiological regeneration of the preoptic nucleus. I.
Ventricular zone cell proliferation. Cell Tissue Res. 271, 341-350.
Dawley, E. M., Fingerlin, A., Hwang, D., John, S. S. and Stankiewicz, C. A. (2000).
Seasonal cell proliferation in the chemosensory epithelium and brain of red-backed
salamanders, Plethodon cinereus. Brain Behav. Evol. 56, 1-13.
Delgado-Gonzalez, F. J., Alonso-Fuentes, A., Delgado-Fumero, A., GarciaVerdugo, J. M., Gonzalez-Granero, S., Trujillo-Trujillo, C. M. and DamasHernandez, M. C. (2008). Seasonal differences in ventricular proliferation of adult
Gallotia galloti lizards. Brain Res. 1191, 39-46.
Diamond, M. C., Law, F., Rhodes, H., Lindner, B., Rosenzweig, M. R., Krech, D.
and Bennett, E. L. (1966). Increases in cortical depth and glia numbers in rats
subjected to enriched environment. J. Comp. Neurol. 128, 117-126.
Dunlap, K. D., Pelczar, P. L. and Knapp, R. (2002). Social interactions and cortisol
treatment increase the production of aggressive electrocommunication signals in
male electric fish, Apteronotus leptorhynchus. Horm. Behav. 42, 97-108.
Dunlap, K. D., Castellano, J. F. and Prendaj, E. (2006). Social interaction and
cortisol treatment increase cell addition and radial glia fiber density in the
diencephalic periventricular zone of adult electric fish, Apteronotus leptorhynchus.
Horm. Behav. 50, 10-17.
Dunlap, K. D., McCarthy, E. and Jashari, D. (2008). Electrocommunication signals
alone are sufficient to increase neurogenesis in the brain of adult electric fish,
Apteronotus leptorhynchus. Dev. Neurobiol. 68, 1420-1428.
Fabel, K., Wolf, S. A., Ehninger, D., Babu, H., Leal-Galicia, P. and Kempermann,
G. (2009). Additive effects of physical exercise and environmental enrichment on
adult hippocampal neurogenesis in mice. Front. Neurosci. 3, 50.
Franchina, C. R. (1997). Ontogeny of the electric organ discharge and the electric
organ in the weakly electric pulse fish Brachyhypopomus pinnicaudatus
(Hypopomidae, Gymnotiformes). J. Comp. Physiol. A 181, 111-119.
Galea, L. A. and McEwen, B. S. (1999). Sex and seasonal differences in the rate of
cell proliferation in the dentate gyrus of adult wild meadow voles. Neuroscience 89,
955-964.
Gavassa, S., Silva, A. and Stoddard, P. K. (2010). Reproductive output of
Brachyhypopomus gauderio decreases throughout the breeding season. Abstracts
Int. Cong. Neuroethol, vol. 11. Salamanca, Spain.
Goldman, S. A. (2008). Neurogenesis in the adult songbird: a model for inducible
striatal neuronal addition. In Adult Neurogenesis (ed. F. H. Gage, G. Kempermann
and H. Song), pp. 593-617. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press.
Hansen, A. and Schmidt, M. (2004). Influence of season and environment on adult
neurogenesis in the central olfactory pathway of the shore crab, Carcinus maenas.
Brain Res. 1025, 85-97.
Hoshooley, J. S. and Sherry, D. F. (2004). Neuron production, neuron number, and
structure size are seasonally stable in the hippocampus of the food-storing blackcapped chickadee (Poecile atricapillus). Behav. Neurosci. 118, 345-355.
Kawasaki, M. and Heiligenberg, W. (1989). Distinct mechanisms of modulation in a
neuronal oscillator generate different social signals in the electric fish Hypopomus. J.
Comp. Physiol. A 165, 731-741.
Keller, C. H., Zakon, H. H. and Sanchez, D. Y. (1986). Evidence for a direct effect of
androgens upon electroreceptor tuning. J. Comp. Physiol. A 158, 301-310.
Kempermann, G., Kuhn, H. G. and Gage, F. H. (1997). More hippocampal neurons
in adult mice living in an enriched environment. Nature 386, 493-495.
