Studies have emphasized the role of the hippocampus in learning and different kinds of memory. Spatial memory was impaired after bilateral hippocampal lesion in dogs1,2. The previous reports indicated that the asymmetry in the temporal lobe of the human brain might be related to laterality of the hippocampus3,4. In rats, early reports addressed morphological asymmetry in the hippocampus5. Recent studies investigated the asymmetry of the total number of neurons in rat hipocampal formation6. Our group counted pyramidal cells in hippocampal sectors and addressed significant sexual

Total neuron numbers in CA1-4 sectors of the dog hippocampus

Murat Çetin Rağbetli, Atıf Aydınlıoğlu*, Necat Koyun*, Recep Yayıcı & Kadir Arslan**

Departments of Histology & Embryology & *Anatomy, Medical Faculty, Yüzüncü Yıl University, Van & **Department of Anatomy Faculty of Veterinary, Kafkas University, Kars, Turkey

Received May 21, 2009

Background & objectives: Early reports addressed morphological asymmetry in the cross-sectional width of the rat hippocampus. The present study was aimed at counting total number of neurons in CA1-4 sectors and the subiculum of the dog hippocampus as well as investigating possible left /right and male/female asymmetry. Methods: Adult mongrel dogs (8 female and 5 male) were assessed by the right and left pawedness and sacrificed by exsanguinations. In each hippocampus dissected, the total neuron numbers of CAs and subiculum were estimated by the physical fractioning method. Results: Significant hemispheric asymmetries were found in the number of pyramidal cells of CA1, CA3/2, CA4 and the subiculum. Sex difference was also found in the subiculum, in favour of the males. Interpretation & conclusion: Our study indicated a left dominant asymmetry in males and right dominancy in females as well as no functional asymmetry in specific regions of the dog hippocampus. Further investigations are necessary to verify the hypothesis that hippocampal morphological asymmetries in normal subjects are functionally related in memory or in cognitive skills.

Key words Cornu ammonis - hippocampus - sex differences - sterelogy - subiculum

dimorphism in rat hippocampus6. In two studies with rats exposed to brain injury, the numbers of neurons in sham groups showed no dimorphism within each subregion of the hippocampal formation7,8. In the old male rats normally nourished or malnourished prenatally, hemispheric asymmetry was addressed in the CA1 and CA3/CA2 subfields of hippocampal formation9.

The reports addressing hippocampal asymmetry in dogs are scarce. A study with dogs reported hemispheric asymmetry of electrical activity in the hippocampus10. Our group addressed the significant sex and paw

Indian J Med Res 131, June 2010, pp 780-785

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differences and no right-left asymmetry related to hippocampal volume in dogs11.

The present study was undertaken to count total number of neurons in CA1-4 sectors and the subiculum of the dog hippocampus and investigate the presence of the paw, sex and right/left differences.

Material & Methods

The Animal Experiments and Ethic committee of Kafkas University, Turkey, approved the study protocol. Eight female and five male adult mongrel dogs were used. Paw preference was assessed by observing the right and left paw movements as mentioned elsewhere11-13. Briefly, an individual dog was allowed to access to preferred food with either its left or right paw. The animals exhibited a reliable number of left and right paw reaches. After assessment of paw preference the animals were anaesthetized with pentobarbital sodium (25 mg/kg; intravenous injection) and, in order to provide a better tissue fixation, sacrificed by exsanguination from the left carotid artery via a polyethylene cannula.

Dissection of the hippocampus: With the incision passing through the septum pellucidum, the brain was bisected in the sagittal plane. By way of the medial surface of each hemisphere, it was entered into the lateral ventricle. Each hippocampus was exposed and dissected carefully by using fine surgical devices. Each hippocampus was cut into the slices gently by a custom made slicing apparatus11. Sufficient number of slices was obtained from this procedure for physical fractioning technique of stereology. Each slice was laid on a tissue processing cassette at same position and labelled. After routine histological procedures, the tissues were embedded in paraffin.

