A morphologically distinct granule cell type in the dentate gyrus of the red fox correlates with adult hippocampal neurogenesis

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A morphologically distinct granule cell type in the dentate gyrus of the red fox correlates with adult hippocampal neurogenesis Irmgard Amrein⁎, Lutz Slomianka Institute of Anatomy, University of Zürich, Winterthurerstr 190, 8057 Zürich, Switzerland

A R T I C LE I N FO

AB S T R A C T

Article history:

Wild red foxes, proverbially cunning carnivores, are investigated for adult hippocampal

Accepted 25 February 2010

neurogenesis and morphological characteristics of the dentate gyrus. Adult red foxes harbor

Available online 4 March 2010

almost 15-times more young, doublecortin-positive neurons in their dentate gyrus than domesticated dogs. The number of doublecortin-positive cells corresponds to 4.4% of the

Keywords:

total granule cell number, whereas dividing cells amount to only 0.06%. Compared to

Carnivore

laboratory mice, proliferating (Ki67-positive) and dying cells are rare, but the percentage of

Cytoarchitecture

new neurons is quite similar. The numbers of proliferating cells, young cells of neuronal

Immunohistochemistry

lineage and dying cells correlate. Resident granule cells can be divided into two types with

Stereology

strikingly different morphologies, staining patterns and distinct septotemporal

Proliferation

distributions. Small sized granule cells with a nuclear diameter of 7.3 μm account for ∼ 83% of all granule cells. The remaining granule cells are significantly larger with a nuclear diameter of 9.4 μm diameter and stain heavily for NeuN. Septally and mid-septotemporally, densely packed small cells dominate. Here, only few large granule cells are scattered throughout the layer. Temporally, granule cells become more loosely packed and most of the cells are of the large type. High rates of neurogenesis are observed in foxes with high numbers of large granule cells, whereas the number of small granule cells does not correlate with any of the neurogenesis-related cell counts. Staining for parvalbumin, glutamate receptor 2/3, GAP-43 and dynorphin shows an anatomical context that is a composite of features common also to other mammalian species. In summary, we report a morphologically distinct granule cell type which correlates with adult hippocampal neurogenesis in the fox. Furthermore, the maturation phase of the young neurons may be prolonged as in other long living species such as primates. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Red foxes (Vulpes vulpes) are the most widespread carnivore species of the world. Even though humans have hunted larger carnivores for centuries, foxes survived in their

ancestral habitats. Foxes are proverbially cunning and show exceptional adaptability to natural and manmade environmental changes. This flexibility has been explained by non-specialized food and habitat preferences of this species, as ‘… the most important requirement within any

⁎ Corresponding author. E-mail address: [email protected] (I. Amrein). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.02.075

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habitat to a fox, is another fox’ (Lloyd, 1980). Group-living foxes form highly sophisticated hierarchies with one male, one breeding female and female helpers (Macdonald, 1979). The suppression of reproduction in subordinate females is associated with socially-mediated stress, population density and food availability (Macdonald, 1979). In rodents, social stress, mating and maternal-related factors can influence adult hippocampal neurogenesis (for review see Gheusi et al., 2009). Foxes display additional behavioral traits which have been associated with adult hippocampal neurogenesis in rodents. Foxes show highly developed spatial memory skills, and successfully retrieve cached food (Macdonald, 1976; MacDonald et al., 1994). In rodents, successful retrieval of food in baited radial mazes has been positively correlated with neurogenesis (Veena et al., 2009). Red foxes have also been shown to be more curious and exploratory than other carnivores kept in zoos (Kusak and Huber, 1991), however, when living within cities, they remain shy and alert towards humans. In rodents, explorative behavior in elevated plus maze does not correlate with neurogenesis (Shors et al., 2002), but the level of neurogenesis correlates with the behavioral pattern in an open field test (Naylor et al., 2008), and object recognition (Bruel-Jungerman et al., 2005; Jessberger et al., 2009). In short-living rodents, high neurogenic activity in young animals declines rapidly while aging, both in wild and laboratory mice (Amrein et al., 2004a; Ben Abdallah et al., 2010). In a comparative study (Amrein et al., 2004a) we could show that basal proliferation rates differ between rodent taxa. Increased species-specific complexity of the habitat can be compensated by increased survival of the newly born cells or by increasing total granule cell number. Long life expectancy and late development of the hippocampal formation however might require other strategies. We have shown that in long living bats hippocampal neurogenesis in adulthood is the exception (Amrein et al., 2007). In other long living species such as marmoset monkeys, proliferation occurs on a comparatively low level and also decreases with age (Leuner et al., 2007). However, marmosets might compensate for a low rate of cell division by an extended maturation of the adult born neurons, as has been shown for rhesus monkeys (Ngwenya et al., 2006). Reports about adult neurogenesis in other carnivores such as cats and dogs suggest that we can expect newborn neurons in the adult fox hippocampus as well (Altman, 1963; Siwak-Tapp et al., 2007). Since we argue that foxes show exceptional high behavioral plasticity, and adult hippocampal neurogenesis adds to the structural and functional plasticity of the brain, we expected neurogenesis in the fox hippocampus to be higher than in other long living species. In order to compare our findings with other species all measurements are normalized to total granule cell number. To provide an anatomical context for these findings, we briefly describe the basic morphology of the fox dentate gyrus and the distributions of some antigens used in studies of the functional anatomy of this part of the brain. The extent of newly born cells in the adult fox dentate gyrus is compared to data in other carnivores and rodents, and the findings are discussed in a comparative context.

