Calbindin and Fos in and above the SCN
Pergamon
PII: S0306-4522(00)00212-8
Neuroscience Vol. 99, No. 3, pp. 565–575, 2000
565
䉷 2000 IBRO. Published by Elsevier Science Ltd
Printed in Great Britain. All rights reserved
0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
CALBINDIN AND Fos WITHIN THE SUPRACHIASMATIC NUCLEUS AND THE
ADJACENT HYPOTHALAMUS OF ARVICANTHIS NILOTICUS AND RATTUS
NORVEGICUS
M. M. MAHONEY,* A. A. NUNEZ†‡ and L. SMALE*†‡§
Departments of *Zoology, †Psychology and ‡Neuroscience Program, Michigan State University,
East Lansing MI, 48824, USA
Abstract—The suprachiasmatic nucleus is the site of the primary circadian pacemaker in mammals. The lower sub paraventricular
zone that is dorsal to and receives input from the suprachiasmatic nucleus may also play a role in the regulation of circadian
rhythms. Calbindin has been described in the suprachiasmatic nucleus of some mammals, and may be important in the control of
endogenous rhythms. In the first study we characterized calbindin-expressing cells in the suprachiasmatic nucleus and lower subparaventricular zone of nocturnal and diurnal rodents. Specifically, Rattus norvegicus was compared to Arvicanthis niloticus, a
primarily diurnal species within which some individuals exhibit nocturnal patterns of wheel running. Calbindin-immunoreactive
cells were present in the suprachiasmatic nucleus of Arvicanthis and were most concentrated within its central region but were
relatively sparse in the suprachiasmatic nucleus of Rattus. Calbindin-expressing cells were present in the lower sub-paraventricular
zone of both species. In the second study we evaluated Fos expression within calbindin-immunoreactive cells in nocturnal Rattus
and in Arvicanthis that were either diurnal or nocturnal with respect to wheel-running. All animals were kept on a 12:12 light/dark
cycle and perfused at either 4 h after lights-on or 4 h after lights-off. In the suprachiasmatic nucleus in both species, Fos expression
was elevated during the day relative to the night but less than 1% of calbindin cells contained Fos in Arvicanthis, compared with
13–17% in Rattus. In the lower sub-paraventricular zone of both species, 9–14% of calbindin cells expressed Fos, and this
proportion did not change as a function of time. Among Arvicanthis, the number of calbindin expressing neurons in the lower
sub-paraventricular zone was influenced by an interaction between the wheel running patterns (nocturnal vs diurnal) and time of
day. Thus, the number of calbindin-positive cells within the suprachiasmatic nucleus differed in Arvicanthis and Rattus, whereas the
number of calbindin-positive cells within the lower sub-paraventricular zone differed in nocturnal and diurnal Arvicanthis.
Our examination of R. norvegicus and A. niloticus suggests potentially important relationships between calbindin-containing
neurons and whether animals are nocturnal or diurnal. Specifically, rats had more Fos expression in calbindin containing cells in the
suprachiasmatic nucleus than Arvicanthis. In contrast, Arvicanthis exhibiting diurnal and nocturnal patterns of wheel-running
differed in the number of calbindin-containing cells in the lower sub-paraventricular zone, dorsal to the suprachiasmatic nucleus.
䉷 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.
Key words: nocturnal, diurnal, rhythm, circadian, paraventricular.
Nocturnal and diurnal species typically differ with respect to
the temporal organization of a wide range of physiological
and behavioral parameters including locomotor activity,
copulatory behavior, 10,24 parturition, 39 body temperature, 33
and sleep. 13,27 Little is known about how the underlying
neural mechanisms that mediate these various rhythms differ
in diurnal animals because most research has focused on
nocturnal laboratory animals such as rats, mice and hamsters.
In both nocturnal and diurnal animals, the suprachiasmatic
nucleus (SCN), located within the mammalian hypothalamus,
is responsible for the production and synchronization of many
circadian rhythms. 26,31,40,48 With respect to some functional
characteristics, the SCN appears to be similar in nocturnal
and diurnal animals. For example, uptake of 2-deoxyglucose
indicates that the SCN is metabolically most active during the
day, regardless of an animal’s activity pattern. 43 However,
with respect to other aspects of its function there are some
indications that the SCN may differ in diurnal and nocturnal
animals. Light is more likely to have inhibitory than excitatory effects on firing rates of SCN neurons in diurnal squirrels
and degus compared with nocturnal rats. 11,25 Furthermore,
features of the expression of the product of the immediate
early gene c-fos (Fos) appear to differ in various ways in
diurnal rodents from those reported in nocturnal ones. 1,14,18
However, the patterns of Fos expression vary from one
diurnal species to another, and thus their functional relationship to diurnality remains unclear. The hypothalamic region
immediately dorsal to the SCN, the lower sub-paraventricular
zone (LSPVZ), may also be involved in the regulation of
circadian rhythms. This region receives input from both the
retina 12 and the SCN 28,51 and exhibits rhythms in Fos that
differ in nocturnal rats compared to the diurnal species,
Arvicanthis niloticus. 30
In the studies presented here we used Fos to evaluate the
hypothesis that one subpopulation of cells within and above
the SCN might function differently in nocturnal and diurnal
animals. Specifically, we examined Fos expression within
cells containing the calcium-binding protein calbindinD28K (CALB). This protein has been found in the SCN of
several nocturnal 5,34,46 and diurnal species. 7,9,23 CALB
belongs to a group of structurally related proteins of the EFhand family, which includes parvalbumin and calretinin. 35
These proteins are involved in the regulation of intracellular
calcium concentrations and calcium transport. 3 Several lines
§To whom correspondence should be addressed: Tel.: ⫹1-517-432-1632;
fax: ⫹1-517-353-1652.
