Microbats appear to have adult hippocampal neurogenesis, but post-capture stress causes a rapid...

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Highlights

Neuroscience xx (2014) xxx

! Clear adult neurogenesis was observed in three species of microchiropterans. ! Microchiropteran adult neurogenesis is similar to that of othermammals. ! Capture stress causes a rapid decline in detectable hippocampal neurogenesis. ! Adult hippocampal neurogenesis appears to be acommon mammalian neural trait. ! 2014 Published by Elsevier Ltd. on behalf of IBRO.

NSC 15603 No. of Pages 1

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Neuroscience xxx (2014) xxx–xxx

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2 MICROBATS APPEAR TO HAVE ADULT HIPPOCAMPAL3 NEUROGENESIS, BUT POST-CAPTURE STRESS CAUSES A RAPID4 DECLINE IN THE NUMBER OF NEURONS EXPRESSING DOUBLECORTIN

5 R. CHAWANA, a A. ALAGAILI, b N. PATZKE, a

6 M. A. SPOCTER, a,c O. B. MOHAMMED, b C. KASWERA, d

7 E. GILISSEN, e,f,g N. C. BENNETT, h A. O. IHUNWO a AND8 P. R. MANGER a*

9 aSchool of Anatomical Sciences, Faculty of Health Sciences,10 University of the Witwatersrand, 7 York Road, Parktown,11 2193 Johannesburg, South Africa

12 bKSU Mammals Research Chair, Department of Zoology, College13 of Sciences, King Saud University, Box 2455, Riyadh 11451,14 Saudi Arabia

15 cDepartment of Anatomy, Des Moines University, Des Moines,16 Iowa, USA

17 dFaculte des Sciences, University of Kisangani, B.P 123218 Kisangani, Congo

19 eDepartment of African Zoology, Royal Museum for Central20 Africa, Leuvensesteenweg 13, B-3080 Tervuren, Belgium

21 fLaboratory of Histology and Neuropathology, Universite Libre22 de Bruxelles, 1070 Brussels, Belgium

23 gDepartment of Anthropology, University of Arkansas,24 Fayetteville, AR 72701, USA

25 hDepartment of Zoology and Entomology, University of26 Pretoria, Pretoria 0002, South Africa

27 Abstract—A previous study investigating potential adulthippocampal neurogenesis in microchiropteran bats failedto reveal a strong presence of this neural trait. As microchir-opterans have a high-field metabolic rate and a small bodymass, it is possible that capture/handling stress may leadto a decrease in the detectable presence of adult hippocam-pal neurogenesis. Here we looked for evidence of adult hip-pocampal neurogenesis using immunohistochemicaltechniques for the endogenous marker doublecortin (DCX)in 10 species of microchiropterans euthanized and perfu-sion fixed at specific time points following capture. Ourresults reveal that when euthanized and perfused within15 min of capture, abundant putative adult hippocampalneurogenesis could be detected using DCX immunohisto-chemistry. Between 15 and 30 min post-capture, the detect-able levels of DCX dropped dramatically and after 30 minpost-capture, immunohistochemistry for DCX could notreveal any significant evidence of putative adult hippocam-pal neurogenesis. Thus, as with all other mammalsstudied to date apart from cetaceans, bats, including both

microchiropterans and megachiropterans, appear to exhibitsubstantial levels of adult hippocampal neurogenesis. Thepresent study underscores the concept that, as with labora-tory experiments, studies conducted on wild-caught ani-mals need to be cognizant of the fact that acute stress(capture/handling) may induce major changes in the appear-ance of specific neural traits. ! 2014 Published by ElsevierLtd. on behalf of IBRO.

Key words: adult neurogenesis, doublecortin, Chiroptera,free-living animals, capture stress, hippocampus.

