RESEARCH ARTICLE Environmental complexity, seasonality and ... · environmental complexity also influences cell proliferation and neurogenesis in the adult brain. For example, rodents

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INTRODUCTIONThe brains of animals living in complex environments differstructurally from those of animals living in simplified laboratoryenvironments. Beginning in the 1960s, researchers showed thatenvironmental stimuli modify many features of brain structure,including cortical size, neuronal architecture and synapse formation(Diamond et al., 1966). More recent studies demonstrate thatenvironmental complexity also influences cell proliferation andneurogenesis in the adult brain. For example, rodents housed withconspecifics and enriched physical environments produce moreneurons in the hippocampus than those living alone in standardlaboratory housing (Kempermann et al., 1997). These studies havegreatly contributed to our understanding of specific stimuli that canenhance neurogenesis (Fabel et al., 2009; Kozorovitskiy and Gould,2004; Lu et al., 2003; Stranahan et al., 2006). However, becausethey were conducted in laboratory conditions, their conclusions maynot hold true for animals in nature, where environments are muchmore complex.

When examined in animals living in natural habitats, brain cellproliferation or survival is greatly enhanced by the complex physicaland social stimuli in the wild (Amerin et al., 2008; Barnea, 2010;Barnea and Nottebohm, 1996; Boonstra et al., 2001; Delgado-Gonzalez et al., 2008; Hansen and Schmidt, 2004; LaDage et al.,2010). Most free-living animals face seasonal changes in thephysical and social environment and undergo corresponding drastic

changes in physiology and behavior (e.g. breeding and foodcaching). Such natural seasonal changes affect brain cell proliferation(or survival) in some but not all species (Amerin et al., 2008; Galeaand McEwen, 1999; Hoshooley and Sherry, 2004; Lavenex et al.,2000; Ormerod and Galea, 2003; Sampedro et al., 2008; Thompsonand Brenowitz, 2005).

Despite many studies showing that enriched captive environmentsand complex natural environments can influence brain cellproliferation, the relative importance of season and environmentalcomplexity has not been addressed in depth in tetrapods and not atall in fish. We examined brain cell proliferation in a weakly electricfish, Brachyhypopomus gauderio, in three environments (naturalhabitat, enriched laboratory environment and isolated laboratoryhousing) across two seasons to estimate the relative importance ofseasonal, social and physical features of the environment.

Weakly electric fish, such as B. gauderio, are particularly suitablefor addressing this issue because specific brain regions – those usedfor electrogenesis and electroreception – are directly linked to theways that the fish sense and interact with the physical and socialenvironment. Fish navigate through the environment by sensing hownearby objects distort a self-generated electric signal, the electricorgan discharge (EOD). The electric signal originates in a hindbrainnucleus, the pacemaker nucleus (Pn), which stimulates the dischargeof the electric organ, located primarily in the tail. Electroreceptorsdistributed all over the skin of the fish detect spatial and temporal

The Journal of Experimental Biology 214, 794-805© 2011. Published by The Company of Biologists Ltddoi:10.1242/jeb.051037

RESEARCH ARTICLE

Environmental complexity, seasonality and brain cell proliferation in a weakly electricfish, Brachyhypopomus gauderio

Kent D. Dunlap1,*, Ana C. Silva2,3 and Michael Chung1

1Department of Biology, Trinity College, Hartford, CT 06106, USA, 2Unidad Bases Neurales de la Conducta, Instituto deInvestigaciones 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

SUMMARYEnvironmental complexity and season both influence brain cell proliferation in adult vertebrates, but their relative importance andinteraction have not been directly assessed. We examined brain cell proliferation during both the breeding and non-breedingseasons in adult male electric fish, Brachyhypopomus gauderio, exposed to three environments that differed in complexity: (1) acomplex natural habitat in northern Uruguay, (2) an enriched captive environment where fish were housed socially and (3) asimple laboratory setting where fish were isolated. We injected fish with BrdU 2.5h before sacrifice to label newborn cells. Weexamined the hindbrain and midbrain and quantified the density of BrdU+ cells in whole transverse sections, proliferative zonesand 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 greaterrates of cell proliferation than fish in social or isolated captive environments. For most brain regions, fish in social and isolatedcaptive 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 � environmentinteraction). The regionally and seasonally specific effect of social environment on cell proliferation suggests that addition of newcells to these nuclei may contribute to seasonal changes in electrocommunication behavior.

Key words: brain cell proliferation, season, environmental enrichment, electrocommunication, electric fish, Brachyhypopomus.

THE JOURNAL OF EXPERIMENTAL BIOLOGY

795Brain cell proliferation in electric fish

variation in electric fields and pass information for primary sensoryprocessing to the electrosensory lateral line lobe (ELL) in thehindbrain.

The EOD also is used as the primary means of socialcommunication. EOD rate and waveform are sexually dimorphic,hormone sensitive and seasonally variable and thereby conveyinformation 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 rapidEOD modulations that function in aggression and courtship. SeveralEOD modulations are generated by diencephalic nuclei, such as theprepacemaker nucleus (PPn), that transiently modify the activity ofthe Pn (Perrone et al., 2009; Zakon et al., 2002; Zupanc, 2002).Because these three brain regions – Pn, ELL and PPn – all functiondirectly in interactions with the environment, we were particularlyinterested in their response to environmental change. We examinedwhether these specific brain regions or proliferative zones thatcontribute cells to these regions showed particularly high levels ofcell proliferation when fish were exposed to environments that variedin physical and social complexity.