E-mail address: [email protected] (L. Smale).
Abbreviations: ABC, avidin–biotin complex; CALB, Calbindin; CT,
cholera toxin; DAB, diaminobenzidine; LD, light/dark; LSPVZ, lower
sub-paraventricular zone; NGS, normal goat serum; NHS, normal horse
serum; PBS, phosphate-buffered saline; PLP, paraformaldehyde, lysine,
sodium m-periodate; SCN, suprachiasmatic nuclei; ZT, zeitgeber time.
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M. M. Mahoney et al.
Fig. 1. Actograms of wheel-running of representative diurnal (A) and nocturnal (B) Arvicanthis kept in a 12:12 LD cycle. Each line represents a 24-h period.
Black bar below each actogram indicates when lights were off and open bar indicates when lights were on.
of evidence suggest that CALB-immunoreactive neurons
within the SCN are critical for the maintenance of circadian
rhythms in at least one species, Mesocricetus auratus (the
golden hamster). In this species Fos is expressed primarily
in the CALB-immunoreactive neurons in the SCN in animals
that have been exposed to a light pulse during the subjective
night. 46 Hamsters with partial SCN lesions that spared the
CALB-containing subregion continued to exhibit circadian
locomotor rhythms, whereas hamsters with lesions that abolished the small CALB-immunoreactive subregion failed to
exhibit such rhythms. 20 In SCN lesioned animals given transplants of SCN tissue the recovery of rhythmicity was positively correlated with the amount of CALB within the graft. 20
Fos is expressed in the SCN and LSPVZ under a variety of
conditions. In the SCN, Fos expression is regulated by ambient light, by circadian phase and by an interaction between
these two variables. 2,8,41,44 In the SCN of nocturnal rats kept
on a 12:12 light/dark (LD) cycle, Fos levels reach their peak
soon after lights are on, decline to an intermediate level in the
latter half of the light period, then reach their lowest values at
night. 2,8,16,41,44 A fundamentally similar pattern is seen in the
SCN of mice, 6 and Arvicanthis niloticus, a diurnal murid
rodent from East Africa. 14,30 Fos expression increases dramatically within the SCN of nocturnal rodents kept in constant
darkness when they are pulsed with light during the subjective
night. 6,44 Diurnal rodents are more variable in this respect; in
degus light actually inhibits Fos expression in the dorsal SCN
during the subjective day, 18 and in chipmunks light is as likely
to induce Fos during the subjective day as during the subjective night. 1 Within LSPVZ cells, Fos is also expressed rhythmically. The number of Fos-immunoreactive cells in the
LSPVZ of Rattus kept in a 12:12 LD cycle peaked at zeitgeber
time (ZT) 1 (ZT 0 ˆ lights on) and was relatively low at ZT 5,
13 and 17. 30 By contrast, Arvicanthis housed in the same
conditions had two peaks of Fos production within the
LSPVZ, one at ZT 1 and the other at ZT 17. 30 Pulses of
light given to rats 17 or hamsters 32,41 kept in constant darkness
also induce Fos or its mRNA in this retinorecipient region just
dorsal to the SCN.
The aim of the current research was to explore how the
neural substrates controlling circadian rhythms differ in
nocturnal and diurnal animals. In these studies, the nocturnal
laboratory rat, Rattus norvegicus was compared to the diurnal
rodent, Arvicanthis niloticus. This small gregarious rodent,
which inhabits much of sub-Saharan Africa, 38 has proven to
be a suitable model for the study of circadian rhythms. These
animals breed easily in the lab and are strictly diurnal with
respect to rest, 29 body temperature, mating, 22,24 and the
pattern of general activity. 24,45 Furthermore, trapping data
have recently revealed that these animals also exhibit diurnal
patterns of activity in the wild. 4 Although Arvicanthis are
predominantly diurnal, wheel-running records of animals
from our laboratory colony have revealed that some individuals, when housed in a cage with a running wheel, have
relatively nocturnal rhythms. 4 These nocturnal individuals
remain active in their wheels for 6–8 h after lights-off (Fig.
1). 4,37 However, when the wheels are removed, nocturnal
Arvicanthis exhibit a diurnal pattern of activity. 4 Throughout
this paper we will refer to diurnal wheel runners as diurnal and
animals with nocturnal wheel running patterns as nocturnal (for
a more complete description of these patterns see Ref 4).
Our first objective was to characterize the expression of
CALB and Fos within the SCN and LSPVZ of Arvicanthis
and Rattus. Reports on CALB in the SCN of Rattus have been
somewhat conflicting 5,36,46 and the distribution of this protein
has never been described in Arvicanthis. Our second objective
was to evaluate the hypothesis that differences between
nocturnal and diurnal animals are associated with differences
in the function of CALB-immunoreactive neurons within the
SCN and/or the LSPVZ. We examined patterns of Fos expression within CALB-immunoreactive neurons in the SCN and
LSPVZ of Rattus as well as the nocturnal and diurnal forms of
Arvicanthis during the light and dark portions of a 12:12 LD
cycle.