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29INTRODUCTION

30Studies on adult neurogenesis in free-living mammals are31becoming more numerous due to the need to understand32this biological process in relation to normal life-history33parameters (Amrein et al., 2004, 2011; Bartkowska34et al., 2008, 2010; Epp et al., 2009; Kempermann,352012; Cavegn et al., 2013; Chawana et al., 2013;36Patzke et al. 2013a,b). The investigation of free-living37mammals may provide a broader understanding of the38dynamics and mechanisms influencing adult neurogene-39sis of species in their natural habitat and ultimately40reveal potential reasons for the presence of adult neuro-41genesis in the mammalian brain. Free living mammals42are subject to a number of pressures such as predation,43foraging and varying weather patterns, all of which are44factors that may influence the process of adult neurogen-45esis (Kempermann, 2012).46While working on wild-caught mammals has the47potential advantage to reveal aspects of interest to a48broad understanding of adult neurogenesis, the capture49of these animals from their natural environments may50be considered to be an acute stressor that is di!cult to51control and unpredictable. While chemical capture of52wild animals (using dart guns) appears to lower blood53glucocorticoid levels, physical restraint and translocation54leads to significant increases in the stress-related55release of glucocorticoids (e.g. Widmaier and Kunz,561993; Morton et al., 1995). In terms of adult neurogenesis,57the e"ect of acute stress has been observed to lead to a58reduction in hippocampal neurogenesis in a range of lab-59oratory-kept species (Gould et al., 1998; Tanapat et al.,602001; Falconer and Galea, 2003; Kim et al., 2004;61Dagyte et al., 2009; Hulshof et al., 2012), although in rats62the reduction in the number of proliferating cells was

http://dx.doi.org/10.1016/j.neuroscience.2014.07.0630306-4522/! 2014 Published by Elsevier Ltd. on behalf of IBRO.

*Corresponding author. Tel: +27-11-717-2497; fax: +27-11-717-2422.E-mail address: [email protected] (P. R. Manger).Abbreviations: BSA, bovine serum albumin; DAB, diaminobenzidine;DCX, doublecortin; DCX+, doublecortin immunopositive; GCL,granular cell layer; NRS, normal rabbit serum; PB, phosphate bu"er;SVZ, subventricular zone.

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Please cite this article in press as: Chawana R et al. Microbats appear to have adult hippocampal neurogenesis, but post-capture stress causes a rapid

decline in the number of neurons expressing doublecortin. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.07.063

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http://dx.doi.org/10.1016/j.neuroscience.2014.07.063

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63 observed to occur within 2 h of the acute stressor and64 recovery to baseline levels within 24 h post exposure65 (Heine et al., 2004).66 An earlier study of adult neurogenesis in67 microchiropterans led to the conclusion that the68 hippocampus of the species studied had absent to low69 rates of adult neurogenesis (Amrein et al., 2007). While70 possible reasons for the absence of adult hippocampal71 neurogenesis were raised, it appears that no specific con-72 clusion was reached. One issue that was not raised by73 Amrein et al. (2007) was whether the stress of capture/74 handling of these small mammals may have had an75 important role in the lack of detectable adult hippocampal76 neurogenesis. While Amrein et al. (2007) state the bats77 were ‘‘perfused rapidly after trapping’’, no estimate of78 the time that elapsed between trapping and perfusion79 was provided, thus it is possible that capture stress could80 pose a serious methodological problem; however, this81 does not explain the absence of adult hippocampal neuro-82 genesis in the three neotropical bat species obtained from83 breeding colonies located in Germany, but again, no84 information regarding the handling of these bats prior to85 perfusion was provided. Given that microchiropterans86 have generally lower basal metabolic rates compared to87 other mammals of similar size (Austad and Fischer,88 1991; Neuweiler, 2000), but active or field metabolic rates89 significantly higher than other mammals and even birds90 (Neuweiler, 2000), it is possible that even a short period91 of stress, in the range of minutes, related to capture and92 handling may have a major e"ect on the expression of93 proteins in the microchiropteran brain, and in the case94 of Amrein et al. (2007) may have led to a false-negative95 report of the absence of adult hippocampal neurogenesis96 in the bat species studied, a finding that is becoming97 entrenched in the neurogenesis literature (e.g. Bonfanti98 and Peretto, 2011; Powers, 2013). Given this potential99 confound in the study of Amrein et al. (2007), we sought

100 to analyze the relationship between capture stress and101 adult hippocampal neurogenesis in wild-caught microchir-102 opterans using immunohistochemistry for the doublecor-103 tin protein (DCX), an endogenous marker of putative104 adult hippocampal neurogenesis (Kempermann, 2012;105 Patzke et al., 2013b).