Definition of hippocampal sectors: The cortical band of the hippocampus was divided into four parts according to its width cell size, and cell density (Fig. 1): CA1 contains small pyramidal cells. Field CA2 is characterized by a narrow, dense band of large pyramidal cells and field CA3 by a broad, loose band of large pyramidal neurons. CA4 forms the loosely-structured end zone. It is enclosed by the narrow, dark band of cells of the dentate gyrus. The subiculum is the transitional region between the CA1 subfield and the adjacent entorhinal cortex, where the stratum oriens of CA1 ends and the large, relatively loosely organized pyramidal cells are present (Figs 2, 3)14. Since it is a smaller area, CA2 was included in CA3 for comparison between sexes (and right/left) (Fig. 1).

Fig. 2. Micrographs showing the pyramidal cells of CAs and the subiculum in the right hippocampus. Toluidine blue × 400. Compare with Fig. 3 (the left).

Fig. 1. Micrograph of dog hippocampus showing the CA and the subiculum pyramidal cells. CA, cornu ammonis. Toluidine blue × 25.

Stereological procedure: For cell counting by the physical fractioning method15-17, a pilot study was performed first. The sections with 5µm (total, 4000–4500) were taken by rotary microtome (Leica RM2125-

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China). The first section in the series to be analyzed was chosen at random from the first 11 sections and every consecutive 400th section was collected from the series, which means a 1/400 section-sampling fraction, total 10-13 (ssf). Dissector pairs were taken from the tissue until the tissue sample was exhausted. The section procedure was started from the outside of hippocampus in the series and section sampling from a random point stated above. About 10-13 sections from each hippocampus are adequate to estimate the total neuron number when using the physical fractionator method for cell counting15,16,18. Two consecutive sections were mounted on each slide. Each sampled section containing the pyramidal cells in CA and subiculum regions was collected on slides and then stained with 1 per cent toluidine blue. Photographs of adjacent sections were taken with a digital camera (Sony DSC-S85, Japan) at a magnification of ×40. If a pyramidal cell of the hippocampus was seen in the reference section but not in the look-up section, it was counted as a dissector particle19. An unbiased counting frame was placed on the reference and the look-up sections on the screen of the PC to perform the counting according to the dissector counting method19,20.

For pyramidal cells: counting frame size (316×285µm2), section sampling fraction (ssf, 1/4400 unit) and area sampling fraction (asf, 1/44.4 unit) were determined. The coefficient of error (CE< 10%) and the coefficient of variation (CV) are also valuable data to see whether the number of subjects

in each group is adequate15,18. The preparations were examined with a light microscope system (Zeiss axioskop 40 Gottingen, Germany) including two separate devices for movements (x,y and z) of the stage on the micrometre-scale, step-meter designed by Kaplan et al21 and mechanical microcator designed by M.G. Reed (dial indicator, Mitutoyo-Japan)22. By using a 40× objective (NA 0.65), the whole section images were captured on a computer as JPG files by an attached video camera system. Hippocampal areas were analyzed by using image j programme (NIH; http://rsb.info.nih.gov.ij/) on a personal computer. In the described regions, the nucleus profile of the pyramidal cell was found by the physical dissector systematically-randomly spaced. Calculation the number total neurons was calculated19,22.

The total neuron number of CA and subiculum were estimated by the following formula:

Where N: total neuron number of CA and subiculum, ∑Qˉ: total disector neuron number, ssf: section sampling fraction, asf: area sampling fraction.

N= ∑Qˉ × ssf

1×

asf

1

The mean coefficient of variation for each group and the mean coefficient of error for stereological estimation of neuron number and other stereological parameters from the pilot study were validated.

Statistical analysis: The data resulted from cell count for the groups (gender, left/right side, paw preference) were subjected to logarithmic transformation. Wilcoxon Signed Rank Test (Minitab 14 for Windows) was used to compare the dependent samples (right/left) in the same sex and paw groups. The independent groups, i.e. male/female and right paw preference/left paw preference, were compared by Mann-Whitney U test (Minitab 14 for Windows).