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Calbindin and Fos in and above the SCN
It should be noted that this study differs from most
studies of Fos in the SCN in that we kept animals on a LD
cycle, rather than housing them in constant darkness and
pulsing them with light. The latter, more common approach
has contributed to our understanding of mechanisms underlying light-induced shifts in the phase of endogenous
rhythms. 2,8,17,41,44 However, light during the subjective night
is not the only factor stimulating Fos expression in the SCN,
particularly in diurnal animals. 1,18 Furthermore, our purpose
was not to understand phase-shifting mechanisms, but to
understand the mechanisms that determine phase relationships between the LD cycle and rhythms in behavior and
physiology. Phase relationships between rhythms can be quite
different in constant darkness compared to a LD cycle. 49 To
address the issue of relationships between neural function and
differences between diurnal and nocturnal patterns we therefore
chose to examine animals kept in a LD cycle.
EXPERIMENTAL PROCEDURES
Animals
We used adult (⬎60 days) A. niloticus bred in the laboratory from a
stock descended from 29 individuals originally trapped in Kenya in
1993.15 In addition, we used adult male Rattus (Charles River Laboratories, Cambridge, MA, USA). All animals were kept in a 12:12 LD cycle,
singly housed in Plexiglas cages (38 × 34 × 16 cm) and provided with
water and food (Harlan 8640 Teklad, Madison, WI, USA) ad libitum. A
red light (⬍5 lux) remained on constantly for the purposes of animal care.
All experiments were performed in compliance with the Michigan State
University All-University Committee on Animal Use and Care in accordance with the standards in the NIH Guide for the Care and Use of
Laboratory Animals. All efforts were made to minimize the suffering
and the number of animals used in these experiments.
Single labeling for calbindin and Fos within the suprachiasmatic
nucleus and lower sub-paraventricular zone of Arvicanthis niloticus
and Rattus norvegicus
At 3–5 h after lights-on, animals were deeply anesthetized with
sodium pentobarbital (Nembutal; Abbott Laboratories, 0.5 cm 3/Arvicanthis, 1.0 cm3/Rattus) and perfused transcardially with 0.01 M
phosphate-buffered saline (PBS; pH 7.2, 150–200 ml/Arvicanthis and
250 ml/Rattus) followed by 150–200 ml of fixative (PLP; 4% paraformaldehyde, (Sigma, St. Louis, MO, USA), 1.4% lysine, Sigma, 0.2%
sodium m-periodate, Sigma, in 0.1 M phosphate buffer; pH 7.4). Brains
were post-fixed in PLP (4 h) before being transferred to 20% sucrose
(Sigma) in 0.1 M phosphate buffer. After 24 h in sucrose, brains were
sectioned at 30 mm using a freezing microtome and sections were transferred to cryoprotectant 50 and stored at ⫺20⬚C until processing began.
To determine the pattern of CALB-immunoreactivity, every other
section from Arvicanthis (n ˆ 2 male, n ˆ 2 female) was labeled and
every third section from Rattus (n ˆ 1) was labeled. Free floating tissue
was incubated in (i) 5% normal horse serum (NHS; Vector Laboratories, Burlingame, CA, USA, in PBS with 0.3% Triton-X; Research
Products International) for 1 h at room temperature, followed by (ii)
primary antibody for 24 h at 4⬚C (mouse anti-CALB 1:2000, Sigma; in
PBS with 0.3% Triton-X and 3% NHS), followed by (iii) biotinylated
secondary antibody for 1 h at room temperature (1:200, horse antimouse; Vector Laboratories; in PBS with 0.3% Triton-X and 3%
NHS), followed by (iv) avidin–biotin complex (ABC) for 1 h at
room temperature (0.9% each avidin and biotin solutions; ABC
Vectastain Kit, Vector Laboratories; in PBS with 0.3% Triton-X).
Tissue was then reacted in diaminobenzidine (DAB; 0.5 mg/ml,
(Sigma), in Trizma buffer, pH 7.2) with 30% hydrogen peroxide
(0.35 l/ml buffer). In between each step, tissue was rinsed three
times for 10 min in PBS.
To determine the pattern of Fos-immunoreactivity, every other
section from Arvicanthis (n ˆ 3 female) was labeled as well as every
third section from Rattus (n ˆ 1 male). The procedure was the same
as described above except that normal goat serum (NGS) was used as
the blocking agent, rabbit anti-Fos as the primary antibody (1:5000,
Santa Cruz, CA, USA) and goat anti-rabbit as the secondary (1:200;
Vector Laboratories). Controls for CALB and Fos immunostaining
were performed by omitting the primary antibody from the above
procedure. No staining of cells was observed in any control tissue.
All tissue was mounted onto gelatin-coated slides, dehydrated, and
coverslipped. All sections containing SCN and LSPVZ were examined
under a light microscope (Leitz, Laborlux S, Wetzlar, Germany).
Camera lucida drawings were made of Fos-immunoreactivity and
CALB-immunoreactivity in SCN sections of representative Arvicanthis.
Double labeling for calbindin and Fos within the suprachiasmatic
nucleus and the lower sub-paraventricular zone of Arvicanthis
niloticus and Rattus norvegicus
The Arvicanthis used in this study were adult males (⬎60 days) that
exhibited either nocturnal or diurnal patterns of wheel running. To
make this determination, Arvicanthis were singly housed in Plexiglass
cages with running wheels (26 × 34 × 8 cm) for at least 10 days prior to
perfusion. Numbers of wheel revolutions, recorded when a magnet in
the wheel closed a switch on the wheel housing, were stored in 5-min
bins using the Dataquest III data collection system (Mini-Mitter,
Sunriver, OR, USA). Hourly rates of wheel running were averaged
over the last five days prior to perfusion and used to determine if
animals were nocturnal or diurnal. Animals were considered to be
diurnal if they had become inactive by the end of the third hour after
lights out (range ˆ 1–3 h, Fig. 1). A given hour of wheel running was
defined as inactive if the number of wheel revolutions was less than
10% of the daily peak. Nocturnal animals continued running for ⬎6 h
after lights-off (range ˆ 6–8 h; Fig. 1). Arvicanthis used in this second
experiment were also utilized in Rose et al. and their patterns of
rhythmicity are described more completely in that paper. 37
Animals in this study were perfused at either ZT 4 (Rattus n ˆ 6,
diurnal Arvicanthis n ˆ 6, nocturnal Arvicanthis n ˆ 6) or ZT 16
(Rattus n ˆ 6, diurnal Arvicanthis n ˆ 6, nocturnal Arvicanthis n ˆ 5).