106 EXPERIMENTAL PROCEDURES

107 In the current study we examined 36 brains from 10108 microchiropteran species including Miniopterus109 schreibersii (n= 2) captured from a wild population in110 Gauteng, South Africa, Cardioderma cor (n= 2),111 Chaerephon pumilis (n= 2), Coleura afra (n= 2),112 Hipposideros commersoni (n= 2), and Triaenops113 persicus (n= 2) captured from wild populations in114 coastal Kenya, Hipposideros fuliganosas (n= 2) and115 Nycteris macrotis (n= 2) captured from wild116 populations in the Yoko Forest near Kisangani,117 Democratic Republic of the Congo, and Pipistrellus118 kuhlii (n= 2) and Asellia tridens (n= 18) captured from119 wild populations near Unizah, Saudi Arabia. All animals120 were adults, as judged from epiphyseal closure of the121 metacarpophalangeal joints (Anthony, 1988). Appropriate

122permissions to trap and euthanize the bats were obtained123from the Gauteng Department of Nature Conservation,124South Africa, the Kenya National Museums, Kenya, the125University of Kisangani, DR Congo, and the Saudi Wildlife126Authority, Saudi Arabia. All animals were treated and127used in accordance with the University of the Witwaters-128rand Animal Ethics Committee Guidelines (clearance129number 2008/36/1) which parallel those of the NIH for130the care and use of animals in scientific experiments. All131bats were euthanized (overdose of sodium pentobarbital,132100 mg/kg, i.p.) and perfused through the left ventricle133with 0.9% saline, followed by 4% paraformaldehyde in1340.1 M phosphate bu"er (PB, pH 7.4) at various times fol-135lowing capture. For H. fuliganosas and T. persicus, the136animals were perfusion fixed within 15 min of capture.137For M. schreibersii, C. cor and C. pumilus, the specimens138were fixed between 15 and 30 min of capture, and for139C. afra, H. commersoni, N. macrotis and P. kuhlii, the140specimens were fixed within an hour of capture. For A. tri-141dens, two animals were sacrificed and perfused at each of142the following time points (in minutes) post-capture: 10, 15,14320, 30, 60, 120, 180, 240 and 300. Following perfusion,144the brains were removed and post-fixed in 4% paraformal-145dehyde in 0.1 M PB overnight, cryoprotected in 30%146sucrose in 0.1 M PB at 4 "C and stored in an antifreeze147solution at "20 "C until sectioning and histological pro-148cessing. Before sectioning, the brains were divided into149two halves along the mid-sagittal fissure and the tissue150was allowed to equilibrate in 30% sucrose in 0.1 M PB151at 4 "C. The specimens were cryosectioned in the sagittal152plane into 50-lm-thick sections. A one in three series of153sections was stained for Nissl substance (Cresyl Violet)154to reveal cytoarchitectural features, and immunostained155at two di"erent dilutions of the primary antibody to DCX156(1:300 and 1:600) to reveal immature neurons.157In the current study we used immunolabeling of DCX,158an endogenous marker of putative immature neurons, to159ascertain the potential presence or absence of adult160neurogenesis. While DCX immunopositive neurons161away from the hippocampus may not relate to adult162neurogenesis in these regions, such as the piriform163cortex (Klempin et al., 2011), it has been established that164DCX immunolabeling of granule cells of the dentate gyrus165is a good proxy for the presence of adult hippocampal166neurogenesis (Rao and Shetty, 2004; Couillard-Despres167et al., 2005). The presence of DCX is also thought to168reflect cumulative adult hippocampal neurogenesis over169a period of 2 weeks to 6 months, although this period is170species specific (Rao and Shetty, 2004; Kohler et al.,1712011). Thus, lack of DCX staining should be a reliable172indicator of the absence of adult hippocampal neurogene-173sis (Patzke et al., 2013b) or of a perturbation in the matu-174ration process of newly generated neurons.175Free floating sections were incubated in a 1.6% H2O2,17649.2% methanol, 49.2% 0.1 M PB solution, for 30 min to177reduce endogenous peroxidase activity, which was178followed by three 10-min rinses in 0.1 M PB. To block179non-specific binding sites the sections were then pre-180incubated for 2 h, at room temperature, in blocking181bu"er (3% normal rabbit serum – NRS, 2% bovine182serum albumin, BSA, and 0.25% Triton X-100 in