A second advantage of studying environmental influences onbrain cell proliferation in electric fish is that a number of laboratorystudies on one species (Apteronotus leptorhynchus) have previouslydetailed patterns of cell proliferation and social influences on celladdition. In A. leptorhynchus, cell proliferation is abundantthroughout the brain. In a 2h period, approximately 0.2% of allbrain cells undergo mitosis, a rate ~10–100 times that found inproliferative zones in the adult rodent brain (Zupanc, 2008). Cellbirth is particularly concentrated in proliferation zones in thecerebellum and the periventricular zone (PVZ) but is also scatteredat lower densities throughout the brain (Zupanc and Horschke, 1995).Cell addition in the PVZ that contributes cells to the PPn is enhancedby experimental manipulation of social interactions in the laboratory(Dunlap et al., 2006). Thus, there is good reason to believe thatnatural changes in the social environment may affect cellproliferation, especially in brain regions associated withelectrocommunication.

Among electric fish, B. gauderio is particularly appropriate forassessing the role of natural stimuli and seasonal change on braincell proliferation because of the extensive previous field andlaboratory work documenting seasonal, hormonal and social

influences on their electrocommunication behavior and the key brainnuclei used in electrocommunication (Quintana et al., 2004; Salazarand Stoddard, 2009; Silva et al., 2007). Indeed, more is known aboutthe seasonality, natural behavior and ecology of B. gauderio thanany other South American electric fish. In Uruguay, these fish inhabitshallow freshwater lakes and streams that are commonly coveredby floating vegetation (Silva et al., 2003). During the breeding season(October–February), they live in dense concentrations, with fishusually separated by less than 1m (Miranda et al., 2008). In thenon-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 largeeffect on cell proliferation rates, with environmental complexity(natural habitat vs enriched captivity vs isolated captivity) havinga secondary but still significant effect. Fish during the breedingseason and those living in a natural habitat had much greater ratesof cell proliferation throughout the brain than fish during the non-breeding season and fish in captivity. In addition, fish living inenriched captive environments that allowed for abundant socialinteraction had higher rates of brain cell proliferation than fish inisolation. This effect was specific to the breeding season and presentonly in brain regions that are associated with electrogenesis andelectrosensation. These results suggest that environmentally inducedchanges in brain cell proliferation may contribute to changes in socialsignaling associated with reproduction.

MATERIALS AND METHODSWe 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) fishhoused 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 (mid-January to February) and the non-breeding season (late May to mid-June). The similarities and differences across seasons andenvironments are presented in Tables1 and 2. We examined thebrains of six to eight male fish from each subject group. The lengthof fish ranged from 14 to 19cm, and all subject groups had equivalentmean (±s.e.m.) body length (16.1±0.4cm). A previous study has

Table 1. Comparison of environments in different seasons in each treatment groupGroup comparisons Similarities DifferencesBreeding vs non-breeding (field)

Physical environment Size and complexity of physical environmentWater conductivity

Mean water temperaturePhotoperiod

Biotic environment Unknown UnknownSocial environment Living in aggregations Reproductive signaling

Social spacingAge structure of conspecifics

Breeding vs non-breeding (group)Physical environment Size of physical environment

Water conductivityPresence of retreat sites


Biotic environment Plant coverFood availability

None

Social environment Sex and age composition of groupOverall density of fish

Reproductive signaling and behavior

Breeding vs non-breeding (isolated)Physical environment Size of physical environment

Food availabilityWater conductivity



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reliably shown that fish greater than 12cm in length are adult animals(Franchina, 1997) and thus all fish in our study were well withinthe size range for adults.

Animals and field siteAll 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 havebeen described previously (Miranda et al., 2008), and thereproductive 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 breedingseason, and in late May 2009, during the non-breeding season. Atthe time of sampling, the water temperature was 25–27°C in Januaryand 17–18°C in May. We located fish using a detector thattransduces the electric signal into an audio signal, and captured fishwith scoop nets. We placed fish individually in inflated plastic bagspartially filled with water and floated them on the lake beforebringing them to the shore. At the shore, approximately one-thirdof the fish were injected with bromodeoxyuridine (BrdU) and bled(see below) to measure cell proliferation and androgen levels in thefield; the remaining fish were packaged for transport to thelaboratory.

Captive conditionsDuring both the breeding and non-breeding seasons, fish werehoused in captivity under one of two conditions: social groups insemi-natural outdoor pools or isolated in a controlled environmentalchamber. During each season, we created four pools (250l,80�80�40cm length�width�diameter) on the roof of thelaboratory at Instituto Investigaciones Biologicas Clemente Establein Montevideo, Uruguay (Perrone et al., 2009). Each pool housed12–16 adult fish. The social composition was three to four malesand nine to 12 females per group to maintain a constant sex ratioof 1:3, which mimicked the adult sex ratio in the natural breedingpopulation (Miranda et al., 2008). Each pool contained floatingplants (Eichhornia crassipes, Pistia stratiotes) native to the naturalhabitat of the fish and six to eight PVC tubes for shelter. Duringthe breeding season, the mean water temperature of the pools was27–30°C; during the non-breeding season, it was 16–17°C. Inprevious experiments, fish living in these conditions showedseasonal changes in reproductive physiology and behavior thatparallel the seasonal changes in the wild (Silva et al., 2007;Quintana et al., 2004).

K. D. Dunlap, A. C. Silva and M. Chung

Isolated fish were housed in individual 10–12l tanks with a sheltertube and kept at a constant light cycle and temperature thatmimicked field conditions during each season. During the breedingseason, the light cycle was 14h:10h light:dark and the temperaturewas a constant 28°C. During the non-breeding season, the light cyclewas 10h:14h 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 2–4weeks before theywere terminally anesthetized in late February and mid-June.

BrdU injection, and processing of brains and plasma samplesTo label newborn brain cells from fish in the field, we injected fishwith BrdU (Sigma B-9285; 20gg–1 body weight) 15–60min aftercapture and held them in plastic boxes filled with lake water (1–2l)and placed in the shade. Water temperature was 24–27°C duringthe period of BrdU incorporation. Approximately 2.5h(135–165min) following BrdU injection, fish were killed with 2-phenoxyethanol (0.075%). Brains were dissected and placed on icein buffered paraformaldehyde (4%, 90–120min) followed by a seriesof washes in PBS (3�30min). Brains were then placed in sucrose(30%, 4–8h) and a 1:1 mixture of sucrose and mounting mediumfor cryoprotection (4–8h), placed in mounting medium alone andquickly frozen by covering them with pulverized dry ice.