Animals killed during the dark phase were fitted with light-tight
hoods prior to and during the perfusion. Procedures for perfusion
and tissue preparation were the same as in the first study except that
the brains remained in fixative for 9 h prior to being placed in sucrose
and tissue was sectioned at 40 mm. Every third section from each
animal was processed for Fos using a nickel enhanced DAB procedure
as follows: free floating tissue was incubated in (i) 5% NGS (in PBS
with 0.3% Triton-X) for 1 h at room temperature, followed by (ii)
primary antibody for 24 h at 4⬚C (rabbit anti-Fos 1:5000, Santa
Cruz; in PBS with 0.3% Triton-X and 3% NGS), followed by (iii)
biotinylated secondary antibody for 1 h at room temperature (1:200,
goat anti-rabbit; Vector Laboratories; in PBS with 0.3% Triton-X and
3% NGS), followed by (iv) ABC for 1 h at room temperature. Tissue
was then reacted by mixing DAB (0.5 mg/ml Trizma buffer, Sigma),
3% hydrogen peroxide (0.825 l/ml buffer) and 2.5% nickel sulfate (in
0.175 M sodium acetate buffer, Sigma). Following the Fos reaction,
tissue was rinsed and processed for detection of CALB-immunoreactivity as described in the first study. Negative controls were done
by performing the above procedures with the exception that CALB,
Fos or both CALB and Fos primary antibodies were omitted.
Tissue was mounted, dehydrated, coverslipped and examined under
a light microscope (Leitz, Laborlux S). A single section through the
central SCN was selected from each animal and a camera lucida drawing was made of SCN cells positive for Fos, CALB, and for both Fos
and CALB. We mapped and counted the same three types of cells in a
rectangular region (215 mm × 160 mm) of the LSPVZ immediately
dorsal to the SCN section selected for analysis. All labeling was
confirmed by examining the tissue with a high-power oil objective.
Data from Arvicanthis were analysed using a two-way analysis of
variance followed by Tukey honestly significant difference test 52 when
necessary and data from Rattus were analysed using independent ttests (SYSTAT, Chicago, IL, USA). Data in the form of proportions
were arc sine transformed prior to analysis. 52 Differences were considered
significant if P ⬍ 0.05 (two-tailed tests were used when appropriate).
RESULTS
Single labeling for calbindin and Fos within the suprachiasmatic nucleus and lower sub-paraventricular zone of
Arvicanthis niloticus and Rattus norvegicus
Suprachiasmatic nucleus of Arvicanthis niloticus. CALBimmunoreactive expression within a section through the
568
M. M. Mahoney et al.
Fig. 2. Representative examples of staining for calbindin-immunoreactivity within a coronal section through the central suprachiasmatic nucleus of (A)
Arvicanthis and (B) Rattus. The lower sub-paraventricular zone is the area immediately dorsal to the suprachiasmatic nucleus: 3V, third ventricle: OC, optic
chiasm. Scale bar ˆ 50 mm.
central SCN of a representative Arvicanthis is depicted in Fig.
2A. There was a very distinct pattern of CALB-immunoreactivity within the SCN; the most centrally located
CALB-immunoreactive neurons were more densely concentrated and darkly stained than the CALB-immunoreactive
neurons in the more peripheral regions of the nucleus (Fig.
2A). We saw no obvious differences between males and
females with respect to the distribution of CALB-immunoreactive cells.
The variation of density and distribution of CALB expression in the rostrocaudal extent of the SCN of Arvicanthis is
shown in Fig. 3A. In the rostral SCN, CALB-immunoreactive
cells were relatively sparse (Fig. 3A). In sections through the
middle of the SCN, CALB-immunoreactive cells increased in
number and became localized within the central region of the
nucleus (Fig. 3A). In the caudal SCN, CALB-immunoreactive
cells became less clustered and were most darkly stained
within the dorsal portion of the nucleus (Fig. 3A).
The distribution of Fos-immunoreactivity within the rostrocaudal extent of the SCN of Arvicanthis is depicted in Fig. 3B.
In sections through the rostral SCN, Fos-immunoreactive
cells were evenly distributed across the nucleus (Fig. 3B).
In sections through the central and caudal SCN Fos-immunoreactivity was primarily expressed outside of the dense
CALB-immunoreactive portion of the nucleus; Fos-immunoreactive nuclei were more heavily concentrated within
ventral, medial and dorsal regions, and were relatively sparse
within the central region (also see Ref. 14).
Lower sub-paraventricular zone of Arvicanthis niloticus.
In Fig. 2A, darkly stained CALB-immunoreactive cells can
be observed in the region immediately dorsal to the SCN.