Kozorovitskiy, Y. and Gould, E. (2004). Dominance hierarchy influences adult
neurogenesis in the dentate gyrus. J. Neurosci. 24, 6755-6759.
LaDage, L. D., Roth, T. C., 2nd, Fox, R. A. and Pravosudov, V. V. (2010).
Ecologically relevant spatial memory use modulates hippocampal neurogenesis.
Proc. Biol. Sci. 277, 1071-1079.
Lavenex, P., Steele, M. A. and Jacobs, L. F. (2000). Seasonal pattern of cell
proliferation and neuron number in the dentate gyrus of wild adult eastern grey
squirrels. Eur. J. Neurosci. 12, 643-648.
Lipkind, D., Nottebohm, F., Rado, R. and Barnea, A. (2002). Social change affects
the survival of new neurons in the forebrain of adult songbirds. Behav. Brain Res.
133, 31-43.
Lu, L., Bao, G., Chen, H., Xia, P., Fan, X., Zhang, J., Pei, G. and Ma, L. (2003).
Modification of hippocampal neurogenesis and neuroplasticity by social
environments. Exp. Neurol. 183, 600-609.
Maler, L., Sas, E., Johnston, S. and Ellis, W. (1991). An atlas of the brain of the
electric fish Apteronotus leptorhynchus. J. Chem. Neuroanat. 4, 1-38.
Marsat, G., Proville, R. D. and Maler, L. (2009). Transient signals trigger
synchronous bursts in an identified population of neurons. J. Neurophysiol. 102, 714723.
Metzner, W. and Juranek, J. (1997). A sensory brain map for each behavior? Proc.
Natl. Acad. Sci. USA 94, 14798-14803.
Miranda, M., Silva, A. C. and Stoddard, P. K. (2008). Use of space as an indicator of
social behavior and breeding systems in the gymnotiform electric fish
Brachyhypopomus pinnicaudatus. Environ. Biol. Fishes 83, 379-389.
Ormerod, B. K. and Galea, L. A. (2003). Reproductive status influences the survival
of new cells in the dentate gyrus of adult male meadow voles. Neurosci. Lett. 346,
25-28.
Peñafiel, A., Rivera, A., Gutiérrez, A., Trías, S. and De La Calle, A. (2001).
Temperature affects adult neurogenesis in the lizard brain. Int. J. Dev. Biol. 45, 8384.
Perrone, R., Macadar, O. and Silva, A. (2009). Social electric signals in freely moving
dyads of Brachyhypopomus pinnicaudatus. J. Comp. Physiol. A 195, 501-514.
Pouso, P., Quintana, L., Bolatto, C. and Silva, A. C. (2010). Brain androgen receptor
expression correlates with seasonal changes in the behavior of a weakly electric
fish, Brachyhypopomus gauderio. Horm. Behav. 58, 729-736.
Quintana, L., Silva, A., Berois, N. and Macadar, O. (2004). Temperature induces
gonadal maturation and affects electrophysiological sexual maturity indicators in
Brachyhypopomus pinnicaudatus from a temperate climate. J. Exp. Biol. 207, 18431853.
Quintana, L., Pouso, P., Fabbiani, G. and Macadar, O. (2010). A central pacemaker
that underlies the production of seasonal and sexually dimorphic social signals:
anatomical and electrophysiological aspects. J. Comp. Physiol. A 197, 75-88.
Radmilovich, M., Fernandez, A. and Trujillo-Cenoz, O. (2003). Environment
temperature affects cell proliferation in the spinal cord and brain of juvenile turtles. J.
Exp. Biol. 206, 3085-3093.
Ramirez, C., Nacher, J., Molowny, A., Sanchez-Sanchez, F., Irurzun, A. and
Lopez-Garcia, C. (1997). Photoperiod-temperature and neuroblast proliferationmigration in the adult lizard cortex. NeuroReport 8, 2337-2342.
Rieder, C. L. and Cole, R. W. (2002). Cold-shock and the mammalian cell cycle. Cell
Cycle 1, 169-175.