2.

Results

2.1.

Morphology

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The morphology of the fox dentate gyrus (Fig. 1) closely resembles that of the dog (Hof et al., 1996a). The granule cell layer (Fig. 2a) is typically 6 to 8 granule cell bodies deep and well-delimited from the molecular layer and polymorphic layer of the hilus. There is no clear cell sparse zone between the granule cell layer and the polymorphic cell layer of the hilus (Fig. 1c), which is seen in rabbits or primates (Geneser, 1987; Rosene and van Hoesen, 1987). A narrow continuation of the stratum radiatum separates the polymorphic cell layer of the hilus from CA4 (as used by Rosene and van Hoesen, 1987; the reflected blade of the CA3 pyramidal cell layer), which is continuous with the polymorphic cell layer close to its infrapyramidal tip (Fig. 1c). In contrast to the dog, the cells of CA4 do not form a compact layer but a very loosely organized band (Fig. 1c) similar to that in pigs or primates (Holm and Geneser, 1991; Rosene and van Hoesen, 1987).

2.1.1.

Two distinct granule cell types in the fox dentate gyrus

The number of total granule cells in the fox hippocampi varies by a factor of two, with the animal identified as being the oldest (approximately four years) having the highest score (Table 1). Within the population of granule cells, two clearly distinct cell types were found and analyzed separately (Figs. 2a,b). The prevalent cell type is round, with a mean nuclear diameter of 7.3 µm, evenly distributed fine heterochromatin granules and a narrow rim of cytoplasm. This cell type accounts for most cells in the septal and mid-septotemporal part of the dentate gyrus and represents 83% (min 71%; max 89%) of the total granule cell population. The other type consists of larger, ovoid-shaped granule cells with a mean nuclear size of 9.4 µm, scarce heterochromatin and one distinct nucleolus. They are found scattered in the septal and mid-septotemporal granule cell layer and prevalent in the temporal part. The cytoplasm of these large cells is wider and extends into a prominent axon hillock. Cells are oriented perpendicular to the polymorphic layer in the septal and medial part of the dentate gyrus, while in the temporal part the orientation of the axonal hillock is more variable. Nuclear diameters of the two cell type are significantly different (p < 0.0001, Fig. 3). Total granule cell number is largely determined by the number of small granule cells (r Total GC - small GC = 0.998, p < 0.0001), whereas the number of large granule cells does not correlate with the number of granule cells (rTotal GC-large GC = −0.58, ns., Fig. 4).

2.2.

Neurogenesis

2.2.1. Ki67-, DCX-, PSA-NCAM- and NeuroD-immunoreactivity The morphological appearance of the Ki67-positive cells is equal to that seen in rodents. Positive cells often appear in clusters in the subgranular layer, while single positive cells are comparatively rare (Figs. 2c,d). Most DCX-stained cells are located in the subgranular layer. In the granule cell layer, they

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Fig. 1 – (a) Fox hippocampus in situ viewed laterally. The overlying cortices and white matter are removed. Scale bar = 1 cm. (b) Fox dentate gyrus in the context of the entire hippocampal region at the point of the first appearance of the entorhinal cortex. Scale bar = 1 mm. (c) Fox dentate gyrus. The rectangular inset marks the approximate location of Figs. 2h and 7c–g. Scale bar = 0.5 mm. Abbreviations: EC, entorhinal cortex; PaS, parasubiculum; PrS, presubiculum; Sub, subiculum; DG, dentate gyrus; pcl, polymorphic cell layer; gcl, granule cell layer. are most common in the deep part, with some cells spreading out into more superficial positions (Fig. 2f). In the crest region of the granule cell layer they are also seen in the subjacent polymorphic layer of the hilus. All DCX-positive cells are small sized, and stained axons and dendrites can be observed frequently (Fig. 2g). The antibody against PSA-NCAM shows a more extensive staining of neuronal processes than the DCX antibody (Fig. 2h). NeuroD very faintly stains cells throughout the extent of the granule cell layer. Very intensely stained small cells are restricted to the subgranular layer.

