2013

Chronobiology International, 2013; 30(9): 1089–1100! Informa Healthcare USA, Inc.ISSN: 0742-0528 print / 1525-6073 onlineDOI: 10.3109/07420528.2013.800090

Photoperiod affects the diurnal rhythm of hippocampal neuronalmorphology of siberian hamsters

Tomoko Ikeno, Zachary M. Weil, and Randy J. Nelson

Department of Neuroscience, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA

Individuals of many species can regulate their physiology, morphology, and behavior in response to annual changesof day length (photoperiod). In mammals, the photoperiodic signal is mediated by a change in the duration ofmelatonin, leading to alterations in gene expressions, neuronal circuits, and hormonal secretion. The hippocampus isone of the most plastic structures in the adult brain and hippocampal neuronal morphology displays photoperiod-induced differences. Because the hippocampus is important for emotional and cognitive behaviors, photoperiod-driven remodeling of hippocampal neurons is implicated in seasonal differences of affect, including seasonal affectivedisorder (SAD) in humans. Because neuronal architecture is also affected by the day-night cycle in several brain areas,we hypothesized that hippocampal neuronal morphology would display a diurnal rhythm and that day length wouldinfluence that rhythm. In the present study, we examined diurnal and seasonal differences in hippocampal neuronalmorphology, as well as mRNA expression of the neurotrophic factors (i.e., brain-derived neurotrophic factor [Bdnf],tropomyosin receptor kinase B [trkB; a receptor for BDNF], and vascular endothelial growth factor [Vegf]) and a circadianclock gene, Bmal1, in the hippocampus of Siberian hamsters. Diurnal rhythms in total length of dendrites, the numberof primary dendrites, dendritic complexity, and distance of the furthest intersection from the cell body were observedonly in long-day animals; however, diurnal rhythms in the number of branch points and mean length of segmentswere observed only in short-day animals. Spine density of dendrites displayed diurnal rhythmicity with different peaktimes between the CA1 and DG subregions and between long and short days. These results indicate that photoperiodaffects daily morphological changes of hippocampal neurons and the daily rhythm of spine density, suggesting thepossibility that photoperiod-induced adjustments of hippocampal neuronal dynamics might underlie seasonaldifference of affective responses. Bmal1 mRNA showed a diurnal rhythm and different expression levels between longand short days were observed. However, there were no strong effects of day length on Bdnf, trkb, and Vegf geneexpression, suggesting that these genes are not involved in the photoperiodic effects on hippocampal neurons.

Keywords: Animal models, BDNF, circadian clock, day length, diurnal rhythm, seasonal affective disorder, seasonality,Siberian hamsters, trkb, VEGF

INTRODUCTION

Many organisms are able to respond to annual cycles of

day length (photoperiod) by physiological, morpho-

logical, and behavioral changes in anticipation of

approaching seasons (Goldman, 2001). In response to

short day lengths, many small rodents cease reproduct-

ive activities to avoid producing offspring during the

winter when temperatures and food availability are

challenging. The photoperiodic response is mediated by

a change in the duration of nocturnal hormone, mela-

tonin (Pevet, 1988). Melatonin secretion from the pineal

gland is regulated by a circadian clock, which is an

internal daily timekeeping system, and is directly sup-

pressed by light (Goldman, 2001).

In addition to the reproductive traits, seasonal

changes of brain morphology (such as brain volume

and neuronal architecture) have also been reported for

individuals of some vertebrate species. In songbirds, for

example, the volumes of brain regions that control song

production increase dramatically in anticipation of the

breeding season (Brenowitz et al., 1997; Smith et al.,

1997; Tramontin et al., 2001). These volumetric changes

are mediated by increases in neuronal size, number,

and spacing (reviewed in Tramontin & Brenowitz, 2000).

In mammals, seasonal differences have been observed

in various brain regions, especially in the hippocampus,

which is one of the most plastic sites in adult mamma-

lian brains (Breedlove & Jordan, 2001). Wild-captured

rodents of many species display seasonal variation in

Correspondence: Tomoko Ikeno, Department of Neuroscience, The Ohio State University, Columbus, OH 43210, USA. Tel. +1 614688 4674; Fax: +1 614 292 3464; E-mail: tomoko.ikeno@ousmc.edu

Submitted March 6, 2013, Returned for revision April 12, 2013, Accepted April 24, 2013

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hippocampal volume; exposure to short days in the

laboratory reduces whole-brain and hippocampal

volume (Burger et al., 2013; Galea & McEwen, 1999;

Perrot-Sinal et al., 1998; Pyter et al., 2005). In white-

footed mice, Peromyscus leucopus, and Siberian ham-

sters, Phodopus sungorus, short days change neuronal

architecture, including soma size, dendritic complexity,

and dendritic spine density in the hippocampus

(Pyter et al., 2005; Workman et al., 2011).

The hippocampus has been considered a key area

for emotion and cognition (Small et al., 2011). The

hippocampus plays an inhibitory role in the regulation

of the hypothalamic-pituitary-adrenal (HPA) axis activ-

ity, which is the major neuroendocrine system that

regulates reactions to stressors and regulates several

biological and psychological processes, including mood

and emotions (Jacobson & Sapolsky, 1991). Reduction in

hippocampal volume, atrophy of hippocampal neurons,

altered neuronal morphology, or loss of hippocampal

dendritic spines is often observed in depressed patients

(Cole et al., 2011; Neumeister et al., 2005; Stockmeier

et al., 2004) and in nonhuman animals displaying

depressive-like responses (Hajszan et al., 2009, 2010;

Magarinos & McEwen 1995; Watanabe et al., 1992).