For plasma androgen measurements, fish were bled from thecaudal vein using a heparinized syringe and needle (25G) within3h after capture and 3min after anesthesia. Blood samples wereplaced on ice for 4–8h and then centrifuged. All brain and plasmasamples were stored on dry ice in the field for 1–2d until they wereshipped to the laboratory, where they were stored at –80°C.

Fish were measured for body length and dissected to determinesex and gonadal state. During the non-breeding season, when thesex of fish was sometimes ambiguous, gonads were preserved inparaformaldehyde and later examined under a dissecting scope.Testes were scored according to three phases in the annual cycle ofthis species: 1regressing or recovering gonads, 2maturing gonadsand 3mature gonads (Quintana et al., 2004). Visual inspection didnot allow us to discriminate between regressing and recoveringgonads, so they were pooled in a single group.

For captive fish, the procedures for BrdU injection, brainprocessing and plasma sampling were identical to those for the fieldfish, except for the following: in the laboratory, fish were injectedwith BrdU within 15–20min after capture, water temperature duringthe period of BrdU incorporation was 23–24°C and brains werefrozen in 2-methylbutane (–80°C) after sucrose cryoprotection.

Table 2. Comparison of environments in different treatment groupsGroup comparisons Similarities DifferencesIsolated vs group

Physical environment Mean water temperaturePhotoperiodWater conductivity

Size of physical environment

Biotic environment None Presence of plantsSocial environment None Presence of conspecifics

Group vs fieldPhysical environment Mean water temperature

PhotoperiodWater conductivityAvailability of retreat sites

Size and complexity of habitatDiversity of retreat sites

Biotic environment Presence of plants Density and diversity of plants, predators, prey and pathogensSocial environment Presence of conspecifics in a female-biased sex

ratioDensity of conspecificsStability of social groupPresence of juveniles



BrdU immunohistochemistryTo identify newborn cells, we used BrdU immunohistochemistrywith a protocol identical to that used in a similar study (Dunlap etal., 2006). In brief, sections were treated with citrate buffer(5mmoll–1, 10min, 95°C) and pepsin solution (2.5% pepsin in0.1moll–1 HCl, 3min, 37°C) to expose the BrdU epitope. Sectionswere treated sequentially with sheep anti-BrdU (1:200, overnight,4°C; Capralogics Inc., Hardwick, MA, USA), biotin-conjugateddonkey anti-sheep IgG (1:800, 2h, room temperature; ChemiconInternational, Temecula, CA, USA) and streptavidin conjugated toAlexa 488 (1:800, 1h, room temperature; Molecular Probes, Eugene,OR, USA).

Quantifying the distribution of BrdU+ cellsIn quantifying our observations, we addressed three generalquestions: (1) what is the overall, global rate of cell proliferation;(2) what fraction of newborn cells are generated in specificproliferation zones; and (3) what is the rate of cell proliferation inbrain regions specifically associated with electrogenesis andelectrosensation? We examined transverse sections of the brain usinga Nikon Eclipse E600 epifluorescence microscope and used the brainatlas 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, tworegions of the hindbrain (sections –9 to –7 and sections –7 to –5 ofthe Maler atlas) and two regions of the midbrain (sections 14–16and sections 17–19, which we term the posterior and anteriormidbrain, respectively). Each region spanned ~500m in axiallength.

To estimate the overall cell proliferation, we counted all theBrdU+ cells in one lateral half of each transverse section anddoubled this value to estimate the number of cells born in the wholesection. To estimate the number of newborn cells in twoproliferative zones, we counted all the BrdU cells in a 100mband surrounding the ventricle (the PVZ) in the hindbrain andmidbrain and a 100m band along the dorsal border of thehindbrain cistern, which contains the eminentia granularis parsmedialis (EGm). To estimate the number of cells born outside ofthese proliferative zones, the ‘background’ area, we subtracted thenumber of BrdU+ cells in the PVZ and EGm from those in thewhole section. To quantify the relative importance of proliferativezones as sites of cell proliferation, we divided the number of BrdU+cells in the PVZ and EGm by the total number of cells in eachtransverse section. To quantify cell proliferation in theelectrocommunication system, we counted BrdU+ cells unilaterallyin the ELL in atlas sections –9 to –6 and bilaterally in the Pn inatlas sections –8 to –5. Cell counts in the ELL were doubled toestimate the total number in the whole section. We did not directlyassess cell proliferation in the PPn. Instead, we used BrdU+ celldensity in anterior midbrain PVZ as an indirect measure of celladdition to the PPn because, in the closely related electric fish A.leptorhynchus, the majority of cells added to the PPn duringadulthood arise from the adjacent PVZ in the anterior midbrain(Zupanc and Zupanc, 1992).

The area of each whole transverse section and brain region wasquantified by capturing an image of the sections and then measuringthe area using NIH Image J software (Bethesda, MD, USA). Thebackground area was calculated by subtracting the area of the PVZand EGm from the area of the whole section. All values weremultiplied by the section thickness (0.030mm) to calculate thevolume of the sample of each region examined, and the density wasexpressed as BrdU+ cellsmm–3. For each individual brain, we

averaged the values of two to five randomly chosen sections in eachaxial level.

Measurement of plasma 11-ketotestoteroneWe measured 11kT in plasma samples (5–10l) of male fish(N5–6) using an enzyme linked immunoassay kit (CaymanChemical Co., Ann Arbor, MI, USA). The detection limit for thisassay is 6pgml–1, and the cross-reactivity of the 11kT antibody is<0.05% with other androgens. We assayed plasma samples induplicate in a single run of the assay. The intra-assay variation was3.3%.