CALB-immunoreactive cells were evenly distributed
throughout the LSPVZ and were not concentrated in a particular subregion. In sections that contained the rostral SCN,
the density of CALB-immunoreactive cells in the LSPVZ was
more sparsely distributed than within the SCN. However, in
LSPVZ regions dorsal to the central and caudal SCN the
density of CALB-immunoreactive cells increased. CALBimmunoreactive cells tended to decrease in number approximately 300 mm dorsal to the SCN, except along the border of
the third ventricle.
In the LSPVZ of Arvicanthis, Fos-immunoreactive cells
were present in a region fanning out dorsally from the SCN.
The distribution of these Fos-immunoreactive cells changed
across the rostrocaudal axis; in sections containing the rostral
SCN cells with Fos-immunoreactive nuclei were sparse and
more lightly stained above than within the SCN. In LSPVZ
regions above the central and caudal SCN Fos-immunoreactive cells became more densely concentrated, and were
more darkly stained than within the SCN itself. Thus, within
the LSPVZ region, the distribution of Fos-immunoreactive
and CALB-immunoreactive cells overlapped while in the
SCN, CALB-immunoreactive and Fos-immunoreactive
distribution did not correspond as well.
Suprachiasmatic nucleus of Rattus norvegicus. As
depicted in Fig. 2B, sparse levels of CALB-immunoreactive
staining were observed in the central SCN of Rattus. CALBimmunoreactive cells were not concentrated within any
particular subdivision of the Rattus SCN. In comparison to
CALB-immunoreactive staining, Fos-immunoreactive cells
Calbindin and Fos in and above the SCN
569
Fig. 3. Camera lucida drawings of sections through the suprachiasmatic nucleus of a representative Arvicanthis. Sections are arranged from rostral to caudal
and the left side of each picture is medial. Sections are labeled for (A) calbindin or (B) Fos expression. OC, optic chiasm. Open circles indicate calbindinimmunoreactive cells and X indicate Fos-immunoreactive cells.
were present and stained most darkly within the ventral region
of the rostral and central SCN sections. However, in the
caudal SCN of Rattus, this regional distribution of Fosimmunoreactive cells became less obvious and the cells
became more scattered.
Lower sub-paraventricular zone of Rattus norvegicus.
CALB-immunoreactive cells were present throughout the
rostrocaudal extent of the LSPVZ but were not heavily
concentrated in this region. Thus, CALB-immunoreactivity
was sparse within the SCN and LSPVZ of Rattus. Cells
expressing Fos-immunoreactivity were less concentrated
and more lightly stained within the LSPVZ than in the
adjacent SCN. Additionally, Fos expression was regionally
distributed within the LSPVZ; Fos-immunoreactive cells
were darker and more densely distributed in the central
and caudal LSPVZ than in the rostral LSPVZ. In comparison,
Fos- immunoreactive cells in the SCN were more concentrated in the rostral and central regions than within the caudal
region.
Double labeling for calbindin and Fos within the suprachiasmatic nucleus and the lower sub-paraventricular zone
of Arvicanthis niloticus and Rattus norvegicus
Suprachiasmatic nucleus of Arvicanthis niloticus. Within
the SCN of Arvicanthis, the distribution of CALB-immunoreactive and Fos-immunoreactive cells was distinctly different. CALB containing cells were most concentrated and
darkly stained within the central portion of the SCN. An
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M. M. Mahoney et al.
Fig. 4. Calbindin-immunoreactivity and Fos-immunoreactivity within the suprachiasmatic nucleus and the lower sub-paraventricular zone of Arvicanthis as
seen under a high-power oil immersion lens (Leitz Laborlux S). Black/purple staining indicates Fos-immunoreactivity and orange/brown indicates calbindinimmunoreactivity. ⫹ indicates a single-labeled calbindin-immunoreactive cell; * indicates a single-labeled Fos-immunoreactive nucleus; arrow indicates a
cell double labeled for both calbindin-immunoreactivity and Fos-immunoreactivity. Suprachiasmatic nucleus is depicted in A–C, and the lower sub-paraventricular zone in D. Scale bar ˆ 20 mm.
area that contained a relatively high number of Fos-immunoreactive cells and fewer CALB-immunoreactive cells
encircled this central CALB-immunoreactive region.
Cells positive for both CALB- and Fos-immunoreactivity
were detectable within the SCN of Arvicanthis (Fig. 4)
but were extremely rare (Table 1). Less than 1% of
CALB-immunoreactivity cells in the Arvicanthis SCN
expressed Fos-immunoreactivity. The few double-labeled
cells observed were not concentrated in any discrete region of
the SCN.
Cells containing Fos-immunoreactivity were more darkly
stained and significantly more numerous in Arvicanthis killed
during the light (ZT 4) compared to the dark (ZT 16) phase
of the LD cycle (Table 1, F ˆ 25.24, d.f. ˆ 1, P ⬍ 0.001);
which confirms previous reports. 14,30 However, time of day
had no effect on the number of CALB containing cells
(Table 1, F ˆ 0.008, d.f. ˆ 1, P ˆ 0.93), or the per cent of
CALB-immunoreactive cells that contained Fos-immunoreactivity (F ˆ 0.59, d.f. ˆ 1, P ˆ 0.45). Nocturnal and diurnal
Arvicanthis did not differ with respect to the numbers of
SCN cells that contained Fos-immunoreactivity (Table 1,
F ˆ 0.51, d.f. ˆ 1, P ˆ 0.484), or CALB-immunoreactivity
(Table 1, F ˆ 0.43, d.f. ˆ 1, P ˆ 0.52), or were double labeled
(F ˆ 0.03, d.f. ˆ 1, P ˆ 0.86).