2.2.2. Quantitative analysis of proliferating cells, young neurons and dying cells In all fox hippocampi we found low numbers of proliferating cells (Ki67), even fewer numbers of pyknotic cells (Fig. 2i) but high numbers of young cells of neuronal lineage (DCX and PSA-NCAM) (Table 2). The number of proliferating cells, differentiating neuronal cells and dying cells are positively correlated (Person's rKi67DCX = 0.799, p = 0.031; rKi67-pyknotic = 0.894, p = 0.007; rDCX-pyknotic = 0.959, p = 0.001, Fig. 4). The number of PCNA-positive cells was estimated in two animals. Estimates were slightly higher than Ki67 positive cell numbers (10,200 or 124% of the number of Ki67 positive cells in VV1 and 13,460 or 159% of the Ki67 numbers in VV7). PSA-NCAM positive cell number was estimated in only one animal. The total number of 450,630

cells corresponds to 70% of the DCX-positive cells in the same animal.

2.2.3. Large granule cells correlate with neurogenesis-related cell counts The number of large granule cells is tightly correlated with the numbers of DCX-positive cells (rlarge GC - DCX = 0.960, p = 0.002) and dying cells (rlarge GC - pyknotic = 0.968, p = 0.001), but only marginally with that of Ki67 positive cells (rlarge GC - Ki67 = 0.795, p = 0.059; Figs. 4 and 5). The total number of granule cells as well as the number of small granule cells does not correlate with that of Ki67-, DCX-positive and dying cells (Fig. 4).

2.2.4.

No sex effect in neurogenesis and granule cell counts

Measurements for neurogenesis (proliferation, neuronal differentiation, and pyknotic cells) and granule cell numbers do not differ between sexes (Fig. 4).

2.2.5. Fewer proliferating and pyknotic cells in the fox dentate gyrus compared to C57Bl/6 Normalized for the total number of granule cells, the numbers of proliferating and dying cells are significant lower in the fox compared to two and three months old C57Bl/6 mice (p < 0.0001 and p = 0.001 respectively; data from Ben Abdallah et al., 2010) (Fig. 6). Although the normalized numbers of cells of the neuronal lineage are not significantly different between foxes

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Fig. 2 – Cytology and markers for proliferation, neuronal differentiation and cell death. (a) Granule cell layer. Two granule cells of the large type are marked with white arrowheads. The small black arrowhead marks the pyknotic cell illustrated in i. (b) Granule cells of the large type (white arrowheads) and small type (dark arrowheads). (c and d) Cluster of Ki67-positive cells in the subgranular zone. At high magnification the cluster resolves into 15 individual cells with morphologies and staining patterns typical also for other mammals. (e) Cluster of PCNA-positive cells in the subgranular zone. (f) Doublecortin-positive cells in the subgranular zone and deep granule cell layer extending neurites both towards the molecular layer and the hilus. (g) Arrowheads mark doublecortin-positive cells that have their haematoxylin stained nuclei within the focal plane. (h) PSA-NCAM-positive cells. (i) Pyknotic cell with heavily condensed chromatin. Scale bars: a,c,f = 20 µm; b,d,e,g,i = 10 µm; h = 100 µm.

and C57Bl/6 (Fig. 6), DCX-positive cells amount to 2.5% of the total granule cell number in mice but to 4.4% in foxes.

2.3.

Neurochemistry

2.3.1.

NeuN

and granule cell layers (Fig. 7a). In the granule cell layer, both large and small granule cells are stained, but the large type typically appeared darker than the small type (Fig. 7b).

2.3.2. In lightly counter-stained sections, we do not observe unstained nuclei in cells with a neuronal morphology in the polymorphic

Parvalbumin

Rare immunoreactive cells in the molecular layer at midseptotemporal levels are of mainly two types. Small round cells are located in the deep one-quarter of the molecular

Table 1 – Summary of optical fractionator data in methacrylate-embedded fox hippocampi. Note that one animal (VV1) was not analyzed for granule cell number due to poor section quality. Abbreviations: GC = granule cells, CE = Coefficient of Error (m = 0), counts = cells counted in the dissector samples/animal, # Sections = number of consecutive sections analyzed (for coronal and horizontal sections every 30th, for sagittal every 10th section). Animal# VV2 VV3 VV4 VV5 VV6 VV7 Mean