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183 0.1 M PB). Thereafter, the sections were incubated for184 48 h at 4 "C in the primary antibody solution (1:300 and185 1:600, goat anti-doublecortin, DCX, SC-18 Santa Cruz186 Biotech) under gentle agitation. The primary antibody187 incubation was followed by three 10-min rinses in 0.1 M188 PB and the sections were then incubated in a189 secondary antibody solution (1:1000 dilution of190 biotinylated rabbit anti-goat IgG, BA 5000, Vector Labs,191 in 3% NRS and 2% BSA in 0.1 M PB) for 2 h at room192 temperature. This was followed by three 10-min rinses193 in 0.1 M PB, after which sections were incubated for 1 h194 in an avidin–biotin solution (1:125; Vector Labs),195 followed by three 10-min rinses in 0.1 M PB. Sections196 were then placed in 1 ml of a solution containing197 0.025% diaminobenzidine (DAB) in 0.1 M PB for 5 min,198 followed by the addition of 3.3 ll of 30% hydrogen199 peroxide per 1 ml of DAB solution. Chromatic200 precipitation was visually monitored under a low power201 stereomicroscope. Staining continued until such time202 that the background stain was at a level that would203 allow for accurate architectonic matching to the Nissl204 sections without obscuring the immunopositive205 structures. Development was arrested by placing206 sections in 0.1 M PB for 10 min, followed by two more207 10-min rinses in this solution. Sections were then208 mounted on 0.5% gelatine-coated glass slides, dried209 overnight, dehydrated in a graded series of alcohols,210 cleared in xylene and coverslipped with Depex. To211 ensure non-specific staining of the immunohistochemical212 protocol, we ran tests on sections where we omitted the213 primary antibody, and sections where we omitted the214 secondary antibody. In both cases no staining was215 observed. It was not possible to undertake Western blot216 control testing due to the nature of the collection of the217 tissue from wild populations. Staining patterns of DCX218 were observed using low power stereomicroscope and219 digital photomicrographs were captured using Zeiss220 Axioshop and Axiovision software. No pixilation221 adjustments or manipulation of the captured images222 was undertaken, except for the adjustment of contrast,223 brightness, and levels using Adobe Photoshop 7.224 For quantifying DCX immunopositive cells, a modified225 unbiased stereological procedure was used as described226 previously (Malberg et al., 2000; Segi-Nishida et al., 2008;227 Noori and Fornal, 2011). All sections stained with the228 1:300 dilution of DCX were coded to ensure that the anal-229 ysis was performed by a blinded observer (M.A.S) and230 immunopositive DCX cells were counted at 40# magnifi-231 cation in the subgranular zone of the left hippocampus232 of all specimens using an Olympus BX-60 light micro-233 scope equipped with a video camera. Cells were included234 if the cells lay within, or touched, the subgranular zone.235 The subgranular zone was defined as the area from one236 cell diameter within the granular cell layer (GCL) from237 the hilus-GCL border and two cell diameters below the238 hilus-GCL border (Eriksson et al., 1998). Cells were239 excluded if the cell was more than two cell diameters from240 the GCL, focusing through the thickness of the section241 (optical dissector principle, see Gundersen et al., 1988;242 West, 1993; Coggeshall and Lekan, 1996) to avoid errors243 due to oversampling. Every section was counted through-

244out the hippocampus and the sum was multiplied by 3 (as245we used a one in three series, see above) to provide an246estimate of the total number of immunopositive DCX cells247in the entire left hippocampus (Table 1).248Statistical analysis was done using STATA software249package version 13.1 (StataCorp. 2013. Stata Statistical250Software: Release 13. College Station, TX: StataCorp251LP). Given our sample sizes, we performed non-252parametric tests (Mann–Whitney tests) to compare253hippocampal DCX cell counts of animals from di"erent254perfusion delay time groups (those perfused within25515 min of capture and those perfused after 15 min of256capture). In addition we undertook Spearman’s257correlation test to measure the strength of correlation of258cell counts obtained in animals belonging to a particular259time group.