It has been suggested that photoperiodic changes

in hippocampal structure possibly underlie seasonal

affective disorder (SAD), which is a specific mood

disorder characterized by depression induced by shor-

tened day lengths in winter (Workman & Nelson, 2011).

In addition to seasonal changes, many processes,

ranging from gene expression to behavior, show daily

rhythms that are regulated by the circadian clock (Ko &

Takahashi, 2006). Diurnal rhythmicity is observed in

mood status both in humans (Adan & Sanchez-Turet,

2001; Owens et al., 2000) and in animal models of

affective disorders (Kaya et al., 2011). In mammals, a

master clock is located in the suprachiasmatic nucleus

(SCN) of the hypothalamus, where circadian clock genes

drive autonomous feedback loops to generate circadian

signals (Ko & Takahashi, 2006). In addition to the SCN,

circadian clock genes are also expressed throughout the

brain, including the hippocampus (Jilg et al., 2010). In

some tissues or cells, neurotrophic factors or their

receptors are implicated in depression; for example,

brain-derived neurotrophic factor (BDNF), tropomyosin

receptor kinase B (TrkB) (a receptor for BDNF and also

called neurotrophic tyrosine kinase receptor type 2

[Ntrk2]), and vascular endothelial growth factor (VEGF)

show circadian or diurnal rhythms in mRNA or protein

expressions (Blanco et al., 2011; Dolci et al., 2003;

Koyanagi et al., 2003; Schaaf et al., 2000). Because these

genes are essential for neuronal development and

survival, synaptic transmission and plasticity, or neuro-

genesis (Fournier & Duman, 2012; Yu & Chen, 2011) and

because the hippocampus expresses these genes (Dolci

et al., 2003; Shaaf et al., 2000; Wang et al., 2005), we

hypothesized that hippocampal neuronal architecture

also shows diurnal differences, as well as seasonal

changes, and that these neurotrophic factors contribute

to hippocampal neuronal plasticity.

In the present study, we examined the diurnal

differences of hippocampal neuronal morphology as

well as mRNA expression of neurotrophic genes, Bdnf,

trkb, and Vegf, and the circadian clock gene, Bmal1, in

the hippocampus of Siberian hamsters exposed to long

or short days. Individuals of this species exhibit many

seasonal adaptations such as a loss of body mass and

the cessation of reproduction during the winter that can

be produced by short-day exposure in the laboratory

(reviewed in Scherbarth & Steinlechner, 2010). Because

individuals of this species also respond to short days

with a depressive-like phenotype, Siberian hamsters

may serve as a potential animal model for SAD

(Prendergast & Nelson, 2005; Pyter & Nelson, 2006;

Workman & Nelson, 2011; Workman et al., 2011). We

compared the diurnal patterns between long days and

short days in Siberian hamsters to determine whether

day length affects hippocampal rhythmicity.

MATERIALS AND METHODS

AnimalsSiberian hamsters (Phodopus sungorus) used in this

study were bred in our colony at The Ohio State

University. Male hamsters were weaned during the

light phase at 21–24 d of age and immediately placed

into either short-day conditions (SD: 8 h light:16 h dark;

N¼ 35) or maintained in long-day conditions (LD: 16 h

light:8 h dark; lights off at 15:00 h Eastern Standard Time

[EST] in all cases; N¼ 37) and at a constant temperature

of 21� 2 �C and relative humidity of 50� 10%. Hamsters

were individually housed in polypropylene cages

(30� 15� 14 cm) and had ad libitum access to food

(Harlan Teklad Rodent Diet 8640; Indianapolis, IN, USA)

and filtered tap water. Hamsters were housed in their

respective photoperiods for 4 wks prior to experiments.

All procedures were approved by the Ohio State

University Institutional Animal Care and Use

Committee and comply with guidelines established

by the National Instituted of Health published in

Guide for the Care and Use of Laboratory Animals

(Institute of Laboratory Animal Resources (U.S.), 2011).

Tissue collection and processingAfter 4 wks of photoperiod exposure, hamsters were

anesthetized with isoflurane vapors at zeitgeber time

(ZT; hours after lights on) 0 (N¼ 7 in LD; 6 in SD), ZT4

(N¼ 5 in LD; 9 in SD), ZT8 (N¼ 5 in LD; 6 in SD), ZT12

(N¼ 5 in LD; 5 in SD), ZT16 (N¼ 5 in LD; 4 in SD), and

ZT20 (N¼ 10 in LD; 5 in SD), and body mass was

measured. Then, hamsters were rapidly decapitated,

and brains were removed, placed in RNAlater (Ambion,

Austin, TX, USA), and stored at 4 �C to maintain mRNA

integrity for gene expression analysis. Five brains

collected at each time point of ZT4, ZT12, and ZT20

were cut along the anterior-posterior axis by using a

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razor blade immediately after removal from a skull,

the half of the brain was placed in RNAlater and

the other half was processed for Golgi impregnation

using the FD Rapid GolgiStain Kit (FD

NeuroTechnologies, Ellicott City, MD, USA) according

to the manufacturer’s instructions. Testes, epididy-

mides, gonadal fat pads, and seminal vesicles were

also removed and weighed.