Results

Statistically significant left/right differences were found in the number of pyramidal cells of CA1, CA2-3, CA4 and subiculum (P<0.05) (Table). Sex differences in CA1-4 sectors were not significant. Neuron number of the subiculum was found to be statistically different between sexes (P<0.05), in favour of the males (Fig. 4). The difference in the pyramidal cell numbers between the left paw and the right paw groups failed to attain significance. In each group, however, left/right differences were found to be statistically significant. In analysis of the subfields; in males, while CA1

Fig. 3. Micrographs showing the pyramidal cells of CAs and the subiculum in the left hippocampus. Toluidine blue × 400. Compare with Fig. 2 (the right).

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subfield showed the right dominancy, CA2/3, CA4 and the subiculum had more neuron number in the left hemisphere than the right. In females, on the contrary, CA1 subfield had the left dominancy. The other CA1-4 sectors and the subiculum had more neurons in the right hemisphere. The left paw preference group had a similar appearance to the females. In the right paw group, while CA1 and CA2/3 sectors had the right dominancy, CA4 subfield and the subiculum had more neurons in the left hemisphere than the right (Table).

Discussion

The present study showed hemispheric asymmetry in CA1-4 sectors and the subiculum of the dog hippocampus. Subiculum, however, was found to be sexually dimorphic.

The lateralization found in this study showed variation among CA1-4 sectors and subiculum. In

general, while the CAs showed the hemispheric asymmetry in favour of the right, the subiculum showed the left lateralization. Lateralization also changed with sex and paw preference in CA subfields and the subiculum as: left dominancy in the males and in the right paw group; right dominancy in the left paw group. Our study reporting number of neurons in dog hippocampal formation is probably the first. No hemispheric asymmetry in the density of Timm staining at the level of the dentate gyrus, in the dendritic layer of CA1 and CA2 fields, in the mossy fiber area, or in the subiculum has been reported in rats23.

Lister et al9 quantified hippocampal asymmetry in neuronal numbers and estimated this in both normally nourished and prenatally malnourished male rats. Their results showed that CA1 and CA3/2 subfields had fewer neurons in the right hippocampus. Since our male group had left dominancy in general, the left dominant asymmetry they found in male rats agrees with our findings. In a previous work, our group examined cell density and reported a greater density of neurons in the CA3 sector of the left hemisphere in both male and female rats6. The study with the two closely related strains of wood mice, Apodemus flavicollis and Apodemus sylvaticus, reported that the male A. flavicollis had a significant asymmetry in the deep subiculum in which the pyramidal layer is significantly larger in the left hippocampus compared to the right24. No significant hemispheric differences in any subfield

Fig. 4. Micrographs showing the subiculum pyramidal cells in male (M) and female (F) dog hippocampi. Toluidine blue × 400. Note that pyramidal cells are more number in male than in the female.

Table. The mean values and comparison results of the variables in relation with estimation of neuron number of CAs and subiculum in dog. Variable N CA1 mean CA3/2 mean CA4 mean Subiculum meanT-RH 13 39861670+ 33556868+ 31420895+ 27310989+

T-LH 13 39581784 33056019 31273586 27369912Male 5 82958233 NS 70472368 62352726 60935618**Female 8 77246716 64200710 62908079 50771703F-RH 8 38324138** 32650921** 31645540** 25924448**F-LH 8 38922578 31549790 31262538 24847255M-RH 5 42321721* 35006383* 31061462* 29529454*M-LH 5 40636512 35465985 31291263 31406164 Rpaw 7 80020061 NS 68475286 64152835 56547510Lpaw 6 78770745 64440087 60993069 52439357 Lpaw-R 6 38842786** 32746671** 30767827** 25341966**Lpaw-L 6 39927959 31693416 30225241 24735546 Rpaw-R 7 40734998** 34251322** 31980667** 28998723**Rpaw-L 7 39285062 34223964 32172168 29627940

T, total; RH, right hemisphere; F, female; M, male; Rpaw, right pawedness group; Lpaw, left pawedness group. +P=0.002; ** P<0.05; *P=0.05

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of the hippocampal formation in control animals7 and also lateralization in their control groups were reported8. We have reported6 left dominant asymmetry in pyramidal cell density of CA3 sector in both sexes. In general, the present study indicated the right dominancy in female dogs. The results of the current study suggest that left dominant asymmetry might be found only in a specific subfield in female hippocampal formation.