Statistical analysisWe examined variation in BrdU+ cell density and plasma 11kT usingmultivariate ANOVA, with region, season and environment asfactors, followed by Bonferroni post hoc tests to identify differencesamong groups. In our initial analysis, we found that the two axialregions of the hindbrain (sections –10 to –8 and –7 to –5) showedno differences in any subject group (F3.0, P>0.05), and so theseaxial regions were combined in subsequent analysis. All data arepresented as means ± s.e.m., and P<0.05 was considered statisticallysignificant.

RESULTSOur quantitative analysis of BrdU labeling allowed us to estimatethe overall rates of cell proliferation across the hindbrain andmidbrain, the relative importance of proliferation zones as sites ofcell birth and the density of newborn cells in specific brain nucleiassociated with electrogenesis (Pn) and electrosensation (ELL). Inaddition, our experimental design allowed us to partition variancein BrdU+ cell density and plasma 11kT levels to season (breedingvs non-breeding), environment (isolated vs group housed vs field)and their interaction.

General patterns of cell proliferationAveraged over all brain regions, environments and seasons,transverse sections had a mean of 135±32BrdU+ cells, a meanvolume of 0.124±0.002mm3 and a mean density of 1088±293BrdU+cellsmm–3. In all subject groups, BrdU+ cell density was greater inthe hindbrain than in the midbrain (F6.9, P<0.005; for field activeanimals, see Fig.3). The overall distribution of BrdU+ cells wassimilar to that reported for A. leptorhynchus (Zupanc and Horschke,1995), with the exception that the cerebellum of B. gauderioappeared to have relatively fewer BrdU+ cells than that of A.leptorhynchus.

HindbrainIn the hindbrain, the EGm was the primary site of cell proliferation,containing ~20–55% of the newborn cells in the transverse sectionand a BrdU+ cell density three to five times greater than that of thebackground area. Although newborn cells were found surroundingthe entire cistern, the vast majority were located in the dorsal borderof the cistern in the EGm (Fig.1). The PVZ of the hindbrain wasalso a proliferative zone, but was quantitatively less important thanthe EGm. The PVZ contained ~5–10% of all the newborn cells inthe transverse section and a BrdU+ cell density two to three timesgreater than that of the background area.

MidbrainThe anterior and posterior midbrain differed according to seasonand environment (F32.2, P<0.0001), and thus these regions werekept separate in our analysis. Cell proliferation was concentrated in


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the PVZ (Fig.2A), which contained 15–40% of all labeled cells inthe transverse section and a BrdU+ cell density three to six timesgreater than background. BrdU+ cells were scattered throughout thetorus semicircularis and along the medial edge of optic tectum andwere in particularly high density in the ventral hypothalamus.

Pn and ELLIn the Pn and ELL, BrdU+ cells were found at approximately thesame density as the background areas, and thus these nuclei cannotbe considered areas of concentrated mitosis. Within the Pn (Fig.2B),labeled cells were located at all axial regions and tended to be moreabundant in the ventral half of the Pn. In field fish during thebreeding season (the subject group with the highest Pn cellproliferation), each 30m section of the Pn contained a mean of4.1±1.1BrdU+ cells per section. Given that the Pn is ~500m inaxial length (Quintana et al., 2010), we estimate that the entirenucleus would generate 60–70 new cells in a 2.5h period. This isa rate approximately half that reported from the Pn in Apteronotus(Zupanc and Horschke, 1995). Labeled cells were found throughoutthe ELL, although they were in much greater density in thepyramidal 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 lateralsegment than the central segments, although this difference was notquantified. This apparent concentration of newborn cells in the lateralsegment deserves future research because, in other electric fishspecies, this is the subsection of the ELL that is specifically involvedwith processing social electric signals (Metzner and Juranek, 1997;Marsat et al., 2009).

Effect of seasonThere was a strong overall effect of season on BrdU+ cell densityfor all regions of the brain (Table3, Figs1, 3), with seasonaccounting for ~30–60% of the overall variation in BrdU+ cell


density. Across all treatment groups and brain regions, fish duringthe breeding season had BrdU+ cell densities three to seven timesgreater than fish during the non-breeding season.

Effect of environmental complexityFor all brain regions, there was a significant effect of environment(isolation vs group housing vs field) on cell proliferation (Table3,Fig.4), with environment accounting for ~10–30% of the overallvariation in BrdU+ cell density. In both seasons and in all brainregions, field active fish had greater (approximately two to fivetimes) BrdU+ cell density than the two captive groups (isolationand group housed). One of the only methodological differences insampling field and captive fish was that, for logistical reasons, fishsampled in the field spent approximately 30min longer in holdingboxes before they were injected with BrdU than captive fish. Suchcapture stress inhibits cell proliferation in some species (Amerin etal., 2008), so it is likely that our report underestimates the truedifference between field and captive fish.

Fig.1. Seasonal variation in brain cell proliferation in the eminentiagranularis pars medialis, the major proliferative zone of the hindbrain, ofadult Brachyhypopomus gauderio. (A)Breeding season; (B) non-breedingseason. The cavity in the lower right of each photograph is the cerebro-medullary cistern. Scale bar, 100m.

Fig.2. BrdU labeling in the brain of adult Brachyhypopomus gauderio. In allphotographs, dorsal is towards the top of the figure. (A)Periventricular zoneof the anterior midbrain. The cavity at the center is the ventricle. Scale bar,100m. (B)Pacemaker nucleus. Arrows point to BrdU+ cells. The largecells are relay cells, which only show background labeling. Scale bar,50m. (C)Electrosensory lateral line lobe (ELL). Figure shows one lateralhalf of the ELL, with lateral toward the left. Scale bar, 50m.



Season � environment interactionThe effect of environment varied by season in some brain regions(Table3, Fig.5). During the breeding season, group-housed fish hadgreater BrdU+ cell density in the Pn, ELL and the anterior midbrainPVZ than isolated fish, but lower BrdU+ cell density than field activefish. However, during the non-breeding season, group-housed andisolated fish had equivalent BrdU+ cell densities (P>0.05; Fig.5).Thus group housing elevated cell proliferation in these regions toan intermediate level during the breeding season, but had no effectduring the non-breeding season. There were no significantenvironment � season interactive effects on BrdU cell density inthe whole transverse sections and background regions in any axialregion, the hindbrain PVZ and EGm, or the posterior midbrain PVZ(Table3). Thus these regions were enhanced by field conditions butnot group housing during both seasons.