cDNA cloningTotal RNA was isolated using Trizol (Life Technologies,

Carlsbad, CA, USA) from two brains according to the

manufacturer’s instructions. cDNA was synthesized

from total RNA with Moloney murine leukemia virus

(M-MLV) reverse transcriptase (Life Technologies) and

random primers (Life Technologies). Fragments of Bdnf,

trkb, Vegf, and 18 S ribosomal RNA genes were obtained

by polymerase chain reaction (PCR) using PCR

Supermix (Life Technologies) according to the manu-

facturer’s instruction. Primers used were bdnf-1 F

(50-ACT CTG GAG AGC GTG AAT GG-30) and bdnf-1 R

(50-TGT CCA ATA AAT AGA TTG TAG AAC CA-30) for

Bdnf, and 18 S-1 F (50-GTC TAA GTA CGC ACG GCC

GG-30) and 18 S-1 R (50-CAT GCA CCA CCA CCC ACG

GA-30) for 18 S rRNA. For trkb, the primary PCR was

performed using trkb-1 F (50-TCT CCW GGC ATC GTG

GCA TT-30) and trkb-1 R (50-CCA GGC CRG CCC ATG

AAG TG-30) and the secondary PCR was performed with

10% of the primary PCR product using trkb-2 F (50-CCT

AAC CTC CAN GTG GAG GAA GG-30) and trkb-1 R.

For Vegf, the primary PCR was performed using vegf-1 F

(50-GCC AGC ACA TAG GAG AGA TGA-30) and vegf-1 R

(50-GGG CAG AGC TGA GTG TTA GC-30) and the

secondary PCR was performed with 10% of the primary

PCR product using vegf-1 F and vegf-2 R (50-CGA TCN

GGG AGA GAG AGA TTG G-30). PCR products were

cloned into plasmids using pGEM-T Easy Vector System

(Promega, Fitchburg, WI, USA) and sequenced at the

Plant-Genomics Center at the Ohio State University. The

determined partial sequences of 595, 496, 350, and

1239 bp for Bdnf, trkb, Vegf, and 18 S rRNA, respectively,

showed high similarities of more than 90% to the

corresponding genes in Mus musculus. Accession num-

bers (DDBJ/GenBank/EMBL) are AB794380 for Bdnf,

AB794381 for trkb, AB794382 for Vegf, and AB794386 for

18 S rRNA.

Quantitative real-time PCRThe temporal expression patterns of Bdnf, trkb, Vegf,

and Bmal1 in the hippocampus were investigated. The

hippocampus was dissected from one brain hemisphere

by gently separating it from the overlying cortex and the

underlying thalamus, and then total RNA was isolated as

mentioned above. DNAs in RNA samples were digested

with deoxyribonuclease I, Amplification Grade (Life

Technologies). cDNAs were synthesized from 1 mg of

RNA using High Capacity cDNA Reverse Transcription

Kit (Applied Biosystems, Foster City, CA, USA). For

real-time PCR analysis, 1% of the cDNA was used at a

final concentration of 1�Power SYBR Green PCR

Master Mix (Life Technologies) and 0.05 mM of each

primer using a 7500 Fast Real-Time PCR System (Life

Technologies) according to the manufacturer’s instruc-

tions. Each reaction was performed in duplicates.

Primers used were bdnf-3 F (50-TTA CCT GGA TGC

CGC AAA C-30) and bdnf-3 R (50-GCG GGC TGG GTC

AGA GT-30) for Bdnf, trkb-3 F (50-CCA CTC CCC ACG

ATG CA-30) and trkb-3 R (50-TTC ATT CGT GTG TTT GA

AAC C-30) for trkb, vegf-3 F (50-GGG TGA GAT TCC GGC

AGA A-30) and vegf-3 R (50-TTC CAT GAG GGA CCA TGC

TT-30) for Vegf, bmal1-1 F (50-GGC AGC GAT GGC TGT

CA-030) and bmal1-1 R (50-TCC ACC CAG GCC TGC AT-

30), which were designed from the P. sungorus Bmal1

sequence (DDBJ/GenBank/EMBL, AY316534), for

Bmal1, and 18 S-3 F (50-AGA AAC GGC TAC CAC ATC

CAA-30) and 18 S-3 R (50-GGG TCG GGA GTG GGT AAT

TT-30) for 18 S rRNA. In all the reactions, the generation

of only a single expected amplicon was confirmed by

melting analysis. Each reaction was done in duplicate.

Quantification of cDNAs was performed by the standard

curve methodology.

Analysis of neuronal morphologyBrains were sliced at 100mm on a cryostat and counter-

stained with cresyl violet (Sigma-Aldrich, MO, USA).