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Calbindin and Fos in and above the SCN
Table 1. Fos-immunoreactivity and calbindin-immunoreactivity in diurnal and nocturnal wheel-running forms of A. niloticus at zeitegeber time 4 and
16 (ZT 0 ˆ lights-on, mean ^ S.E.M.)
ZT 4
ZT 16
Diurnal
Nocturnal
Diurnal
Nocturnal
SCN
Fos-immunoreactive cells*
CALB-immunoreactive cells
% CALB-immunoreactive cells positive for Fos
424.0 ^ 48.5
282.5 ^ 47.1
0.5 ^ 0.3
496.5 ^ 83.9
197.7 ^ 56.7
0.5 ^ 0.2
146.3 ^ 35.7
235.0 ^ 29.9
0.4 ^ 0.2
161.0 ^ 65.0
254.0 ^ 63.9
0.3 ^ 0.1
LSPVZ
Fos-immunoreactive cells
CALB-immunoreactive cells†
% CALB-immunoreactive cells positive for Fos
71.5 ^ 9.5
174.7 ^ 15.8
11.6 ^ 2.2
100.7 ^ 14.6
129.3 ^ 12.9
11.9 ^ 2.5
82.8 ^ 13.0
141.7 ^ 7.7
13.3 ^ 1.3
96.8 ^ 13.0
168.0 ^ 22.0
14.0 ^ 2.2
*Significant effect of time, P ⬍ 0.001.
†Significant effect of interaction, P ⬍ 0.05; diurnal ⬎ nocturnal at ZT 4 (P ⬍ 0.05).
Suprachiasmatic nucleus of Rattus norvegicus. In doublelabeled tissue, CALB-immunoreactive cells were sparse but
evenly distributed throughout the SCN and Fos-immunoreactive cells were found throughout the SCN but were
most heavily concentrated within its ventral region. Doublelabeled cells were detectable in the SCN of Rattus (Fig. 5).
Approximately five to 10 double-labeled cells were seen in
each section through the SCN, which represented approximately 13–17% of CALB-immunoreactive cells (Table 2).
These double-labeled cells were not concentrated within
any particular subdivision of the SCN.
As previously reported (e.g. see Refs 2, 8, 16 and 30) cells
with Fos-immunoreactive nuclei stained more darkly and
were significantly more numerous in animals killed during
the light portion of the LD cycle (Table 2; t ˆ 7.392,
d.f. ˆ 10, P ⬍ 0.0001). There was no significant effect of
time on the total number of cells that contained CALBimmunoreactivity (t ˆ ⫺0.8, d.f. ˆ 10, P ˆ 0.43), the number
of double-labeled cells (t ˆ ⫺1.55, d.f. ˆ 10, P ˆ 0.15), or the
proportion of CALB-immunoreactive cells that contained
Fos-immunoreactivity within the Rattus SCN (Table 2;
t ˆ ⫺1.23, d.f. ˆ 10, P ˆ 0.25).
Lower sub-paraventricular zone of Arvicanthis niloticus.
CALB-immunoreactive and Fos-immunoreactive cells were
evenly distributed through the LSPVZ region that was
sampled in Arvicanthis. CALB-immunoreactive and Fosimmunoreactive cells were more darkly stained but were
less densely concentrated in the LSPVZ than in the SCN. In
nocturnal and diurnal forms of Arvicanthis approximately
11–12% of CALB-immunoreactive cells in the LSPVZ
region expressed Fos-immunoreactivity (Table 1). No differences existed between these forms of Arvicanthis with respect
to the number of cells that contained Fos-immunoreactivity
(Table 1, F ˆ 2.8, d.f. ˆ 1, P ˆ 0.1), or CALB-immunoreactivity (F ˆ 0.40, d.f. ˆ 1, P ˆ 0.531), or were doublelabeled (F ˆ 0.05, d.f. ˆ 1, P ˆ 0.82). In this area time of
day had no effect on the number of cells that expressed
CALB-immunoreactivity (F ˆ 0.03, d.f. ˆ 1, P ˆ 0.85), Fosimmunoreactive (F ˆ 0.08, d.f. ˆ 1, P ˆ 0.772), or on the
percent of CALB-immunoreactive cells that contained Fos
(F ˆ 0.826, d.f. ˆ 1, P ˆ 0.375). However, the number of
CALB-immunoreactive cells present in the LSPVZ was influenced by an interaction between time of day and whether an
animal was nocturnal or diurnal (Table 1; F ˆ 5.8, d.f. ˆ 1,
P ⬍ 0.026). Specifically, the number of CALB-immunoreactive
neurons in the LSPVZ was significantly higher in diurnal
Arvicanthis at ZT 4 (P ⬍ 0.05). A trend in the opposite direction was seen at ZT 16 but that difference failed to reach
significance (P ⬎ 0.05).
Lower sub-paraventricular zone of Rattus norvegicus.
CALB-immunoreactive and Fos-immunoreactive cells were
evenly distributed through the LSPVZ region that we sampled
in Rattus. Double-labeled cells were clearly identifiable in
this region (Fig. 5) and approximately 13–17% of CALBimmunoreactive cells expressed Fos (Table 2). Time of day
had no significant effect on the number of CALB-labeled cells
(t ˆ 0.433, d.f. ˆ 10, P ˆ 0.0.67), or the percent of CALBimmunoreactive cells that contained Fos-immunoreactivity
within the nucleus (t ˆ 0.42, d.f. ˆ 10, P ˆ 0.68). Animals
killed during the light part of the day had a significantly
higher number of cells that contained Fos-immunoreactivity
in the LSPVZ than did animals killed at night (t ˆ 2.82,
d.f. ˆ 10, P ˆ 0.018).