Relative importance of proliferation zones as a function ofseason and environment

For almost all comparisons, season and environment had nosignificant effect on the fraction of BrdU+ cells located inproliferative zones (PVZ and EGm). Thus, overall, it appears thatenvironmental stimuli affect cell proliferation uniformly inproliferative and background regions. There is one exception to thisgenerality. In the hindbrain, the relative importance of the PVZ asa site of new cell birth was affected by season: the percentage ofBrdU+ cells in the PVZ compared with the whole transverse sectionat the same axial level was significantly lower during the breedingseason (5.3±0.7%) than during the non-breeding season (12.8±1.3%)(F15.9; P<0.001). The hindbrain PVZ appears less responsive toseasonal change than other hindbrain regions: the PVZ only showedan approximately threefold increase in BrdU+ cell density from thenon-breeding to the breeding season whereas the whole brain section,background, EGm, ELL and Pn showed an approximately five- toseven-fold increase.

Testis maturity and plasma 11-ketotestosterone levelsIn our January sampling, most fish captured from the field (five ofeight) and those in group housing (six of nine) had fully maturetestes (gonadal score3) and the remaining fish had testes ofintermediate maturity (gonadal score2). The mean gonadal maturity

Table 3. Results of ANOVA showing the effects of season, environment and their interaction on BrdU+ cell density in three axial regions ofthe brain

Season Environment Season � Environment

Brain region % Var F P % Var F P % Var F P

HindbrainWhole 58.7 53.3 <0.0001 7.3 3.3 <0.05 1.8 0.8 >0.05Background 56.7 49.5 <0.0001 12.0 5.2 <0.05 3.3 1.5 >0.05PVZ 33.7 20.7 <0.0001 10.1 3.4 <0.05 6.6 2.0 >0.05EGm 60.4 66.0 <0.0001 8.1 4.4 <0.05 4.6 2.5 >0.05Pn 28.6 24.2 <0.0001 25.3 10.7 <0.0005 11.1 4.6 <0.05ELL 48.1 176.5 <0.0001 30.5 55.0 <0.0001 20.4 37.1 <0.0001

Posterior midbrainWhole 43.8 36.8 <0.0001 12.9 5.7 <0.01 6.2 2.8 >0.05Background 56.0 77.9 <0.0001 17.6 12.2 <0.0005 9.4 3.5 >0.05PVZ 45.1 38.3 <0.0001 12.6 5.3 <0.01 3.2 1.3 >0.05

Anterior midbrainWhole 57.1 82.2 <0.0001 18.6 13.3 <0.0001 6.8 2.9 >0.05Background 56.9 77.4 <0.0001 21.9 14.9 <0.0001 8.3 2.7 >0.05PVZ 48.5 84.1 <0.0001 18.9 16.3 <0.0001 16.1 13.9 <0.0001

% Var, the percentage of the variation in BrdU+ cell density that is attributable to each factor, as determined by ANOVA.

0

4000

8000Posterior midbrain

Whole section

Back-ground

PVZ

Whole section

Back-ground

PVZ EGm ELL Pn0

4000

8000

12,000

16,000Hindbrain

Cel

l pro

lifer

atio

n (B

rdU

+ c

ells

mm

3 )

Breeding season

Non-breeding season

0

4000

8000 Anterior midbrain

Fig.3. Effect of season on cell proliferation in different brain regions of fieldactive Brachyhypopomus gauderio. The anterior midbrain comprisessections 17–19 and the posterior midbrain comprises sections 14–16,according the Maler brain atlas (Maler et al., 1991). EGm, eminentiagranularis pars medialis; ELL, electrosensory lateral line lobe; Pn,pacemaker; PVZ, periventricular zone. Whole section is the density ofBrdU+ cells in the whole transverse section. Background is the density ofBrdU+ cells in the transverse section outside of proliferative zones (PVZand EGm). Cell proliferation was significantly higher (P<0.0001) during thebreeding season than during the non-breeding season in all brain regions.N6–8 fish per sample.


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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 allfish during the non-breeding season had fully regressed gonads(gonadal score1).

Plasma 11kT concentrations varied significant by season (F7.58,P<0.01), environment (F3.64, P<0.05) and their interaction(F2.82, P<0.05). Concentrations of 11kT were higher during thebreeding season than during the non-breeding season (Fig.4).During the breeding season, field fish had significantly higher 11kTlevels than fish in either captive group, and group-housed fish hadsignificantly higher levels than isolated fish. During the non-breeding season, fish in all environment groups had equivalentvalues (P>0.05). Fish were held in holding boxes for ~3h followingcapture (during the period of BrdU incorporation), and because suchacute stress commonly inhibits androgen secretion, the levels for11kT that we report may be less than those experienced byundisturbed fish. Nevertheless, our values are well within the rangeof those reported in other studies of B. gauderio, in which fish werebled immediately after capture (Gavassa et al., 2010; Salazar andStoddard, 2009). More importantly, fish were treated equivalentlyacross subject groups, allowing us to make comparisons of androgenconcentration among groups.

DISCUSSIONWe found that environmental complexity enhanced the proliferationof brain cells in the weakly electric fish, B. gauderio. Fish in thenatural environment had greater rates of cell proliferation than


captive fish in all the brain regions we examined. This resultcorroborates the findings of other studies (Amerin et al., 2008;Barnea, 2010; Boonstra et al., 2001; LaDage et al., 2010), indicatingthat examining animals in captivity tends to underestimate theproliferative capacity of the adult vertebrate brain. We also foundthat group-housed captive fish had greater rates of cell proliferationthan isolated fish, but only in brain regions associated withelectrosensation and electrogenesis. Thus the degree ofenvironmental complexity is correlated with the specificity of theeffect: the highly complex natural environment had a broad, globaleffect on the brain whereas the limited enhancement of the captiveenvironment had a more regionalized, specific effect.