Hippocampal cell morphology was assessed in the CA1

and dentate gyrus (DG) fields in the dorsal hippocampus

(Figure 1A). Sections were visualized using a Nikon E800

bright-field microscope and intact neurons were recon-

structed using Neurolucida software (MicroBrightField,

Burlington, VT, USA) with a 20� objective. Six repre-

sentative neurons were traced per area, from each

animal. The criteria for neuronal selection were (1)

neurons had to be fully impregnated; (2) dendrites could

not be truncated; and (3) for the CA1 region, neurons

had to be pyramidal cells with somata lying within the

pyramidal cell layer and for the DG, neurons had to be

granule cells with somas lying within the granule cell

layer (Figure 1A). One brain sample collected at ZT12 in

long-day conditions was removed from morphological

analysis of dendrites, because the sections of this

sample were in a poor state and it was impossible to

trace the entire neuron. The cell traces were analyzed

using the accompanying NeuroExplorer software

(MicroBrightField). Cell body perimeter and size, the

number of branch points, total length of dendrites,

length of dendritic segments (intervals between soma

origin and the first branch point, between two succes-

sive branch points, or between the last branch point and

the branch end), length of segments, and the number of

primary dendrites were calculated (see Figure 1B). Sholl

analyses were also conducted. This method is used for

measuring the complexity of neurons by counting the

number of dendritic intersections with concentric cir-

cles centered on the cell body (Figure 1B). In our

analyses, the distance between concentric Sholl circles

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was set to 10 mm. The number of intersections of

dendrites and Sholl circles was counted. The distance

between the cell body and the furthest intersection was

also measured (Figure 1B).

For spine density analysis, six neurons (in both the

pyramidal cell layer of the CA1 and granule cell layer of

the DG) were selected from each animal. Spines were

counted on dendrites longer than 20 mm if they were

beyond at least one branch point using Neurolucida

software with a 100� objective. Spine density (spines/

1 mm) was calculated for each trace in NeuroExplorer

software and averaged per neuron.

Statistical analysisSomatic and reproductive measures were analyzed

by unpaired two-tailed t test with photoperiod as the

independent variable. Gene expressions and neuronal

measures were analyzed by two-way analysis of variance

(ANOVA; photoperiod�ZT). Bonferroni post hoc test

was used to further evaluate group differences. Statistics

were performed using GraphPad Prism 4 (GraphPad

Software, San Diego, CA, USA).

RESULTS

Somatic and reproductive measuresShort days significantly reduced body mass (p50.0001,

two-tailed t test) (Figure 2A), paired testes mass

(p50.0001, two-tailed t test) (Figure 2B), paired epidi-

dymides mass (p50.0001, two-tailed t test) (Figure 2C),

fat pad mass (p50.0001, two-tailed t test) (Figure 2D),

and paired seminal vesicle mass (p50.0001, two-tailed

t test) (Figure 2E) compared with long days.

Temporal analysis of gene expressionsIn the hippocampus, the expression level of Bdnf

showed a diurnal pattern (Figure 3A). Statistical analysis

FIGURE 2. Somatic and reproductive

responses to photoperiod. Long-day (LD)

hamsters had larger (A) body mass, (B)

paired testes mass, (C) paired epididy-

mides mass, (D) paired fat pad mass, and

(E) paired seminal vesicle mass than short-

day (SD) hamsters. Data represent

mean� SEM values. **p50.01 (t test).

FIGURE 1. Schematic illustration of (A) the hippocampus and (B)

a virtual neuron with two primary dendrites and dendritic

parameters used for the morphological analysis in the present

study. Open small circles show branch points. Arrows represent

dendritic segments. Dotted circles arranged concentrically are

Sholl circles used for Sholl analysis. Closed small circles represent

intersections, where dendrites cross Sholl circles. Here, interval of

Sholl circles was set to 10mm, so distance between the furthest

intersection and the cell body is 30mm in this neuron.

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with two-way ANOVA (photoperiod�ZT) revealed a

significant main effect of ZT (p50.05). Further analysis

with Bonferroni post hoc tests showed that the Bdnf

mRNA abundance was higher at the beginning of the

light phase (ZT0) than that at the beginning of the dark

phase (ZT16) in long days (p50.05). A similar pattern

of fluctuation was observed in the trkb expression

level (Figure 3B); however, the effect of ZT was statis-

tically not significant (p¼ 0.054, two-way ANOVA

(photoperiod�ZT)). Although two-way ANOVA did not

detect any main effects of photoperiod and ZT on the

mRNA level of Vegf, Bonferroni post hoc tests showed

that the Vegf mRNA expression was higher at ZT0 than at

ZT16 in long days (p50.05) (Figure 3C). A clear diurnal

rhythm was observed in the expression of Bmal1 mRNA

(Figure 3D). Two-way ANOVA (photoperiod�ZT)

revealed significant main effects of photoperiod

(p50.05) and of ZT (p50.01). Further analysis with

Bonferroni post hoc tests showed that Bmal1 mRNA

expression was higher during the light phase (ZT0 and

ZT4) than at the beginning of the dark phase (ZT8) in

short days (p50.05). The results of the two-way ANOVA

are listed in Table 1.

Temporal analysis of cell body morphologyRepresentative tracings of hippocampal neurons are

shown in Figure 4A. Neither photoperiod nor ZT

nor an interaction between the two factors was

significant on the cell body perimeter or the cell

body area in CA1 pyramidal cells or DG granule cells

(p40.05, two-way ANOVA, for all cases) (Figure 4B,

C). The results of the two-way ANOVA are listed

in Table 1.

Temporal analysis of dendritic morphologyBasilar dendrites of pyramidal cells in the CA1 regionIn basilar dendrites of CA1 pyramidal cells, the num-

ber of branch points underwent a diurnal change

only in short days (Figure 5A). Two-way ANOVA

(photoperiod�ZT) revealed a significant main effect of

ZT (p50.01). Further analysis with Bonferroni post hoc

tests showed that the number of branch points signifi-

cantly increased at ZT20 in short days (p50.01).