Cutting direction

Small GC

CE small GC

Counts small GC

Large GC

CE large GC

Counts large GC

Total GC

CE GC

# Sections

Horizontal Coronal Horizontal Sagittal Coronal Coronal

5,037,611 7,907,677 14,194,820 10,285,492 11,146,971 9,047,275 9,603,308

0.07 0.07 0.05 0.03 0.05 0.06

295 478 857 1902 777 622 822

2,100,428 1,803,215 1,706,028 1,373,562 1,635,463 1,890,910 1,751,601

0.10 0.10 0.10 0.08 0.10 0.09

123 109 103 254 114 130 139

7,138,039 9,710,893 15,900,848 11,659,054 12,782,433 10,938,185 11,354,909

0.06 0.06 0.05 0.03 0.05 0.05

22 27 23 25 26 27 25

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darker inner one-quarter of the layer. Staining is darkest immediately superficial to the granule cells layer and between the adjacent granule cells. Within the granule cell layer (but not elsewhere) high magnification resolved a punctate staining and very fine fibers with occasional varicosities. Staining gradually decreases across the granule cell layer and continues as a light band immediately below the layer. The polymorphic cell layer of the hilus appeared unstained. Immunoreactive cell bodies cannot be distinguished. Staining associated with the granule cell layer and hilus remain rather constant along the septotemporal axis of the dentate gyrus. Temporally, a clear division of the molecular layer into three zones that are sharply delimited from each other is apparent (Fig. 7f). The outer one stains darkest while the innermost one, with the exception of the part immediately superficial to the granule cell layer, is the lightest one. Fig. 3 – Small and large granule cells differ significantly in their diameter as calculated from the area measurements of the nucleator (StereoInvestigator) using 4 beams of measurements (GC = granule cells; NSmallGC = 132; NlargeGC = 66, p < 0.0001, bars = standard deviation SD).

layer. Their dendrites can be followed for only a short distance in the immediate vicinity of the soma. Larger, bipolar cells, oriented vertically to the granule cell layer, extend longer dendrites throughout the depth of the molecular layer, which may penetrate the granule cell layer into the hilus. Smooth and rarely branching, thick-caliber dendrites extend towards the pial surface of the molecular layer (Fig. 7c). In the upper molecular layer, they generate a number of finer, beaded branches that extend towards the pial surface or obliterated hippocampal fissure. Terminal staining is virtually absent from the upper two-thirds of the molecular layer, but a dense varicose fiber plexus is seen immediately superficial to the granule cell layer and extends between the granule cells in the upper one-third to one-half of the layer (Fig. 7c). The apparent density of fibers decreases rather abruptly in deeper parts of the layer and does not extend into the hilus. Smooth, thickcaliber dendrites in the molecular layer are seen to originate from pyramidal-shaped cells just below or embedded into the deep border of the granule cell layer. The basal dendrites of these cells extended throughout the hilus. Many smooth but only a few beaded dendrites are seen in the polymorphic layer of the hilus (Fig. 7c). The predominant immunoreactive cells in the hilus are either round or bipolar with their long axis oriented parallel to the granule cell layer. Their dendrites can be followed into the molecular, where they appear beaded. A few large, weakly immunoreactive multipolar neurons are seen in the polymorphic cell layer. The apparent density of immunoreactive cells and dendrites generally increases along the septotemporal axis of the dentate gyrus. The density of the fiber plexus associated with the granule cell layer however remains rather constant.

2.3.3.

GAP-43

The molecular layer is divided into three zones (Fig. 7e). The outer three-quarters of the layer are divided into roughly equally wide and lightly staining parts by a narrow slightly darker band. A sharp border delimits this part of the molecular layer from the

2.3.4.

Glutamate receptor 2/3

Neuropil staining intensity is much lower than in sections of mouse brain processed in parallel with the fox sections. Although some differential staining of cortical layers is seen, hippocampal layers stain evenly throughout and do not show the layer specific pattern visible in mice. The only exception is a narrow almost unstained band at the base of the granule cell layer that is visible in both species (Fig. 7d). Cells also stained weaker than in mouse sections, but their distribution appeared similar to that seen in mice — both within the

Fig. 4 – Matrix of all measurements, females and males are indicated. Pyknotic cells, DCX-positive and proliferating cells (Ki67) are all correlated. Total granule cell numbers as well as small granule cells do not correlate with any of the neurogenesis-related measurements. Large granule cells are borderline correlated with proliferating cells (Ki67) and significantly correlated with pyknotic and DCX-positive cells. Total number of granule cells is given by the number of small granule cells. Note that in the data involving granule cell counts one male could not be included due to poor tissue quality in the HMA embedded material.

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Table 2 – Summary of neurogenesis-related cell counts. All data were collected in 40 micron thick frozen sections. Ki67 and pyknotic cells were counted in each section containing the hippocampus using an oil 100× lens on a Zeiss microscope. For DCX, the sections were analyzed using the optical fractionator. Abbreviations: # Sections = number of sections analyzed, CE DCX = Coefficient of Error (m = 0) for DCX estimates. Animal# VV1 VV2 VV3 VV4 VV5 VV6 VV7 Mean

Sex

Brain-weight (g)

Ki67

# Section Ki67

Pyknotic cells

# Section pyknotic

DCX

CE DCX

# Section DCX

M F M F F F F

52.1 46.0 43.2 48.1 44.2 45.9 48.5 46.9

8200 7760 4820 6860 3740 4560 8460 6343

25 30 29 32 28 25 31 29

780 1020 600 620 240 400 860 646

30 35 32 33 32 31 41 33

640,225 900,315 436,545 402,650 59,200 173,520 500,670 444,732

0.14 0.08 0.18 0.08 0.14 0.17 0.08

31 34 26 33 30 30 44 33

hippocampus and in adjacent cortical layers. The polymorphic cell layer is filled with large polygonal multipolar cell bodies among a network of what appears to be large-caliber dendrites (Fig. 7d). Staining intensity does not change along the septotemporal axis of the dentate gyrus.

2.3.5.