260RESULTS

261Doublecortin immunopositive (DCX+) cells in the262microchiropteran hippocampus

263Of the 10microchiropteran species examined in the current264study, we found DCX+ cells clearly present in the265subgranular zone of the dentate gyrus of three species266(A. tridens, H. fuliganosas and T. persicus) which were267perfused within 15 min of capture, partially present in268three species (M. schreibersii, C. cor, C. pumilus) which269were perfused between 15 and 30 min of capture, and270low to absent in four species (C. afra, H. commersoni, N.271macrotis and P. kuhlii) all of which were perfused272between 30 and 60 min post-capture (Figs. 1–3; Table 1).273The number of DCX-labeled cells in the hippocampus for274all animals perfused more than 15 min (n= 28) after275capture declined nine times (median = 536 cells and276range = 345–3294 cells) when compared to those277perfused within 15 min of capture (n= 8; median = 4787278cells and range = 1380–6945 cells) (Mann–Whitney test279z = 3.08 and p= 0.0001). In addition, the species where280DCX+ cells were observed, the subgranular zone at the281base of the granule cell layer was populated by282immunopositive cells that had a small soma size with283dendritic processes that extended through the granule284cell layer to ramify into the molecular layer. Furthermore,285the mossy fibers that emanate from these cells were also286observed with DCX immunohistochemistry, indicating287that they are likely to be in the process of becoming288functionally integrated into the hippocampal circuitry289(Fig. 2B). The morphology of the DCX+ cells, when290present, in the dentate gyrus of the microchiropterans291was similar to that seen in other mammals studied with292the same technique.293In the A. tridens time series (Table 1), DCX+ cells294were readily observed at the 10 min post-capture time295point (Figs. 2B, 3B and 4A), showing the full range of296normal morphology of these immature neurons,297including the presence of DCX+ mossy fibers. By29815 min post-capture, the number of DCX+ cells was299dramatically reduced (to around 20% of the pre-15 min300specimens, Table 1), as were the number of DCX+301dendrites and mossy fibers emanating from these cells302(Figs. 3B and 4B). At 20 min post-capture there was a

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303 further reduction in the number of DCX+ cells (to around304 6% of the pre-15 min specimens, Table 1), dendrites and305 mossy fibers (Figs. 3B and 4C). By 30 min post-capture306 (Figs. 3B and 4D; Table 1), only a few DCX+ cells307 remained, and the DCX+ dendrites and mossy fibers308 were almost absent. The remaining time points309 examined, up to 300 min post-capture, evinced DCX

310immunostaining similar to that seen in the 30 min post-311capture time point, with only a few persistent DCX+312cells, dendrites and mossy fibers (Figs. 3B and 4;313Table 1). Similar to the trend observed with all species314provided above, our comparison of DCX-labeled cells in315A. tridens only showed that those animals perfused316more than 15 min after capture (n= 14) was associated

Table 1. Counts of DCX immunopositive neurons in the left hippocampi of the microchiropterans studied that were sacrificed and perfusion fixed atvarious time points following capture

Species Perfusion delay DCX cells, specimen 1 DCX cells, specimen 2

Hipposideros fuliganosas Less than 15 min 3762 3963

Triaenops persicus Less than 15 min 5610 5721

Miniopterus schreibersii Between 15–30 min 2067 2262

Cardioderma cor Between 15–30 min 2718 2568

Chaerephon pumilis Between 15–30 min 3294 3072

Coleura afra Between 30–60 min 558 603

Hipposideros commersoni Between 30–60 min 1059 1026

Nycteris macrotis Between 30–60 min 1188 1134

Pipistrellus kuhlii Between 30–60 min 1254 1335

Asellia tridens 10 min 6621 6945









Fig. 1. Photomicrographs of doublecortin immunoreacted sections of the dentate gyrus of the hippocampus of the various species ofmicrochiropteran examined in the current study. Doublecortin immunoreactive immature neurons are clearly present in (A) Hipposiderosfuliganosas, (B) Asellia tridens and (C) Triaenops persicus, which were euthanized and perfusion fixed within 15 min of capture, partially present in(D) Miniopterus schreibersii, (E) Cardioderma cor and (F) Chaerophon pumilus, which were perfusion fixed between 15 and 30 min post-capture,and very low to absent in (G) Coleura afra, (H) Hipposideros commersoni, (I) Nycteris macrotis and (J) Pipistrellus kuhlii, which were perfusion fixedmore than 30 min post-capture. The scale bar in J = 50 lm and applies to all images.



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317 with a 10 times decline in cell number (median = 401318 cells and range = 345–513 cells) when compared to319 those perfused within 15 min of capture (n= 4;320 median = 4078 cells and range = 1380–6945 cells)321 (Mann–Whitney test z= 2.974 and p= 0.0029).