Total length of dendrites increased in the dark phase

(Figure 5B). Two-way ANOVA (photoperiod�ZT)

revealed a significant main effect of ZT (p50.01) and

Bonferroni post hoc tests showed that length of den-

drites was significantly longer in the dark phase than

that at the light phase in long days (p50.05). Although a

similar apparent increase of dendritic length at ZT20

was observed in short days, it was not statistically

significant (p40.05, Bonferroni post hoc test). A signifi-

cant interaction between photoperiod and ZT was

detected in mean length of segments (p50.01, two-

way ANOVA) (Figure 5C). Bonferroni post hoc tests

showed that mean length of segments decreased at ZT20

only in short days (p50.05). Two-way ANOVA

(photoperiod�ZT) detected a significant main effect

of ZT (p50.05) in the number of primary dendrites

(Figure 5D). Bonferroni post hoc tests showed that

primary dendrites increased at ZT4 and decreased

at ZT12 only in long days (p50.05). A diurnal rhythm

was also observed in the total number of intersections

in the Sholl analysis (Figure 5E). Two-way ANOVA

(photoperiod�ZT) revealed a significant main effect of

ZT (p50.01) and Bonferroni post hoc tests showed that

intersections significantly increased at ZT20 in long days

(p50.05). Two-way ANOVA (photoperiod�ZT) revealed

FIGURE 3. Temporal expression patterns

of hippocampal genes in long days (closed

circles and solid line) and short days (open

circles and broken line). (A) For Bdnf there

was a main effect of ZT (p50.05, two-way

ANOVA [photoperiod�ZT]). Bonferroni

post hoc tests revealed that the Bdnf

mRNA expression was higher at ZT0 than

that at ZT16 in long days (p50.05). (B) For

trkb there were no effects of photoperiod

and ZT, and no interaction between the

two factors (p40.05, two-way ANOVA). (C)

For Vegf two-way ANOVA showed no main

effects of photoperiod and ZT, and no

interaction between the two factors

(p40.05). However, Bonferroni post hoc

tests revealed that the Vegf mRNA expres-

sion was higher at ZT0 than that at ZT16 in

long days (p50.05). (D) For bmal1 there

were main effects of photoperiod and ZT

(p50.05 and p50.01, respectively, two-

way ANOVA). Bonferroni post hoc tests

showed that bmal1 mRNA expression was

higher at ZT0 and ZT4 than that at ZT8 in

short days (p50.05). The results of the

two-way ANOVA are listed in Table 1.

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a significant interaction between the two factors when

we analyzed the distance of the furthest intersection

with the Sholl circles from the cell body (p50.05)

(Figure 5F). The distance at ZT20 was longer than that at

ZT4 and that at ZT12 only in long days (p50.01 and

p50.05, respectively, Bonferroni post hoc tests). The

results of the two-way ANOVA are listed in Table 1.

Apical dendrites of pyramidal cells in the CA1 regionIn apical dendrites of CA1 pyramidal cells, neither

photoperiod nor ZT affected the number of branch

points (Figure 6A) or total length of dendrites (p40.05,

two-way ANOVA, for both cases) (Figure 6B). Two-way

ANOVA (photoperiod�ZT) revealed a significant inter-

action between the two factors in mean length of

segments (p50.01) (Figure 6C). Further analysis with

Bonferroni post hoc tests showed that the mean length

of segments at ZT20 was longer than that at ZT12 only in

long days (p50.05). The number of primary dendrites

displayed diurnal rhythms (Figure 6D). Two-way

ANOVA (photoperiod�ZT) detected a significant main

effect of ZT (p50.05), and Bonferroni post hoc tests

showed that branch points increased at ZT4 and

decreased at ZT20 only in long days (p50.01). The

total number of intersections with the Sholl circles was

not statistically significant (p40.05, two-way ANOVA)

(Figure 6E). The distance of the furthest intersection

with the Sholl circles from the cell body did not display

any diurnal changes (Figure 6F) (p40.05, two-way

ANOVA). The results of the two-way ANOVA are listed

in Table 1.

Dendrites of granule cells in the DG regionIn DG granule cells, the effect of photoperiod or ZT, or

an interaction between the two factors, was not signifi-

cant on most dendritic morphology examined here,

including the number of branch points (Figure 7A), total

length of dendrites (Figure 7B), mean length of

TABLE 1. Statistical results.

p values from the two-way ANOVA

Variable Photoperiod ZT Interaction

Gene expressions (Figure 3)

Bdnf 0.984 0.027* 0.781

trkb 0.370 0.054 0.863

Vegf 0.992 0.221 0.826

Bmal1 0.027* 0.009** 0.963

Cell body morphology (Figure 4)

CA1

Cell body perimeter 0.478 0.158 0.431

Cell body area 0.444 0.361 0.297

DG

Cell body perimeter 0.081 0.060 0.561

Cell body area 0.075 0.131 0.319

Dendritic morphology (Figures 5, 6, 7)