Although the effect of environmental complexity was significant,it was quantitatively surpassed by the effect of season. The overalleffect of season on the brain was likely attributable to seasonalchanges in temperature or photoperiod whereas regionally specificchanges were likely due to changes in reproductive physiology andbehavior. Thus the role of external stimuli in promoting brainplasticity must be understood in the context of annual cycles of theenvironment and reproduction.

Seasonality in cell proliferation, reproductive state andphysical environment

Across all brain regions and treatment groups, fish producedapproximately three to seven times more new cells during thebreeding season than during the non-breeding season. Seasonalityin brain cell proliferation has been documented in other vertebrate

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Fig.4. Effect of environmental complexity on cell proliferationin whole brain, background regions and proliferation zones[eminentia granularis pars medialis (EGm) andperiventricular zone (PVZ)] of Brachyhypopomus gauderio.Data are presented only from the breeding season. Note thedifference in y-axis scale. *, significant difference from bothisolated and group fish; **, significant difference fromisolated fish and field fish. N6–8 fish per sample.



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 seasonalityin brain cell proliferation in a teleost fish. Such seasonal variationin cell proliferation could be attributable to physiological and/orbehavioral changes in the annual reproductive cycle of the animalor to changes in physical environment, such as temperature andphotoperiod. In considering both possibilities, we conclude that,although reproductive physiology and behavior may influenceseasonal changes in brain cell proliferation, annual cycles oftemperature or photoperiod explain most of the seasonal variationin cell proliferation.

To assess the reproductive state of fish during each season, weexamined testis maturity and plasma levels of the primary teleostandrogen, 11kT. In our breeding season sample, most field activeand group-housed fish had fully mature testes, but some had testesof intermediate maturity. Plasma levels of 11kT were also elevatedin field active and group-housed fish, although group-housed fishhad significantly lower 11kT levels than field active fish. Thepresence of some fish with partially regressed testes and low 11kTlevels combined with other data from this same population (Gavassaet al., 2010) indicate that our sampling period was toward the endof the breeding season.

In many vertebrates, brain cell proliferation peaks during or justprior to the breeding season (Chetverukhin and Polenov, 1993;Dawley et al., 2000; Delgado-Gonzalez et al., 2008; Vidal Pizarroet al., 2004), and some of our results are consistent with the ideathat high levels of cell proliferation are causally related to seasonalchanges 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 incell proliferation. Conversely, 11kT levels were highest in breedingfish from the field, which also had the highest levels of cellproliferation.

However, a closer look at our results indicates that cellproliferation is only partially tied to reproductive physiology andbehavior. The fact that season affected cell proliferation rates evenin isolated fish – which had regressed gonads, low 11kT levelsand no social interaction during both seasons – demonstrates thatseasonal changes in reproductive physiology and social signalingare not the sole factors driving the seasonal change in cellproliferation. Similarly, the fact that, during the non-breedingseason, field active fish had higher rates of cell proliferation thancaptive fish yet all fish had equivalently low levels of 11kTindicates that cell proliferation is not strictly attributable toseasonal changes in androgens. Finally, during the breedingseason, field active fish had higher rates of cell proliferation thangroup-housed fish even though their testes were at equivalentstages of maturity, and thus proliferation rates are not strictlycorrelated to testes maturity. Thus, some factor beyondreproductive state must play a large role in seasonal changes incell proliferation rate.

For temperate species, the breeding season typically has hightemperatures and a long photoperiod. These features of the physicalenvironment commonly stimulate changes in reproductivephysiology and behavior. Indeed, for B. gauderio, seasonal changesin temperature are likely the crucial zeitgeber stimulatingreproductive activity (Quintana et al., 2004). However, changes inthe thermal and photic environment can also have direct effects oncellular processes. For example, mitotic rate increases dramaticallywith temperature (Rieder and Cole, 2002) and, for many ectotherms,mean body temperature is higher during the breeding season. Thus,

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Fig.5. Season � environment interactive effect onbrain cell proliferation in brain regions ofBrachyhypopomus gauderio associated withelectrogenesis and electrosensation. Note thedifference in y-axis scale. The pacemaker nucleus(Pn) initiates the production of the electric organdischarge, the electrosensory lateral line lobe(ELL) is the primary electrosensory processingregion and the anterior midbrain periventricularzone (PVZ) likely contributes cells to theprepacemaker nucleus, which regulates theproduction of social electric signals. *, significantdifference from both isolated and group fish; **,significant different from isolated fish and field fish.N6–8 fish per sample.


802

the seasonal changes in brain cell proliferation we found are likelyinfluenced directly by temperature.

When using BrdU to assess cell proliferation, temperature couldinfluence the basal mitotic rates and/or the transport and metabolismof BrdU during the period following BrdU injection. In oursampling, the temperature of the water that fish inhabited variedfrom approximately 27°C during the breeding season to 17°C duringthe non-breeding season. We kept water temperature during theperiod of BrdU incorporation within a narrower range, but itnonetheless varied from 23 to 27°C. To estimate the effect of thistemperature variation during the period of BrdU incorporation, weconducted an experiment on A. leptorhynchus in which all fish werehoused at the same temperature (27°C) but were then shifted to 23or 27°C for 2.5h during the period of BrdU incorporation. We foundno effect of temperature on BrdU+ cell density in the midbrain PVZ(4858±1367 BrdU+ cellsmm–3 at 23°C vs 4539±1359 at 27°C;t0.16, P>0.05). Thus it is likely that any effect of temperature onBrdU labeling in our B. gauderio study is related to mitotic rate inthe environment where they were living, rather than to the dynamicsof BrdU uptake and metabolism.