Dynorphin B

Homogeneous staining in the molecular and granule cell layer without structural definition most likely represents background. The polymorphic cell layer stains darker. Here the staining is granular and appears to outline dendritic profiles (Fig. 7g). A lightly stained zone separates the polymorphic cell layer from the CA4 pyramidal cell layer. A granular-appearing band extends superficial to and in between the pyramidal cells into CA3. Staining intensity increases towards CA1. There are no apparent changes along the septotemporal axis of the dentate gyrus

2.3.6.

C-Fos

Staining with both c-fos antibodies produced only few very faintly stained cells and, very rarely, a cell which, in comparison to mouse sections processed in parallel, is lightly to moderately stained (Fig. 7h). The latter were seen exclusively in temporal sections of the granule cell layer.

3.

Discussion

We provide a morphological description of the red fox dentate gyrus and a quantitative analysis of adult hippocampal neurogenesis in this medium-sized carnivore. The granule cell population consists of two morphologically distinct cell types that show a type-specific distribution along the septotemporal axis of the dentate gyrus. The number of large granule cells correlates with the extent of neurogenesis, whereas the number of small sized granule cells does not. Cell proliferation occurs at a comparatively low rate, but, in comparison to rodents, the maturation phase of young neurons appears extended.

3.1.

Neurochemistry

3.1.1.

Parvalbumin

Our description of the distribution and morphology of neuronal somata is surprisingly close to that for the Macaca mulatta (Seress et al., 1991), but a distinct infragranular fiber plexus seen

in Macaca fascicularis (Pitkänen and Amaral, 1993) is not present in the fox. Instead, it shares its neuropil staining pattern with rats, dogs (Celio, 1990; Hof et al., 1996a; Kosaka et al., 1987) and gerbils (Nitsch et al., 1995), with the exception of the lateral perforant path staining seen in gerbils. The largest labeled cell were weakly immunoreactive in contrast to the strong staining of such cells in dogs (Hof et al., 1996a). A septotemporal increase in the density of parvalbumin-containing cells has also been observed in the mouse (Jinno and Kosaka, 2006) but not in the rat (Nomura et al., 1997). Parenthetically, we did not observe parvalbumin-positive CA3 pyramidal cells which, so far, appear unique to dogs (Hof et al., 1996a).

3.1.2.

GAP-43

The distribution of GAP-43 in the septal and mid-septotemporal dentate gyrus corresponds to that of the rat (Benowitz et al., 1988; Oestreicher and Gispen, 1986). There are no other descriptions of changes in the distribution of GAP-43 along the septotemporal axis of the dentate gyrus. Fox and rat differ from the strong reaction seen throughout the molecular layer of the human dentate gyrus (Benowitz and Perrone-Bizzozero, 1989).

3.1.3.

Glutamate receptor 2/3

Cellular staining in the polymorphic layer was similar, albeit much weaker than that seen using Glu2(4) antibodies in the dog (Hof et al., 1996b). Otherwise, the staining pattern resembled that seen for Glu2/3 receptors in rats, monkeys and humans (Ikonomovic et al., 1995; Leranth et al., 1996).

3.1.4.

Dynorphin

Similar to other species, dynorphin marks the terminal fields of the dentate granule cells in the hilus and mossy fiber zone of the hippocampus (Drake et al., 1994; Khachaturian et al., 1982). The staining pattern in the polymorphic cell layer corresponds to that seen in the human hippocampus (Houser et al., 1990).

3.1.5.

c-Fos

While c-Fos is constitutively expressed also in the fox dentate gyrus, the low signal obtained with both antibodies did not allow the relation of the expression of this immediate early gene to the granule cell types or a comparative assessment. In summary, as far as the markers that we have investigated suggest, neurogenesis in the dentate gyrus of the fox

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findings agrees with reports in laboratory (Lagace et al., 2007) and wild mice (Amrein et al., 2004a), but contrasts with findings in voles and rats, in which hormone levels can influence neurogenesis in a sex-dependent manner (Barker and Galea, 2008). The complex social structure of foxes, which is in interplay with habitat and ontogenetic factors, would require a much larger sample size to address sex or hormone dependent alterations of adult hippocampal neurogenesis.

3.3. Resident granule cells and their relation to neurogenesis

Fig. 5 – (a) The numbers of small and large granule cells in foxes. The percentage of large granule cells varies between 11 and 29% of the total granule cell number. (b) The numbers of DCX-positive cells and large granule cells. In both a and b the animals have been sorted by decreasing numbers of large granule cells. The number of large granule cells is correlated with the number of differentiating, DCX-positive cells (rlarge GC-DCX = 0.960, p= 0.002), but not with the number of small granule cells.

proceeds in an anatomical context common to other mammalian species.

3.2.