CA1 basilar

Number of branch points 0.339 0.005** 0.282

Total length of dendrites 0.260 0.003** 0.653

Mean length of segments 0.412 0.679 0.004**

Number of primary dendrites 0.369 0.029* 0.112

Total number of intersections 0.245 0.007** 0.692

Distance of the furthest intersection 0.457 0.126 0.018*

CA1 apical

Number of branch points 0.870 0.839 0.668

Total length of dendrites 0.715 0.584 0.422

Mean length of segments 0.744 0.808 0.004**

Number of primary dendrites 0.318 0.017* 0.321

Total number of intersections 0.838 0.295 0.406

Distance of the furthest intersection 0.260 0.256 0.552

DG

Number of branch points 0.660 0.841 0.497

Total length of dendrites 0.590 0.947 0.221

Mean length of segments 0.852 0.670 0.077

Number of primary dendrites 0.694 0.148 0.750

Total number of intersections 0.608 0.933 0.190

Distance of the furthest intersection 0.661 0.535 0.041

Spine density (Figure 8)

CA1 basilar 0.268 0.331 0.021*

CA1 apical 0.298 0.076 0.001**

DG 0.356 0.318 0.003**

Significant differences: *p50.05 and **p50.01.

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segments (Figure 7C), number of primary dendrites

(Figure 7D), and total number of intersections

(Figure 7E) (p40.05, two-way ANOVA, for all cases).

A significant interaction between photoperiod and ZT

was detected only in the distance of the furthest

intersection with the Sholl circles from the cell body

(p50.05, two-way ANOVA) (Figure 7F). The results of

the two-way ANOVA are listed in Table 1.

Temporal analysis of spine densityBasilar dendrites of pyramidal cells in the CA1 regionRepresentative pictures of spines on dendrites are shown

in Figure 8A. The spine density of basilar dendrites

of CA1 pyramidal cells showed a diurnal rhythm

(Figure 8B). Two-way ANOVA (photoperiod�ZT)

revealed a significant interaction between the two factors

(p50.05) and Bonferroni post hoc tests showed that

the density was elevated at ZT12 and decreased at ZT20

in long days (p50.05). The results of the two-way

ANOVA are listed in Table 1.

Apical dendrites of pyramidal cells in the CA1 regionA significant interaction between photoperiod and

ZT was detected in the spine density in apical dendrites

of CA1 pyramidal cells (p50.01, two-way ANOVA)

(Figure 8B). Bonferroni post hoc tests showed that the

spine density was increased at ZT12 compared with that

at ZT4 and that at ZT20 in long days (p50.01 and

p50.05, respectively), but was elevated at ZT20 in short

days (p50.05). The results of the two-way ANOVA are

listed in Table 1.

Dendrites of granule cells in the DG regionTwo-way ANOVA (photoperiod�ZT) revealed a signifi-

cant interaction between the two factors in the spine

density of dendrites of DG granule cells (p50.01)

(Figure 8B). Further analysis with Bonferroni post hoc

tests detected significant differences among ZTs only in

short days: spine density was higher in the light phase

than that at ZT12 and that at ZT20 (p50.01 and p50.05,

respectively). The results of the two-way ANOVA are

listed in Table 1.

DISCUSSION

The present study demonstrated that Bmal1 expression

in the hippocampus displays a diurnal rhythm, con-

firming previous reports showing the rhythmic expres-

sion of circadian clock genes (e.g., Golini et al., 2012;

Jilg et al., 2010). Notably, Bmal1 mRNA was highly

expressed with elevated amplitude in short days

compared with long days. The effects of photoperiod

on expression levels of multiple circadian clock genes

have been demonstrated in the pars tuberalis, where the

FIGURE 4. Neuronal morphology of hip-

pocampal neurons. (A) Representative

tracings of CA1 pyramidal cells and DG

granule cells. Scale bar: 100 mm. Temporal

analyses of (B) cell body perimeter and (C)

cell body area of CA1 pyramidal cells and

DG granule cells. Data represent

mean� SEM values. Two-way ANOVA

(photoperiod�ZT) detected no main

effects of photoperiod and ZT, and no

interaction between the two factors for

both cell body perimeter and cell body

area (p40.05, for all cases). The results of

the two-way ANOVA are listed in Table 1.

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seasonal signal of melatonin is decoded, of several

seasonal mammals including Siberian hamsters; it has

thus been proposed that regulation of circadian clock

genes may play a key role for decoding of melatonin

signal (Johnston et al., 2005; Lincoln et al., 2002;

Messager et al., 1999, 2000). Therefore, different expres-

sion levels of Bmal1 revealed here suggest a possibil-

ity that photoperiodic information in the form of

hormonal or other signals might be processed also in

the hippocampus via expression patterns of circadian

clock genes.

VEGF and BDNF are the most prevalent neurotrophic

factors, which are thought to stimulate neuronal

reorganization in the adult brain (Fournier & Duman,

2012; Yu & Chen, 2011). In contrast to the short-day

elevation of Bmal1 amplitude, the diurnal oscillation of

Vegf expression was detectable only in long days.