322 DCX+ cells in other regions of the microchiropteran323 brain

324 In all the microchiropterans studied, varying densities of325 DCX+ cells were observed in the subventricular zone326 of the lateral ventricle (SVZ). From the SVZ, which327 appeared to occupy the majority of the ventricular wall328 adjacent to the caudate nucleus, these cells migrated329 through the rostral migratory stream to the olfactory330 bulb. We observed a stream of DCX+ cells arising from331 the inferior portion of the SVZ that appeared to migrate332 to the piriform cortex and amygdala. A small stream of333 DCX+ cells appeared to migrate dorsally from the334 anterior portion of the rostral migratory stream to335 populate the cerebral neocortex anterior to the primary336 somatosensory cortex.337 In the A. tridens time series, the rostral migratory338 stream was readily evident in individual animals339 perfused within 60 min of capture (Fig. 5A); however,340 the strength of labeling of both cells and fibers declined

341during this first 60 min and the rostral migratory stream342was not evident in individuals perfused from 120 min343post-capture. DCX+ cells in the frontal neocortex were344observed in the individual animals perfused within34510 min of capture (Fig. 5B), but after this time point we346could find no evidence for these cells. In contrast,347DCX+ cells were observed in the piriform cortex in all348individual animals at all of the time points examined349(Fig. 5C), with no significant drop in apparent DCX+350cell number, or expression of DCX in the dendrites351emanating from these cells.

352DISCUSSION

353The present study, demonstrating the likely presence of354adult hippocampal neurogenesis in microchiropterans,355and that detecting this presence is dependent on the356level of post-capture stress/handling to which these357animals are exposed, contrasts with a previous report358detailing the absence of adult hippocampal359neurogenesis in microchiropterans (Amrein et al., 2007).360The absence of adult hippocampal neurogenesis in micro-361chiropterans reported by Amrein et al. (2007) has been362referred to extensively in the literature (e.g. Bonfanti and363Peretto, 2011; Kempermann, 2012) to the point that the364idea that chiropterans in toto, both microchiroptera and365megachiroptera, do not exhibit adult hippocampal neuro-366genesis is becoming ‘‘accepted knowledge’’ (e.g.367Powers, 2013). It should be noted here that Amrein368et al. (2007) only studied species from the microchiropter-369an suborder of bats, and not the megachiropteran subor-370der, for which two recent reports have detailed the371presence of adult hippocampal neurogenesis in a range372of megachiropteran species (Gatome et al., 2010;373Chawana et al., 2013), making the title and conclusions374of the Amrein et al. (2007) paper misleading as they use375only the generic term bats.376The present study indicates that the potential problem377encountered by Amrein et al. (2007), leading to a false-378negative report regarding potential adult hippocampal379neurogenesis in the microchiropterans, was the length380of post-capture stress and handling experienced by the381animals prior to euthanasia and fixation of the neural tis-382sue. The four species for which we did not observe signif-383icant evidence of adult hippocampal neurogenesis were384those that were perfused at a time point greater than38530 min post-capture. The three species in which partial386evidence for adult hippocampal neurogenesis was387observed were perfused between 15 and 30 min post-388capture, but the three species for which we saw signifi-389cant evidence for adult hippocampal neurogenesis were390all perfused within 15 min of capture. The idea that post-391capture stress and handling leads to a rapid decline of392detectable adult hippocampal neurogenesis using DCX393immunohistochemistry in the microchiropterans is sup-394ported by the time series study of A. tridens undertaken395herein, where at 10 min post-capture DCX immunostain-396ing revealed extensive evidence for adult hippocampal397neurogenesis, but that by 15 min post-capture, the extent398of staining had decreased dramatically and was very low399to near absent in subsequent time points. Similar e"ects

Fig. 2. Photomicrographs of adjacent Nissl stained (A) and double-cortin immunoreacted (B) sections of the dentate gyrus of thehippocampus of Asellia tridens at 10 min post-capture. Notethe presence of doublecortin immunopositive cells at the base ofthe granular layer (GL), dendrites throughout molecular layer (ML) ofthe entire dentate gyrus, and mossy fibers (mf) exiting the dentategyrus by passing through the polymorphic layer (PL). In both imagesdorsal is to the top and rostral to the left. The scale bar in B = 500 lmand applies to both images.