The effect of the thermal environment and photoperiod on braincell proliferation has received little attention, but several studiesindicate they have a potent influence. Radmilovich et al. examinedcell proliferation in the brains of turtles acclimated to warm andcold temperatures (Radmilovich et al., 2003). Turtles held at 20°Cproduced approximately six times more cells in brain proliferativezones than those held at 11°C. Similarly, Peñafiel et al. found thatlizards (Psammodromus algirus) maintained at 20°C hadapproximately three to 10 times more BrdU-labeled cells than lizardsmaintained at 10°C (Peñafiel et al., 2001). Ramirez et al. found noeffect of temperature (10 vs 25°C) on brain cell proliferation inanother lizard species (Podarcis hispanica), but long daylength (16vs 8h) increased brain cell proliferation by approximately threefold(Ramirez et al., 1997). Our results from isolated laboratoryconditions showed that fish during the breeding season housed at28°C with long daylengths (14h) produced approximately three toeight times more newborn cells than those held at 17°C at shortdaylengths (10h) during the non-breeding season. For most brainregions (those except Pn, ELL and anterior midbrain PVZ), fish inall three environments had similar magnitudes of seasonal change(i.e. no season � environment interaction). Because temperatureand photoperiod were the only common features shared by theseenvironments, we believe that seasonal changes in temperature andphotoperiod are sufficient to explain seasonal differences in cellproliferation in most brain regions. However, as discussed below,season likely affects cell proliferation in the Pn, ELL and anteriormidbrain PVZ through seasonal changes in reproductive physiologyand/or associated changes in electrocommunication behavior.

Cell proliferation and environmental complexitySimple vs enriched captive environment

Compared with isolated fish, group-housed fish had both abundantopportunity for social interaction and larger, more complex physicalenvironments (Table2). During the breeding season, they were alsoin a more advanced reproductive state, as evidenced by testismaturity and androgen levels (Fig.6). Yet, for most brain regions,cell proliferation rates were equivalent between these two captivegroups. This indicates that, for most of the brain, the size andcomplexity of the physical environment, the presence of plants andconspecifics, and the reproductive status of the fish do not affectbrain cell proliferation. However, for the Pn, ELL and anteriormidbrain PVZ, group housing enhanced cell proliferation. This effect


only occurred during the breeding season, when the reproductivesystem is active and most of the social signaling occurs (Fig.5).The structure of the physical environment did not change from thebreeding to the non-breeding seasons (Table1) and cannot accountfor this season � environment interaction. Thus, the higher ratesof cell proliferation in group-housed fish than in isolated fish duringthe breeding season is most closely associated with differences insocial environment and/or reproductive physiology.

Our study does not enable us to determine whether elevated ratesof cell proliferation are the cause or effect of socially inducedchanges in behavior and reproductive physiology. Moreover, ourstudy only examined the birth of cells, and we do not know thelong-term fate of these cells. While recognizing these limitations,we hypothesize that social stimuli and/or activation of thereproductive system enhance the production of cells in brain regionsinvolved with electrocommunication. Addition of cells to thesenuclei and their differentiation into neurons may then contribute tosocially induced changes in electrocommunication behavior.

The Pn drives the production of electric signals forelectrocommunication and reproductive signaling and, in B.gauderio, its activity is modulated by social interactions andbreeding conditions (Pouso et al., 2010; Quintana et al., 2010; Silvaet al., 2007). For example, the nocturnal EOD rate of males is greaterduring the breeding season than during the non-breeding season andis further elevated when males are housed with females (Silva etal., 2007). The Pn expresses androgen receptors, which likely playa role in reproductive and social changes in Pn activity (Pouso etal., 2010). One possibility is that the cells born under the influenceof social interactions and breeding conditions differentiate intoneurons and incorporate into the Pn circuitry. These new neuronscould then modify Pn activity either by providing sites for novelinputs to the Pn or by directly changing its intrinsic firing rate. Suchincorporation of new neurons could be mediated by androgenbinding to receptors on these new cells or on adjacent cells of thePn.

The ELL also shows elevated cell proliferation in response togroup housing and is crucial for electrocommunication. However,less is known about how the ELL changes functionally in responseto social interaction and reproductive state. In other electric fish(Sternopygus), electroreceptors in the periphery, which send inputsdirectly to the ELL, change their tuning properties in response toandrogens (Keller et al., 1986). In addition, the ELL of Gymnotusshows accelerated cell proliferation as fish enter adulthood (Castelloet al., 2008). These observations indicate that examining

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Fig.6. Plasma 11-ketotesterone (11kT) levels in Brachyhypopomusgauderio according to season and environment. *, significant differencefrom both isolated and group fish; **, significant different from isolated fishand field fish. N5–6 fish per sample.



reproductive influences on ELL function and cell proliferation maybe a fruitful area for future research.

Group housing also enhances cell proliferation in the anteriormidbrain PVZ. The fate of cells born in this proliferation zone isunknown in B. gauderio. In the closely related fish, Eigenmanniasp., some of these cells migrate laterally and become neurons in thePPn, which controls an important electrocommunication signaltermed chirping (Zupanc and Zupanc, 1992). In another electric fish,A. leptorhynchus, approximately two-thirds of the cells born in thisregion differentiate into neurons (Dunlap et al., 2008). Cell additionto the PVZ adjacent to the PPn increases in response to socialhousing and correlates with socially induced changes in chirpingbehavior (Dunlap et al., 2002; Dunlap et al., 2006). Male B. gauderioalso exhibit chirping behavior (Perrone et al., 2009), and in othermembers of the genus, chirping is controlled by the PPn (Kawasakiand Heiligenberg, 1989). If the anterior midbrain PVZ alsocontributes cells to the PPn in B. gauderio, then social enhancementof cell addition to the PPn may also participate in socially inducedchanges in chirping behavior. However, it is important to recognizethat the anterior midbrain PVZ may also contribute cells to othernearby regions of the brain (e.g. the hypothalamus), so, withoutfurther study, it is difficult to assess whether the social effect onthe PVZ is restricted to the electrocommunication system.