DISCUSSION
The distribution of CALB-immunoreactive cells within
the SCN of Arvicanthis was similar in some respects to that
described in M. auratus. Specifically, in both species the
central portion of the SCN contained the highest concentration of darkly stained CALB-immunoreactive cells. 46
These two species differed slightly in that more lightly stained
CALB-immunoreactive cells were found outside the central
portion of the SCN in Arvicanthis (Figs 2A, 3A) than in
M. auratus. 46 Far more cells positive for CALB were seen
in the Arvicanthis SCN than in the rat SCN. Low levels of
CALB-immunoreactivity in the rat SCN have also been
reported in other studies. 5,46 CALB-immunoreactivity has
also been described in the SCN of three diurnal primate
species. In the SCN of humans, CALB-immunoreactive
neurons are relatively concentrated in a somewhat central
region of the SCN 23 as they are in Arvicanthis, whereas in
the SCN of squirrel monkeys and common marmosets CALBimmunoreactive neurons are distributed throughout the
SCN. 7,9 Thus, the distribution and density of CALB-immunoreactive neurons in the SCN varies among species, but this
variability is unrelated to whether animals are nocturnal or
diurnal.
Assuming that CALB-immunoreactivity is an important
component of the circadian clock, 20 the present observations
572
M. M. Mahoney et al.
Fig. 5. Calbindin-immunoreactivity and Fos-immunoreactivity within the suprachiasmatic nucleus and lower sub-paraventricular zone of Rattus as seen under
a high power oil immersion lens (Leitz Laborlux S). Black/purple staining indicates Fos-immunoreactivity and orange/brown indicates calbindin-immunoreactivity. ⫹ Indicates a single-labeled calbindin-immunoreactive cell; * indicates a single-labeled Fos-immunoreactive cell; arrow indicates a cell doublelabeled for both calbindin-immunoreactivity and Fos-immunoreactivity. Suprachiasmatic nucleus is depicted in (A–C), and the lower sub-paraventricular
zone in (D). Scale bar ˆ 20 mm.
help to interpret the results of lesion studies with rats and
hamsters. In the hamster CALB-immunoreactive cells are
predominantly seen in a central subregion of the SCN, and
destruction of this area is sufficient to disrupt behavioral
rhythms. 20 In the rat, CALB-immunoreactive neurons are
more evenly distributed across the SCN, and in this species
partial lesions of the SCN are compatible with relatively
normal rhythms (Nunez, unpublished observations). The
effects of partial SCN lesions restricted to the area of high
concentration of CALB-immunoreactive cells have not been
examined in Arvicanthis.
Evidence from several species 7,46 suggests that CALB
may play a role in the processing of photic input to the
SCN. CALB expression in the hamster SCN is dependent
on photic conditions. 21 Retinal terminals, identified through
intraocular cholera toxin (CT) injections, were dense in the
CALB-immunoreactive rich region of the hamster SCN. 46
Similarly, intraocular CT injections in marmosets revealed
retinal terminals within the ventral region of the SCN, an
area with a high concentration of CALB-immunoreactive
neurons. 7 Furthermore, when hamsters were pulsed with
light during a photosensitive phase 75.4% of CALB-immunoreactive cells within the SCN expressed Fos indicating
that these cells receive information with respect to light. 46
In the current study, we observed relatively low levels of
Fos expression within CALB-immunoreactive cells in the
Calbindin and Fos in and above the SCN
Table 2. Fos-immunoreactivity and calbindin-immunoreactivity in Rattus
norvegicus at zeitegeber time 4 and 16 (ZT 0 ˆ lights-on, mean ^ S.E.M.).
ZT 4
ZT 16
SCN
Fos-immunoreactive cells*
CALB-immunoreactive cells
% CALB-immunoreactive
cells positive for Fos
410.5 ^ 38.7
43.0 ^ 5.2
13.7 ^ 3.7
107.3 ^ 13.5
51.2 ^ 8.7
21.5 ^ 5.17
LSPVZ
Fos-immunoreactive cells†
CALB-immunoreactive cells
% CALB-immunoreactive
cells positive for Fos
39.3 ^ 5.7
33 ^ 4.3
10.1 ^ 2.1
21.3 ^ 2.8
30.8 ^ 2.4
9.0 ^ 1.7
*Significant effect of time, P ⬍ 0.001.
†Significant effect of time, P ⬍ 0.001.
SCN of Rattus and almost no double-labeled cells in the SCN
of Arvicanthis, even though retinal fibers reach the regions of
the SCN that contain CALB-immunoreactive cells in these
animals. 12,47 However, the experimental paradigms used to
study these species differed. Hamsters were pulsed with
light during their photosensitive period 46 whereas Rattus and
Arvicanthis in the current study were kept on a 12:12 LD cycle.
It therefore remains possible that Fos expression within CALBimmunoreactive cells in the SCN is high in Rattus and
Arvicanthis when the animals are kept in DD and pulsed with
light, and low in M. auratus kept on a LD cycle.
Rattus and Arvicanthis differed in the current study with
respect to the proportions of CALB-immunoreactive neurons
that expressed Fos-immunoreactivity. In rats, CALBimmunoreactive cells in the SCN and LSPVZ were indistinguishable with respect to the proportions that expressed Fosimmunoreactive (Table 2). By contrast, these two regions
were quite different in Arvicanthis. In this species a considerably higher proportion of CALB-immunoreactive cells
expressed Fos in the LSPVZ (approximately 10%) than
within the SCN (⬍1%; Table 1). These data suggest that in
these two species CALB-immunoreactive neurons may function similarly in the LSPVZ but differently within the SCN.