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400 in the decrease in the detectable presence of DCX in neu-401 rons have been observed in the dentate gyrus (Dagyte402 et al., 2009; Hulshof et al., 2012) and the retrosplenial cor-403 tex of the rat after exposure to acute stress (Kutsuna404 et al., 2012). The observation that post-capture stress405 rapidly diminishes the detectable presence of adult hippo-406 campal neurogenesis in the microchiropterans may also407 explain other unusual results in field-caught species, such408 as the low proliferation rate, but high di"erentiation rate409 seen in wild-caught South African rodents (Cavegn410 et al., 2013), where capture stress may have reduced411 the detectable presence of newly born neurons using Ki-412 67 immunohistochemistry, but had no specific e"ect on413 the di"erentiating neurons, as the DCX immunohisto-414 chemistry used to detect di"erentiating neurons can be415 present in these neurons over a much longer period.416 That the detectable presence of adult hippocampal417 neurogenesis with DCX immunohistochemistry in the418 microchiropterans disappeared so rapidly is of interest.

419As mentioned, the microchiropterans have a very high-420field metabolic rate in comparison to most other421mammals and birds (Neuweiler, 2000), and it is possible422that the stress associated with capture or handling of423free-living animals, or animals not accustomed to being424handled, when combined with a high-field metabolic425rate, may lead to the rapid non-genomic corticosterone-426induced proteolysis of proteins associated with cell427di"erentiation/maturation such as DCX, but perhaps not428cell death or the cessation of cell proliferation (Kutsuna429et al., 2012). This is particularly so because, like the cells430in the CA1 region, granule cells show quick enhancement431of miniature excitatory potential post-synaptic currents432(mEPSC) and prolongation of N-methyl-D-aspartate433receptor (NMDAR)-mediated influx of calcium ions after434exposure to a wave of corticosteroids (Takahashi et al.,4352002; Pasricha et al., 2011). The rapid calcium influx acti-436vates a calcium-dependant enzyme, calpain, which437breaks down the cytoskeleton (Vanderklish et al. 2000;

Fig. 3. Bar graphs showing the results of our quantitative analysis of the number of doublecortin immunopositive neurons in the left hippocampus ofa range of microchiropteran species. (A) This bar graph shows significant levels of DCX immunoreactive cells in the hippocampus of two species(H. fuliganosas and T. persicus) that were sacrificed and perfused within 15 min of capture from their natural environment. The three speciesperfused between 15–30 min of capture (M. schrebersii, C. cor and C. pumilis) showed lower numbers of DCX immunoreactive cells, while thoseperfused between 30–60 min after capture (C. afra, H. commersoni, N. macrotis and P. kuhlii) all showed very low numbers of DCX immunoreactivecells. (B) This bar graph shows the results of the quantification of DCX immunopositive neurons in A. tridens from specimens that were perfused at arange of time points following capture. Note the significant presence of DCX immunoreactive cells when the animals were perfused 10 min followingcapture, but that this is substantially reduced at 15 min following capture and settles at a low level for longer time points. Two individuals of eachspecies and at each time point were assessed (specimens 1 and 2).



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438 Andres et al., 2013). Given that the corticosterone-439 induced changes in calcium currents occur with 10 min440 of exposure to the steroid (Wiegert et al., 2006) it is pos-441 sible that in this study, this mechanism could have been442 activated, resulting in breakdown of the cytoskeleton443 and DCX, which integrates linkages between the cyto-444 skeleton in neuronal cells and axons (Tint et al., 2009).445 The decline in DCX-labeled cells in the hippocampus446 of the A. tridens may be related to age, given that age-447 related decline of mammalian adult hippocampal448 neurogenesis is well documented (reviewed by Klempin449 and Kempermann, 2007). Thus, it could be argued that

450all the microbats that we caught and perfused within45115 min of capture were substantially younger than those452perfused more than 15 min after capture, with this latter453group being made up entirely of microbats in their senility.454Though possible, it is highly unlikely for this to be the case455in this study despite it being di!cult to accurately deter-456mine the exact chronological age of the microbats caught457from wild populations. Firstly, given that our own observa-458tions in this study yielded repetitive results for the two459specimens in each of the ten time groups as evidenced460by Spearman’s rho = 0.9938, it is more likely that the cell461counts are related to the perfusion delay rather than the