Salazar, V. L. and Stoddard, P. K. (2009). Social competition affects electric signal
plasticity and steroid levels in the gymnotiform fish Brachyhypopomus gauderio.
Horm. Behav. 56, 399-409.
Sampedro, C., Font, E. and Desfilis, E. (2008). Size variation and cell proliferation in
chemosensory brain areas of a lizard (Podarcis hispanica): effects of sex and
season. Eur. J. Neurosci. 28, 87-98.
Silva, A., Quintana, L., Ardanaz, J. L. and Macadar, O. (2002). Environmental and
hormonal influences upon EOD waveform in gymnotiform pulse fish. J. Physiol. Paris
96, 473-484.
Silva, A., Quintana, L. and Galeano, M. (2003). Biogeography and breeding in
gymnotiformes from Uruguay. Environ. Biol. Fishes 66, 329-338.
Silva, A., Perrone, R. and Macadar, O. (2007). Environmental, seasonal, and social
modulations of basal activity in a weakly electric fish. Physiol. Behav. 90, 525-536.
Silva, A., Quintana, L., Perrone, R. and Sierra, F. (2008). Sexual and seasonal
plasticity in the emission of social electric signals. Behavioral approach and neural
bases. J. Physiol. Paris 102, 272-278.
Stoddard, P. K., Zakon, H. H., Markham, M. R. and McAnelly, L. (2006). Regulation
and modulation of electric waveforms in gymnotiform electric fish. J. Comp. Physiol.
A 192, 613-624.
Stranahan, A. M., Khalil, D. and Gould, E. (2006). Social isolation delays the positive
effects of running on adult neurogenesis. Nat. Neurosci. 9, 526-533.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Brain cell proliferation in electric fish
Tanapat, P., Hastings, N. B., Rydel, T. A., Galea, L. A. and Gould, E. (2001).
Exposure to fox odor inhibits cell proliferation in the hippocampus of adult rats via an
adrenal hormone-dependent mechanism. J. Comp. Neurol. 437, 496-504.
Thompson, C. K. and Brenowitz, E. A. (2005). Seasonal change in neuron size and
spacing but not neuronal recruitment in a basal ganglia nucleus in the avian song
control system. J. Comp. Neurol. 481, 276-283.
van Praag, H. (2008). Neurogenesis and exercise: past and future directions.
Neuromolecular Med. 10, 128-140.
Vidal Pizarro, I., Swain, G. P. and Selzer, M. E. (2004). Cell proliferation in the
lamprey central nervous system. J. Comp. Neurol. 469, 298-310.
von Krogh, K., Sorensen, C., Nilsson, G. E. and Overli, O. (2010). Forebrain cell
proliferation, behavior, and physiology of zebrafish, Danio rerio, kept in enriched or
barren environments. Physiol. Behav. 101, 32-39.
805
Zakon, H., Oestreich, J., Tallarovic, S. and Triefenbach, F. (2002). EOD
modulations of brown ghost electric fish: JARs, chirps, rises, and dips. J. Physiol.
Paris 96, 451-458.
Zupanc, G. K. (2002). From oscillators to modulators: behavioral and neural control of
modulations of the electric organ discharge in the gymnotiform fish, Apteronotus
leptorhynchus. J. Physiol. Paris 96, 459-472.
Zupanc, G. K. H. (2008). Adult neurogenesis in teleost fish. In Adult Neurogenesis
(ed. F. H. Gage, G. Kempermann and H. Song), pp. 571-592. Cold Spring Harbor,
NY: Cold Spring Harbor Laboratory Press.
Zupanc, G. K. and Horschke, I. (1995). Proliferation zones in the brain of adult
gymnotiform fish: a quantitative mapping study. J. Comp. Neurol. 353, 213-233.
Zupanc, G. K. and Zupanc, M. M. (1992). Birth and migration of neurons in the
central posterior/prepacemaker nucleus during adulthood in weakly electric knifefish
(Eigenmannia sp.). Proc. Natl. Acad. Sci. USA 89, 9539-9543.
THE JOURNAL OF EXPERIMENTAL BIOLOGY