One explanation that should be considered for the low levels
of Fos-immunoreactivity observed in CALB-immunoreactive
cells in the Arvicanthis SCN is that double-labeled cells may
have been difficult to detect with the methods employed.
Several considerations suggest that this does not account for
low levels of double labeling observed in this study. First,
using the same procedures as those employed here we have
been able to observe substantial Fos-immunoreactive expression within other peptidergic SCN cells. Fos-immunoreactive
nuclei were seen in approximately 20% of vasopressinimmunoreactive neurons 37 and 10% of neurons containing
vasoactive intestinal polypeptide-immunoreactivity (Smale,
unpublished observations). The double-labeled tissue was as
clear in the current study as in the earlier ones, suggesting that
had there been more Fos expression in CALB-immunoreactive cells, we would have seen it. Secondly, we were
able to clearly identify some doubled cells (Fig. 4), so the
basic procedure was adequate. Finally, using the same procedures we detected relatively high levels of double labeling in
the SCN of Rattus. The staining for CALB-immunoreactivity
and for Fos-immunoreactivity was similarly clear in doublelabeled tissue from the two species (Figs. 4 and 5). The lack of
double-labeled cells is consistent with the general pattern
573
observed in single-labeled sections through the Arvicanthis
SCN. That is, a complementarity in the staining was apparent
such that the central region where CALB-immunoreactive
cells were concentrated contained relatively few Fosimmunoreactive cells (Fig. 3).
No differences were found between diurnal and nocturnal
Arvicanthis with respect to any aspect of the SCN that we
examined. However, in the LSPVZ, CALB-immunoreactive
cell number was influenced slightly by an interaction between
time of day and wheel running pattern. Specifically, from day
to night the number of CALB-immunoreactive cells
decreased in diurnal Arvicanthis and increased in nocturnal
Arvicanthis. Thus the number of cells expressing CALB
in this zone tended to be highest during periods of
activity with significantly more CALB-immunoreactive
cells seen in diurnal animals at ZT 4. Diurnal Arvicanthis
exhibit diurnal patterns of general activity both with and
without access to a running wheel. 4 However, nocturnal
Arvicanthis are only active during the dark portion of the
LD cycle when they have access to a running wheel; they
switch to a diurnal pattern of general activity when the
running wheel is removed from their cage. 4 The difference
between the two forms of Arvicanthis with respect to LSPVZ
CALB-immunoreactivity cells could therefore be similarly
triggered by access to a wheel. Alternatively, CALBimmunoreactive in the LSPVZ could differ in these two
forms of Arvicanthis even when they are housed without a
wheel and could play a role in determining their differential
response to running wheels. The LSPVZ region receives input
from the retina 12 and the SCN, 28,51 raising the possibility that
either or both of these inputs might influence the number of
cells expressing CALB-immunoreactivity or Fos-immunoreactivity in this region.
The LSPVZ may interact with the SCN to regulate circadian rhythms. In the diurnal Siberian chipmunk (Eutamias
sibiricus) multiple unit activity exhibited a rhythm in the
SCN that was in phase and parallel to the rhythm seen in
adjacent regions outside of the SCN, including the
LSPVZ. 42 However, in nocturnal rats, the multiple unit activity rhythm in regions outside the SCN was 180⬚ out of phase
with the rhythm inside the SCN. 19 Furthermore, a rhythm in
Fos expression has been found in the LSPVZ that differs
between Arvicanthis and rats. 30 In the LSPVZ of rats, Fos
expression peaked at ZT 1 and then decreased across the
day, whereas in the LSPVZ of Arvicanthis, Fos expression
was elevated at ZT 1 and ZT 17 and was low at ZT 5 and 13.
Fos expression in the LSPVZ of Arvicanthis did not differ
between ZT 4 and 16 in the current study, this suggests that
Fos had not yet declined to trough levels at ZT 4 or risen to
peak levels by ZT 16 (Table 1). 30.
In summary, some features of CALB-immunoreactive cells
in the SCN differed in Arvicanthis when compared to Rattus,
but were the same in nocturnal and diurnal forms of Arvicanthis.
Specifically, in all Arvicanthis the SCN contained a substantial
CALB-immunoreactive cell population concentrated in the
center of the nucleus, and these cells almost never expressed
Fos-immunoreactivity, whereas in the SCN of Rattus sparse
diffusely scattered CALB-immunoreactive cells were considerably more likely to express Fos-immunoreactivity. These
species differences in CALB-immunoreactive cell function
within the SCN may contribute to differences in rhythm
patterns of Rattus and Arvicanthis. In the region dorsal to the
SCN, Rattus and Arvicanthis were similar with respect to
574
M. M. Mahoney et al.
CALB-immunoreactive cells, but nocturnal and diurnal Arvicanthis were different. This intraspecific difference raises the
possibility that neural processes related to CALB-immunoreactive cells in the LSPVZ might contribute to differences
between diurnal and nocturnal Arvicanthis.
Acknowledgements—The authors would like to thank Teresa
McElhinny, Betty Gubik, Colleen Novak, and Sandra Rose for their
valuable assistance and advice. The research described in this paper
was supported by NIMH RO1-MH053433 to L.S. and NSF IBN
9514374 to A.A.N.
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(Accepted 28 April 2000)