Fig. 4. Photomicrographs of doublecortin immunoreacted sections of the dentate gyrus of the hippocampus of Asellia tridens at di"erent time pointspost-capture (pc). At 10 min post-capture (A), numerous cells immunopositive for doublecortin are found throughout the entire dentate gyrus. Thesecells exhibit apical dendrites that ramify into the molecular layer and mossy fibers that exit through the polymorphic layer. At 15 min post-capture (B),the number of doublecortin immunopositive cells, dendrites and mossy fibers has decreased dramatically, with a further decrease in number ofthese structures at 20 min post-capture (C). Later time points (D–F) show a similar low number of doublecortin immunopositive cells, dendrites andmossy fibers. In all images dorsal is to the top and rostral to the left. The scale bar in F = 100 lm and applies to all images.



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462 chance of the two animals at each time point being of the463 same age. Secondly, our findings are unlikely to include464 counts of animals from the extremes of ages because

465we used microbats which had closed epiphyseal plates466and very old bats are hardly ever captured (Brunet-467Rossinni and Wilkinson, 2009). Given this, it is likely that468the changes observed with perfusion delay are due to the469e"ects of corticosterone and stress rather than old age.470Studies on standard laboratory animals often seek to471eliminate any potential stressors from the protocol as it472is well known that introduced stress can influence the473experimental outcome (Balcombe et al., 2004). Similar474care should clearly be taken when examining wild-caught475species, as capture, handling and removal from a familiar476environment may lead to high rates of stress (Morton477et al., 1995). In the case of the microchiropterans, it478appears that this has led to a false-negative report regard-479ing the possible presence of adult hippocampal neurogen-480esis (Amrein et al., 2007).481Reports detailing the presence of adult hippocampal482neurogenesis across mammalian species are becoming483more numerous, and in each case, it would appear that484adult hippocampal neurogenesis is present (reviewed in485Kempermann, 2012; see also Chawana et al., 2013;486Patzke et al., 2013a,b). Thus, at this stage, with the likely487presence of adult hippocampal neurogenesis in the micro-488chiropterans, this neural trait may be a common feature of489mammalian brains; however, as mentioned by490Kempermann (2012), certain species, such as cetaceans491that live in homogeneous environments, do need to be492examined to determine whether there is phylogenetic var-493iability in this trait, which appears to be absent in the ceta-494ceans (Patzke et al., 2013b). These variations may help495to understand whether adult hippocampal neurogenesis496relates to either specific aspects of the environment of497the species examined (extreme heterogeneity or extreme498homogeneity), or whether other explanations may499account for this potential variation (Patzke et al.,5002013b). Thus, at present, adult hippocampal neurogene-501sis may be thought of as being a likely standard feature502of mammalian brains and hippocampal function, but vari-503ations as seen for cetaceans (Patzke et al., 2013b) may504shed more light regarding functional aspects of this inter-505esting neural phenomenon in the adult mammal brain.

506Acknowledgments—This work was supported by funding from507the South African National Research Foundation (P.R.M.), the508Swiss-South African Joint Research Program (A.O.I. and509P.R.M.), the Deanship of Scientific Research at the King Saud510University through the research group project number511RGP_VPP_020 (A.A.), the Belgian co-operation service512(D.G.D.) at the Royal Museum for Central Africa (E.G.), and by513a fellowship within the Postdoctoral-Program of the German Aca-514demic Exchange Service, DAAD (N.P.). The authors thank Pres-515cott Musaba for his help with animal capture and species516identification in the Yoko rainforest, DR Congo.

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Fig. 5. Photomicrographs of doublecortin immunoreacted sections indi"erent regions of the brain of Asellia tridens at 10 min post-capture.(A) Doublecortin immunopositive cells and fibers in the rostralmigratory stream (RMS) located between the caudate nucleus (C)and the cerebral neocortex (NEO). In this image dorsal is to the topand rostral to the left. (B) Doublecortin immunopositive cells showingdendritic ramifications in layer III of the frontal cortex. In this imagedorsal is to the top and rostral to the left. (C) Doublecortinimmunopositive cells showing dendritic ramifications in layer II ofthe piriform cortex. In this image dorsal is to the bottom and rostral tothe right. The scale bar in C = 100 lm and applies to all images.

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