However, the different expression patterns between

FIGURE 5. Temporal analyses of morphology of basilar dendrites of CA1 pyramidal cells in the hippocampus. Data represent mean�SEM

values. (A) For the number of branch points, two-way ANOVA (photoperiod�ZT) revealed a main effect of ZT (p50.01). Bonferroni post

hoc tests showed that the number of branch points increased at ZT20 in short days (p50.01). (B) For total length of dendrites, two-way

ANOVA (photoperiod�ZT) revealed a main effect of ZT (p50.01), and Bonferroni post hoc tests revealed that length of dendrites increased

at ZT20 in long days (p50.05). (C) For mean length of segments, a significant interaction between photoperiod and ZT was detected by

two-way ANOVA (p50.01). Bonferroni post hoc tests showed that mean length of segments decreased at ZT20 in short days (p50.05). (D)

For the number of primary dendrites, there was a main effect of ZT (p50.01, two-way ANOVA (photoperiod�ZT)). Bonferroni post hoc

tests showed that the number of primary dendrites was elevated at ZT4 in long days (p50.01). (E) For the total number of intersections with

the Sholl circles, there was a main effect of ZT (p50.01, two-way ANOVA [photoperiod�ZT]), and Bonferroni post hoc tests showed that

the number of intersections increased at ZT20 in long days (p50.05). (F) For the distance of the furthest intersection with the Sholl circles

from the cell body, two-way ANOVA (photoperiod�ZT) revealed a significant interaction between the two factors (p50.05). Bonferroni

post hoc tests showed that the distance increased at ZT20 compared with those at ZT4 and ZT12 (p50.01 and p50.05, respectively).

*p50.05, **p50.01 (Bonferroni post hoc tests). The results of the two-way ANOVA are listed in Table 1.

FIGURE 6. Temporal analyses of morphology of apical dendrites of CA1 pyramidal cells in the hippocampus. (A) Number of branch points,

(B) total length, (C) mean length of segments, (D) the number of primary dendrites, (E) the total number of intersections with Sholl circles,

and (F) the distance of the furthest intersection with the Sholl circles from the cell body was examined. For mean length of segments (D), a

significant interaction between photoperiod and ZT was detected by two-way ANOVA (p50.01). Bonferroni post hoc tests showed that

mean length of segments increased at ZT20 in long days (p50.05). For the number of primary dendrites (D), two-way ANOVA

(photoperiod�ZT) revealed a main effect of ZT (p50.01), and Bonferroni post hoc tests showed that the number of primary dendrites

increased at ZT4 compared with that at ZT20 in long days (p50.01). For further explanation, see Figure 5. The results of the two-way

ANOVA are listed in Table 1.

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long and short days were quite small and a significant

effect of photoperiod was not observed. Therefore, it

seems unlikely that expression of Vegf mRNA is related

to the photoperiod-induced difference of neuronal

plasticity of the hippocampus. Bdnf expression showed

periodic fluctuations rather than a strong diurnal

rhythm, which has been demonstrated in previous

studies (Golini et al., 2012; Schaaf et al., 2000), irre-

spective of photoperiodic conditions. Moreover,

nearly the same pattern of blunted amplitudes was

observed in the expression of trkb, a receptor of BDNF.

A lack of effect of photoperiod on Bdnf expression is

consistent with a previous report in Siberian hamsters

(Walton et al., 2012). These data suggest that as is the

case in Vegf, it is also unlikely that transcriptional

regulation on Bdnf and trkb is involved in photoperiodic

regulation of hippocampal neurons. However, it has

been reported that BDNF activates the downstream

cascade for neuronal plasticity by phosphorylation of

TrkB (Yoshii & Constantine-Paton, 2010). Therefore, it

remains possible that photoperiod affects BDNF-TrkB

signaling, albeit not through mRNA expression, but

through protein expression or phosphorylation states

to cause the seasonal changes in the hippocampus.

Otherwise, mRNA expression of these neurotrophic

factors might differ among hippocampal subregions as

FIGURE 8. Spines on dendrites of hippocampal neurons. (A) Representative pictures of spines on apical dendrites of CA1 pyramidal cells.

Scale bar: 2 mm. (B) Temporal analyses of spine density of basilar and apical dendrites of CA1 pyramidal cells and dendrites of DG granule

cells. Data represent mean� SEM values. Two-way ANOVA (photoperiod�ZT) revealed a significant interaction between the two factors

(p50.05) in basilar dendrites of CA1 pyramidal cells, and Bonferroni post hoc tests showed that the density was higher at ZT12 and lower at

ZT20 in long days (p50.05). For the apical dendrites of CA1 pyramidal cells, there was a significant interaction between photoperiod and

ZT (p50.01, two-way ANOVA). Bonferroni post hoc tests showed that spine density increased at ZT12 in long days (p50.05) and at ZT20 in

short days (p50.05). Two-way ANOVA (photoperiod�ZT) also revealed a significant interaction between the two factors (p50.01) in

dendrites of DG granule cells. Bonferroni post hoc tests showed that the density increased at ZT4 in short days (p50.05). *p50.05,

**p50.01 (Bonferroni post hoc tests). The results of the two-way ANOVA are listed in Table 1.

FIGURE 7. Temporal analyses of morphology of dendrites of DG granule cells in the hippocampus. (A) Number of branch points, (B) total

length, (C) mean length of segments, (D) the number of primary dendrites, (E) the total number of intersections with Sholl circles, and (F)

the distance of the furthest intersection with the Sholl circles from the cell body was examined. For the distance of the furthest intersection

(F), two-way ANOVA revealed a significant interaction between photoperiod and ZT (p50.05). For further explanation, see Figure 5.

The results of the two-way ANOVA are listed in Table 1.

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observed in the morphological change of neurons

and, therefore, we could not detect clear diurnal or

photoperiodic changes because we analyzed the whole

hippocampus. It would be useful to compare the

expression patterns of neurotrophic factors among the

hippocampal subregions in the future.