Neurogenesis in foxes is not gender dependent

In the small sample presented here, we found no sexdependent differences in any of the measurements. Our

The number of small granule cells does not correlate with any of the neurogenesis-related cell counts, but it determines total granule cell number. Interestingly, the average of 11.3 million granule cells in foxes is similar to what has been found in rhesus monkeys (Keuker et al., 2003), pigs (Holm and West, 1994) and in some human data (Korbo et al., 2004), despite its much smaller overall brain size. On the other hand, the number of large granule cells correlates with neurogenesis but not with total granule cell number. The two clearly distinct granule cells types can be differentiated according to their size, shape and staining characteristics. It is well-known that granule cells in the temporal pole of the granule cell layer are larger in diameter than in the septal part (Bayer, 1982; Gaarskjaer, 1978). However, while changes in cell size occur along a septotemporal gradient in rodents, the fox dentate gyrus clearly consists of two distinct granule cell populations. The largesized, strongly NeuN-positive granule cells are the dominant cell type in the fox temporal dentate gyrus too, but, in addition, they persist as interspersed cells among a majority of small granule cells in the mid-septotemporal and septal parts of the dentate gyrus. Interestingly, the existence of two distinct populations of granule cells was recently inferred in GAP-43 overexpressing mice from two populations of mossy fiber boutons that differentially express either GAP-43 and SNAP-25 or zinc tranporter 3 and synaptophysin (Rekart and Routtenberg, 2010). The number of the large granule cells in the fox correlates with the number of DCX-positive cells, i.e. animals with high numbers of large granule cells do show high neurogenesis. We would not expect that large-sized granule cells have a direct interaction with neurogenesis as it has been shown for cells in the neurovascular niche (Palmer et al., 2000; Shen et al., 2004) At the temporal pole, where large granule cells are numerous, neurogenesis is lowest, an observation also made in the rat by Bayer (1982). Furthermore, large granule cells are not preferentially located in the subgranular layer but are distributed throughout the granule cell layer. Their size and location do suggest that the large granule cells belong to the early formed granule cells. In rats, granule cells in the temporal pole are the first to be fully formed (Bayer, 1980). It has been shown that early generated granule cells derive from a secondary dentate matrix following an infrapyramidal–crest–suprapyramidal migratory pathway (Altman and Bayer, 1990). This migration starts at E21 and ceases by P5. Progenitor cells remain in the subgranular layer forming a tertiary matrix between P3 and P10, where they produce the inner (deep) aspect of the dentate gyrus and remain as resident precursors responsible for adult

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Fig. 6 – Normalized numbers in percent for proliferating (Ki67) and pyknotic cells in the fox are significantly lower than in two to three month old laboratory mice (mouse data taken from Ben Abdallah et al., 2010). The number of differentiating, DCX-positive cells is increased in foxes, but misses statistical significance. GC = granule cell, bars = SD.

neurogenesis (Altman and Bayer, 1990). The survival of early formed cells is better than that of later formed cells (Muramatsu et al., 2007). The number of large granule cells may thus reflect neurogenic potential while the survival of later forming small cells may be a function of age and environmental influences. We have argued earlier that the extent of neurogenesis may be linked to average life expectancy and live history events such as reaching sexual maturity (Amrein and Lipp, 2009). Our findings of a distinct granule cell population, which may originate from the same precursor matrix as the precursors in the later subgranular layer, suggests that this relation may be established already by migrational events during late gestation and early prenatal periods, setting the extend of adult hippocampal neurogenesis later on.

3.4.

Large granule cells stain heavily for NeuN

Mullen et al. (1992) point out that high expression of NeuN may correspond to the state of differentiation or function of the neurons. Large granule cells contain scarce heterochromatin and stain more intensely for NeuN, whereas the evenly distributed fine heterochromatin granules in small granule cells stain less intensely for NeuN. This is in accordance with the observation that NeuN is expressed preferentially in areas of low chromatin density (Lind et al., 2005), indicating a higher cell activity in large granule cells. Thus, large granule cells

may be also functionally related to adult born granule cells, as these cells have been shown to be more excitable than the resident granule cells (Schmidt-Hieber et al., 2004; Wang et al., 2000).

3.5. Generation and maturation of adult-generated neurons in the fox In comparison to data we obtained in laboratory and wild rodents, relatively few cells are dividing in the fox hippocampus. In rodents, the number of Ki67-positive proliferating cells corresponds to nearly 1% of that of the granule cells in yellownecked wood mice (Amrein et al., 2004a) and to 0.7% in two to three month old C57Bl/6 mice (Ben Abdallah et al., 2010). Ki67positive cells amount to only 0.06% of the number of resident granule cells in the fox. Low numbers of proliferating cells were confirmed by a second marker, PCNA, in two animals. That PCNA numbers are slightly higher than Ki67 numbers is explained by a significantly longer post-division half-life of PCNA as compared to Ki67 (see Eisch and Mandyam, 2007). Also, we have found a close correlation between the numbers of pyknotic cells and proliferating cells across a variety of species (Amrein et al., 2004a). The very low numbers of pyknotic cell observed in this study again suggest that cell proliferation is indeed very low in the fox when compared to rodents. However, the rate is closer to that of other long living mammals. BrdU-positive cells amount to ∼ 0.04% of the