In humans, little is known about left-right hippocampal asymmetries. Dam25 counted neural cells in normal human hippocampi and found that the mean densities did not differ significantly between two sides in CA1 and the dentate gyrus (DG). Zaidel et al26 described left dominant asymmetry in males and no left-right difference in females. In their study, asymmetry was found in densities of nucleolated pyramidal cells in subfields CA1 and CA4 in hippocampal tissue of patients who underwent temporal lobectomy for alleviation of epilepsy.

Loss of asymmetry of hippocampal formation was observed in posttraumatic stress disorder patients27. However, a consistent left-less-than-right asymmetry pattern was found in the controls, mild cognitive impairment, and Alzheimer’s disease patients28.

The present study showed sex difference in pyramidal cell number in the subiculum, in favour of the males. Our previous study6 in rats showed sex differences in CA1 and CA4 subfields of the hippocampus. In these sectors, the males had the more pyramidal cell density than the females. There was no significant asymmetry between the pawedness groups in any CA1-4 sectors and the subiculum. We earlier reported volumetric paw differences, in favor of left pawedness among the females and the right pawedness among the males11. The present study suggests that sex and paw differences do not occur in stratum pyramidale as a spesific region, but the whole of the hippocampus (i.e., in the inclusion of all layers of each subfield) and the subiculum might show paw and sex differences6,11.

Hippocampal function (often via subicular output) is related to a large range of processes: behavioural inhibition, increased negative affective bias, exploration, risk assessment and various aspects of learning and memory29. The present study did not indicate paw preferences in pyramidal cell number of CA subfields and the subiculum. It is obscured if the morphological asymmetries are related to the function. There is, however, some evidence of the lateralized function in the hippocampal formation30,21.

In human, the hemispheric differentiation might be related to lateralized and/or increased functional linkage to memory systems in the neocortex30. In monkey, increased ipsilateral linkage was shown between hippocampal formation as well as the subiculum and neocortex in evolutionary development, which indicates the development of hemispheric specialization31.

In rats, CA1 and the subiculum project to the contralateral prefrontal cortex32. On the other hand, sex-difference and left-right asymmetries (left over right) in the rat prefrontal cortex during postnatal development have been found33. In dogs, sex differences and paw preference in the corpus callosum as well as the hippocampus might relate to cerebral specialization of function11-13.

It is suggested that these morphological asymmetries might be functionally related. Lister et al9 proposed that the greater number of neurons in the left hippocampus is a reflection of either additional functional capacity on that side or an alternative functional capacity on the left compared to the right hippocampus. The lateralized asymmetrical state-dependent learning was produced by kindled convulsions from the rat hippocampus, in the left rather than right34.

In conclusion, our findings indicated sex (in the subiculum) and hemispheric asymmetry (in the CA subfields and the subiculum) as well as no paw asymmetry in neuronal numbers in dog hippocampus. The present study confirms left dominant asymmetry in males. However, the relationship between morphological asymmetry and functional lateralization is still unclear. Further investigations are necessary to verify the hypothesis that hippocampal morphological asymmetries in normal subjects are functionally related in memory or in cognitive skills.

Acknowledgment This study has been supported by a grant from the Yüzüncü Yıl University research fund (2008-SBE-YL 037). The authors thank to Dr Aydin Him for language correction.

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Reprint requests: Dr Murat Cetin Ragbetli, Tip Fakültesi, Histoloji & Embriyoloji AD, 65300, Van,Turkey e-mail: ragbetli@hotmail.com