The adult brain has a remarkable capacity for struc-

tural plasticity. To cope with dramatic seasonal envir-

onmental changes, reversible remodeling of neural

circuits occurs in response to photoperiod or even

within a shorter period within a single day (Holtmaat

et al., 2005; Magarinos et al., 2006: Perez-Cruz et al.,

2009; Popov et al., 1992; Pyter et al., 2005; Workman

et al., 2011). Here we demonstrated that hippocampal

neuronal morphology displays a diurnal rhythm in a

subregion-specific manner; moreover, photoperiod

affects the morphological changes differently. Many

parameters of dendrite morphology in CA1 neurons

showed diurnal changes both in long and short days.

Although the pattern of diurnal changes is similar in

basilar and apical dendrites, these changes are only

significant mainly in the basilar dendrites, suggesting

that basilar dendrites are more flexible to daily envir-

onmental fluctuations than apical dendrites. Our results

are consistent with the report that diurnal difference in

the dendritic architecture are more pronounced in the

basilar dendrites than apical dendrites of layer III

pyramidal neurons in the rat infralimbic cortex (Perez-

Cruz et al., 2009). In contrast to CA1 neurons, DG

neurons did not display diurnal morphological changes

in either long or short days. Our results suggest that the

effects of time of day and photoperiod are different

among subregions in the hippocampus. Hippocampal

subregions are functionally and molecularly distinct and

are organized in an interconnecting complex circuit

(Small et al., 2011). Because the CA1 region provides the

main hippocampal outflow (van Strien et al., 2009), the

diurnal change of dendritic structure in CA1 neurons

might contribute to the diurnal variation of hippocam-

pal function, including learning and memory

(Valentinuzzi et al., 2004, 2008).

Overt rhythmicity of most dendritic parameters in

CA1 neurons is observed only in long days or only in

short days. These results suggest that photoperiod

affects the diurnal morphological change differently.

In short days, the number of branches increases at ZT20,

indicating that they produce new branches around this

time. Addition of branch points along the dendrite

would cause shortening of an interval between succes-

sive branch points, resulting in a decrease of segment

length, as observed at ZT20 in short days. However,

generation of new branches does not lead to a signifi-

cant increase of dendritic length at ZT20. One possible

interpretation is that the newly produced branches are

too short to increase total dendritic length. This also

could explain that an increase of branches does not

affect the number of intersections with Sholl circles.

No rhythmicity in the furthest intersection distance

suggests that the covered area by dendrites does not

vary during a day. Overall, these data indicate that CA1

neurons increase their morphological complexity with

increased branches mainly in basilar dendrites during

the latter half of the dark phase in short days. In long

days, however, total dendritic length, intersections with

Sholl circles, and the furthest intersection distance

increases at ZT20, suggesting that dendrites become

longer and cover a larger area during the dark phase of

long days. One would expect that increases of these

parameters are attributed to addition of new branches.

However, this is unlikely because the number of branch

points does not significantly change during long days.

Another possibility might be that the changes of total

dendritic length, intersections, and the furthest inter-

section distance are derived from extension of dendrites.

However, this possibility can be dismissed because

segment length at ZT20 was not significantly affected in

long days. Otherwise, the two processes described above

would occur at the same time. In this case, even though

segment length is extended to lead an increase of total

dendritic length at ZT20, a slight, but not significant,

increase of branch points would shorten intervals

between two successive branch points, and hence

there would be no net change in mean segment

length. Although it remains unclear what morphological

changes CA1 neurons undergo at the present, our

present results suggest photoperiod affects diurnal

changes of morphological parameters in CA1 neurons.

In addition to elevated morphological characteristics

during the dark phase, CA1 neurons have more primary

dendrites at ZT4 in long, but not, short days. It has been

reported that most inhibitory synapses in CA1 neurons

are distributed on proximal primary dendrites (Megıas

et al., 2001). Therefore, the morphological changes

revealed here suggest that inhibition of CA1 neurons

increases at morning only in long days.

The present study also demonstrated a diurnal

change of spine density in both CA1 and DG regions.

Different patterns were again observed between long

and short days. In the CA1 region, where rhythmicity is

more robust in apical dendrites than in basilar den-

drites, spine density increases during the latter half of

the light phase in long days but it increases during the

latter half of the dark phase in short days. In the DG

region, dendrites have dense spines during the light

phase, but the diurnal difference is apparent only in

short days. Spines increase the surface area of dendrites,

which increases the excitatory synaptic density and

number of connections between neurons (Sorra &

Harris, 2000). However, spine density and dendritic

complexity showed different peak time in long days,

suggesting that these dendritic properties have distinct

functions and are regulated by different mechanisms.

Taken together, these data indicate that photoperiod

induces a different diurnal change of dendritic pattern-

ing and a different peak time of spine density in a

subregion-specific manner. Hippocampal neuronal

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morphology has been suggested to play an important

role in depressive-like behaviors (Bedrosian et al., 2011;

Workman et al., 2011). Our present results are consistent

with the hypothesis that photoperiod-induced adjust-

ments of hippocampal neuronal dynamics might under-

lie seasonal difference of affective responses.

DECLARATION OF INTEREST

T.I. was supported by a JSPS Postdoctoral Fellowshipsfor Research Abroad.

The authors report no conflicts of interest. Theauthors alone are responsible for the content andwriting of the paper.

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