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Fig. 7 – Immunocytochemical markers in the mid-septotemporal (except f and h) dentate gyrus. (a) NeuN-like immunoreactivity in a lightly counter-stained section. (b) Both small-type and large-type granule cells are NeuN-positive. Granule cells of the large type (white arrowhead) typically stain darker than those of the small type (black arrowhead). (c) Parvalbumin. (d) GluR 2/3. (e and f) Septal, e, to temporal, f, gradient in GAP-43 staining of the molecular layer. Stars mark the obliterated hippocampal fissure. (g) Dynorphin. (h) Rare c-fos immunoreactive cells in the granule cell layer of the temporal dentate gyrus. Scale bars: a, h = 20 µm; b = 10 µm; c,d = 20 µm; e,f,g = 100 µm. Abbreviations: pcl; polymorphic cell layer; gcl, granule cell layer.

number of granule cells in adult Marmoset monkeys (Callithrix jaccus) calculated from BrdU-positive cells after one injection and three week of survival time (Kozorovitskiy et al., 2005; Leuner et al., 2007) or ∼ 0.02% in Rhesus monkeys (M. mulatta, after one to five BrdU injections and variable survival times (Gould et al., 1999; Keuker et al., 2003). Although the primate data has been derived using another marker, this difference alone is unlikely to explain the at least 10-fold differences in the normalized proliferation rates when compared to rodents. Long living animals as represented by the fox and primates appear to share a common trait of reduced cell division in their subgranular layer. Although proliferation amounts to only one-tenth of that seen in rodents, the normalized number of doublecortinpositive cells of neuronal lineage does not differ from that seen in rodents (means are actually higher, albeit nonsignificantly). Species-specific variations in the length of the maturation of newly born neurons have been reported before. For rodents, the time until a new born granule cells shows mature features is around 30 days (Brown et al., 2003; Kempermann et al., 2003), in contrast to possibly more than

3 months in monkeys (Ngwenya et al., 2006, 2008). On the background of low proliferation, requirements for these highly excitable young neurons (Amrein et al., 2004b; Cameron and McKay, 2001; Deng et al., 2009) may be mediated by an extended maturation period. We believe that the proliferation in natural animal population is species-, maybe even family-specific and relatively stable, its extent depending on developmental processes and on the age of a given animal. Requirements for behavioral plasticity as a function of ontogenetic or phylogenetic development may be satisfied by changes of the survival or the duration of cell maturation. In comparison to dogs, foxes have much more young neurons. Indeed, a beagle has a slightly larger brain and weights more than twice as much as a fox (72 g and 14 kg to 42 g and 5.5 kg) but shows at the age of ∼ four years ∼27,000 DCX-positive cells (Siwak-Tapp et al., 2007), whereas a fox has ∼400,000 DCX-positive cells. Although our foxes were younger on average than the dogs, we do not expect an age-dependent decrease in the dogs by a factor of 15. Thus, we conclude that foxes with intrinsic low proliferation activity compensate the need of young, excitable

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neurons by extending the maturation of the young cell population.

4.

Experimental procedures

4.1.

Animals

Seven wild adult foxes (V. vulpes) were caught within 3 weeks in late January to February, well before close season (first of March to Jun 15th), and shot by official rangers of the city of Zürich, Switzerland, in agreement with federal game law and the cantonal regulations for fox population control. Two males and five females were used in this study. Ages were estimated by the ranger. Brains were removed from the scull within ∼ 30 min of death and immersion fixed in three changes of 4% phosphate-buffered paraformaldehyde containing 15% picric acid for a total of 7 days. The right hemispheres were processed for immunohistochemistry (Section 4.2) while the left hemispheres were embedded in hydroxyethylmethacrylate for granule cell counts (Section 4.3).

4.2.

Table 3 – Summary of antibodies applied on free-floating, 40 micron thick sections. Special treatments other than standard immunohistochemical protocols are indicated. Primary antibody Ki67 (NCL-Ki67p)

Source

Concentration Special treatment

Novocastra

1:1000

PCNA Santa Cruz Doublecortin; Santa Cruz DCX

1:1000 1:1000

PSA-NCAM

Chemicon/ Millipore Chemicon

1:10,000

Sigma Chemicon Santa Cruz

1:20,000 1:5000 1:500

Chemicon/ Millipore Abcam Santa Cruz

1:50

NeuN Parvalbumin GAP43 NeuroD (N-19) Glutamate 2/3 Dynorphin B c-Fos (sc 253)

1:1000

1:250 1:5000

Immunohistochemistry c-Fos (sc 52)

Dissected hippocampi and entorhinal cortices were cut perpendicular to the septotemporal axis of the structure into a septal, mid-septotemporal and temporal block and cryoprotected in 30% sucrose. Blocks were frozen with dry ice and cut at 40 μm on a sliding microtom. 20 series of sections were collected and stored in cryoprotectant at −20 °C until further processing. Immunohistochemistry was performed on free-floating sections. Complete series of all animals were stained for Ki67, DCX, PSA-NCAM and parvalbumin, complete series of selected animals for PCNA, GAP-43, NeuroD, dynorphin B, NeuN, c-fos and glutamate 2/3 receptor. Each batch was accompanied by mouse brain tissue as positive control and mouse and fox tissue as negative controls. Details of the immunocytochemical procedures are listed in Table 3.