The regionally specific effect of social interaction on brain cellproliferation in B. gauderio and A. leptorhynchus is also foundin a frog, Hyla cinerea. In this frog, exposure to conspecificacoustic stimuli elevates cell proliferation rates in brain regionsinvolved with calling behavior, but not in control regions that areunrelated to calling behavior (Almli and Wilczynski, 2009).Together, these results indicate that cell proliferation in brainregions involved with communication behavior is particularlyresponsive to social stimuli and may contribute to adaptivechanges in social behavior.

Enriched captive environment vs complex natural environmentFish from the field had higher rates of cell proliferation in all brainregions and during both seasons than captive fish. Even in brainregions that are stimulated by group housing (ELL, Pn and anteriormidbrain PVZ), cell proliferation is further enhanced by fieldconditions during the breeding season.

There are many physical, social and biotic differences betweenthe enriched laboratory environment and the natural environmentthat could contribute to differences in brain cell proliferation(Table2). Compared with group-housed fish, field active fishexperienced a more complex structural environment. Enhancing thestructural complexity of the environment stimulates cell proliferationin the telencephalon of zebrafish (von Krogh et al., 2010), and thegreater number and diversity of objects in the natural habitat of B.gauderio (compared with the pools in captivity) may broadlystimulate brain activity and promote brain cell proliferation in B.gauderio. In addition, the natural environment is simply larger thanthe artificial pools of group-housed fish. At Laguna Lavalle, malesduring the reproductive season move a span of ~4m over a 3–4dperiod (Miranda et al., 2008). This is approximately three timesfarther than the maximum dimension in the pools in our study.Considering that locomotor behavior stimulates brain cellproliferation in some vertebrates (van Praag, 2008), it is possiblethat field active fish have higher rates of cell proliferation becausethey swim more than group-housed fish. However, the only test ofthis idea in fish indicates that, in laboratory environments thatpromote swimming, fish actually have lower rates of brain cellproliferation (von Krogh et al., 2010).

The social environment was also more complex in the field thanin the group laboratory housing. Certain features of the natural socialenvironment, such as the presence of fish of both sexes in a realisticsex ratio, were preserved in group housing, but the composition ofthe social environment was fixed and limited to only adults. Themore dynamic and age-diverse social stimuli fish receive in the wildmight contribute to higher rates of proliferation throughout the brainand/or the particular enhancement of proliferation in the Pn, ELLand anterior midbrain PVZ. In zebra finches, social complexitysignificantly influences cell recruitment into brain regions subservingvocal communication (Lipkind et al., 2002) and, in B. gauderio,the history and complexity of social interactions affectelectrocommunication (Salazar and Stoddard, 2009). Thus, it seemsplausible that the greater social complexity of the naturalenvironment contributes to the elevated rates of cell proliferationin 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 andpathogens. Sensory cues from predators inhibit brain cellproliferation in rats (Tanapat et al., 2001) but, otherwise, nothingis known about how the biotic environment affects brain celldynamics, and it is difficult to evaluate its influence on B. gauderio.

Relative importance of proliferation zones as sites of cellproliferation

In many vertebrate species, most cells added to the adult brainoriginate from discrete proliferation zones (Chapouton et al., 2007).Although fish clearly possess such proliferation zones, some ofwhich are likely homologous to those of other vertebrates (Zupanc,2008; Zupanc and Horschke, 1995), they also generate a substantialnumber of new cells scattered throughout the brain, a phenomenonwe term ‘background proliferation’. In B. gauderio, we found that~15–55% of new cells were born in proliferation zones. Thisproportion was greater in the hindbrain than in the midbrain.However, in almost all cases, the relative importance of theproliferative zones as sites of cell birth was independent of seasonand environment. Thus field conditions and breeding seasonuniformly increase cell proliferation in proliferative and backgroundzones.

The only exception to this generality was that the hindbrain PVZwas relatively less important as a site of cell proliferation duringthe breeding season compared with the non-breeding season. Theoverall quantitative effect of this seasonal difference is small,because only ~10% of all newborns cells in the hindbrain originatein the PVZ. Nevertheless, this result shows that, in some cases, theenvironment can influence the distribution of newborn cells in andout of proliferative zones.

CONCLUSIONSWe found that the natural environment and the breeding season hada large positive effect on the overall proliferative activity of thebrain. We are uncertain about the precise features that stimulate cellproliferation, but we suggest that the structural and social complexityof the natural environment and the warm temperatures and longdaylengths of the breeding season globally enhance brain cellproliferation.

Our study also highlights the importance of regional and seasonalspecificity in the effect of the environment of cell proliferation.Experimental manipulation of the social environment only affectsspecific brain regions – the Pn, ELL and anterior midbrain PVZ –and only during the breeding season. These brain regions areimportant components of the electrocommunication circuitry that


804 K. D. Dunlap, A. C. Silva and M. Chung

contribute to seasonal changes in social signaling. This correlationbetween regionally and seasonally specific brain cell proliferationin the electrocommunication nuclei and changes inelectrocommunication behavior support the idea that selectiveaddition of new cells may be a mechanism contributing to behavioralresponses to environmental change.

LIST OF ABBREVIATIONS11kT 11-ketotestosteroneBrdU bromodeoxyuridineEGm eminentia granularis pars medialisELL electrosensory lateral line lobeEOD electric organ dischargePn pacemaker nucleusPPn prepacemaker nucleus

ACKNOWLEDGEMENTSWe thank R. Perrone, G. Batista, L. Zubizarreta, L. Quintana and T. de losCampos for their generous and invaluable assistance in the field; T. Williams forcomments on the manuscript; and O. Macadar for inspiration. This work wassupported by fellowships from the US Fulbright Scholar program and the CharlesDana 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 inPMC for release after 12 months.

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RESEARCH ARTICLE Environmental complexity, seasonality and ... · environmental complexity also influences cell proliferation and neurogenesis in the adult brain. For example, rodents