4.3.

Cytoarchitecture

Dissected hippocampi and entorhinal cortices were washed in PBS, dehydrated in ascending alcohols over 4 days and embedded in hydroxyethylmethacrylate (HMA). Pre-infiltration with 1:1 solution of 100% alcohol and HMA (Technovit 7100, Heraeus Kulzer GmbH, Wehrheim/Ts, Germany) overnight was followed by three changes of HMA alone for a total period of one month. Hardened blocks were cut at 20 μm. Hippocampi were cut horizontally (n = 2), sagittally (n = 2) or coronally (n = 3). Every 10th section was collected, mounted on slides and dried at 60 °C for 1 h. Sections were incubated for 40 min at room temperature in Giemsa staining solution (Giemsa stock solution 1.09204.0500, Merck, Darmstadt, Germany, diluted 1:10 in 67 mmol KH2PO4 buffer), differentiated 15 s in 1% acetic acid, dehydrated in 3 changes of absolute alcohol, cleared in xylol and cover-slipped. In addition, a series of frozen sections was stained with Giemsa solution following an adjusted protocol to visualize pyknotic cells (Klaus et al., 2009). Sections were mounted and

Epitope retrieval with heat, 95 °C for 40 min in citric puffer, pH 6.0 Identical to Ki67 Epitope retrieval with microwave in citric buffer, pH 6.0 None Identical to doublecortin None None Identical to Ki67 Identical to doublecortin None Epitope retrieval with Methanol:3% H2O2, 4:1

dried over night at room temperature. Slides were incubated for 10 min at 60 °C in Giemsa staining solution, rinsed in buffer for 90 s, differentiated in three changes of 99% alcohol, cleared in xylol and cover-slipped.

4.4.

Quantitative analysis

4.4.1.

Pyknotic and immunohistochemically stained cells

Pyknotic cells, identified by darkly stained, round to C-shaped condensed and fragmented chromatin in the Giemsa-stained frozen sections (Amrein et al., 2004a; Heine et al., 2004), were counted exhaustively in every 20th section and multiplied by 20 to estimate the total number of pyknotic cells. Cells in the top focal plane of the section were omitted from the counts. The number of proliferating cells visualized with Ki67- (all animals) or PCNA-antibody (2 animals) was also estimated in this manner. DCX and PSA-NCAM positive cells were counted using the optical fractionator (West et al., 1991). Counting was done in every 20th section using a counting frame of 40 × 40 µm, placed at 180 µm intervals along the x- and y-axis in septal and medial sections and at 60 µm intervals in temporal sections.

4.4.2.

Granule cells

Total granule cell number was calculated from the estimates of the number of small and large granule cells in Giemsastained, HMA embedded sections, again using the optical fractionator method. Counting frames of 15 × 15 µm, a disector height of 10 µm and x- and y-step 250 µm were used. Total cell number estimates were calculated by multiplying the raw counts with the inverse of the sampling fractions: N=

1 1 1 × × × ∑Q − ssf asf hsf

hsf =

h t

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where ssf is the section sampling fraction, asf is the area sampling fraction, hsf is the height sampling fraction and ∑Q− is the total number of cells counted in the disector samples. Using the Nucleator module of StereoInvestigator software (MicroBrightField, Williston, Vermont), nuclear diameters in the plane of the section were calculated from Nucleator estimates (Gundersen et al., 1988) of cells selected by disector samples in 3 animals. Nucleoli where selected as reference point and distances to the outer rim of the nucleus were measured along four equally spaced lines originating from the reference point.

4.5.

Statistics

Statistical analyses were performed using SPSS 17.0 (SPSS Inc, Chicago, Illinois). Correlations of cell counts relating to adult neurogenesis (Ki67, DCX and pyknotic cells) as well as granule cell numbers were tested using a 2-tailed Pearson's r. One-way ANOVA was used to compare nuclear diameters of the two granule cell types and to detect possible sex effects, although for the latter sample size is too small to allow definite conclusions. General Linear Models were used to compare the data obtained in foxes with those observed in C57Bl/6 mice of two and three months of age from an earlier study (Ben Abdallah et al., 2010). Coefficients of error for the cell number estimates were calculated using the Gundersen–Jensen estimator with m = 0 (Gundersen et al., 1999; Slomianka and West, 2005).

Acknowledgments We gratefully acknowledge the rangers Zweifel and Albisser for providing the animals, B. Kuhn for the GAP-43 antibody, and R. Lang and I. Drescher for their expert technical assistance. This work was supported by NCCR ‘Neural Plasticity and Repair’ and Swiss National Science Foundation (SNF 3100A0122589).

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