An updated investigation on the dromedary camel cerebellum (Camelus dromedarius) with special insight into the distribution of calcium-binding proteins

Studying the cerebella of different animals is important to expand the knowledge about the cerebellum. Studying the camel cerebellum was neglected even though the recent research in the middle east and Asia. Therefore, the present study was designed to achieve a detailed description of the morphology and the cellular organization of the camel cerebellum. Because of the high importance of the calcium ions as a necessary moderator the current work also aimed to investigate the distribution of calcium binding proteins (CaBP) such as calbindin D-28K (CB), parvalbumin (PV) and calretinin (CR) in different cerebellar cells including the non-traditional neurons. The architecture of camel cerebellum, as different mammals, consists of the medulla and three layered-cortex. According to our observation the cells in the granular layer were not crowded and many spaces were observed. CB expression was the highest by Purkinje cells including their dendritic arborization. In addition to its expression by the inhibitory interneurons (basket, stellate and Golgi neurons), it is also expressed by the excitatory granule cells. PV was expressed by Purkinje cells, including their primary arborization, and by the molecular layer cells. CR immunoreactivity (-ir) was obvious in almost all cell layers with varying degrees, however a weak or any expression by the Purkinje cells. The molecular layer cells and the Golgi and the non traditional large neurons of the granular layer showed the strongest CR-ir. Granule neurons showed moderate immunoreactivity for CB and CR. In conclusion, the results of the current study achieved a complete map for the neurochemical organization of CaBP expression and distribution by different cells in the camel cerebellum.

Immunohistochemical staining. Immunohistochemical staining had been performed on the paraffin blocks as mentioned before by Abdel-Maksoud, et al. 20 . Sections were deparaffinized with xylene and hydrated with a descending grade of ethanol then washed with 0.1 M PBS (3 × 10 min). To decrease the masking of antigen epitopes, the antigen retrieval was carried out using 0.1 M sodium citrate buffer solution (pH = 6) for 7 min using a microwave (600 W). Then, sections were cooled to room temperature for 20 min and washed with PBS (pH 7.4) for 10 min. After blocking the endogenous peroxidase activity with 3% H 2 O 2 in H 2 O for 30 min at the room temperature (RT), the sections were washed with PBS (3 × 5 min), then the sections were blocked with 10% normal donkey serum (NDS) + 0.2% Triton-X100/PBS for 2 h at RT. Subsequently, sections were incubated overnight at 4 °C with the following antibodies: rabbit polyclonal anti-CB . Sections were rinsed 3 × 10 min in 0.2% Triton-X 100/PBS and incubated with biotinylated IgG goat anti-rabbit (catalogue NO.E043201-8) and IgG goat anti-mouse secondary antibody (catalogue NO.P0447) (from Dako, Hamburg, Germany) diluted at 1:200 for 2 h at RT, followed by incubation with Vectastain ABC (Avidin-Biotin complex) reagent for 45 min in a humid chamber at room temperature. Visualization of the reaction was carried out with 0.04% 3,3′-diaminobenzidine (DAB) and 0.003% H2O2 in 0.05 M Tris-HCl buffer (pH 7.5) for 5-10 min. The sections were dehydrated in a graded series of ethanol, cleared with xylene and covered with DPX. Negative control were performed by incubating sections without the primary antibodies. Immunohistochemical staining was evaluated by LeitzDialux 20 Microscope and photos were photographed by canon digital camera (Canon Powershot A95) in the department of anatomy and histology, faculty of veterinary medicine, Assiut University, Egypt.
Morphometric and statistical analysis. The morphometric studies were performed on both light and immunohistochemical images of the camel cerebellum using Image-J software. The dimensions and counting of the cells were performed by measuring at least 10 sections from every cerebellum, and the results were presented as the mean of the measurements and considered as representative at these regions. The cross sectional area (CSA) is the measured area of the cells containing the whole view of the nucleus. The diameters are the longest and shortest diameter of the cells, containing the complete view of the nucleus, passing through the nucleus. The cellular density per CSA was performed by counting cells in a defined area and were finally calculated to 0.01 mm 2 . The linear density for PC was performed by counting the PC in a defined length of 1 mm. We chose the linear density for PC because they appear in the sections arranged in a line between the granular and molecular Scientific Reports | (2020) 10:21157 | https://doi.org/10.1038/s41598-020-78192-7 www.nature.com/scientificreports/ layer. To evaluate the ratio of immunopositive PC for different markers, we counted 100 PC manually from 3 different sections per animal. Some measurements for the size and density of PC and granular cells are summarized in Table 1 .

Results
Anatomical observation. The camel cerebellum weighed 49.2 gm and the entire brain weighed 355 gm, therefore, the camel cerebellum averages 13.86% of the brain. The anatomical features of the camel cerebellum were comparable to those of other mammals. The cerebellum lies caudal to cerebral hemispheres. It consists of 2 symmetrical lateral halves, the 2 cerebellar hemispheres which flank the medial part, the vermis. The dorsal surface of camel cerebellum has many fissures of variable depths subdividing the entire cerebellar surface into a considerable number of leaf-like lamellae (cerebellar folia) which separated by sulci (of variable depth) (Fig. 1a). The internal branched white matter is covered by the foliated outer grey matter giving it the appearance of a tree, and hence its older name, the arbor vitae (Fig. 1b).
The microanatomy. W applied some histochemical staining, such as cresyl violet (cell bodies) together with luxol fast blue (myelinated fibers) and silver impregnation (for both cell bodies and processes) on the camel cerebellar tissues to visualize the cytoarchitecture of the different types of neurons, dendrites and axons,. Histologically, the architecture of the camel cerebellum is conserved as different mammals and consists of the medulla covered by 3 layered-cortex; made up of an outer molecular layer, middle ganglionic (Purkinje) cell layer and inner granular layer (Fig. 2a,b). Surprisingly, we noticed a wide region of white matter that reach distally to the pial surface, without covering with cortical tissue (Fig. 2b, supplementary file). The outer molecular layer consisted of relatively few neuronal cell bodies and many unmyelinated fibers (Fig. 2c). Polymorphic neuronal cells were observed in the molecular layer and the outer half showed more stellate cells, than the basket cells found in the deeper half, (Fig. 2d). Notably, the cresyl violet did not show as many cells in the molecular layer compared to the silver stain. Moreover, different processes of numerous cells could be visualized by the silver stain (Fig. 2c,d). The dendrites of the PCs appear to extend through the molecular layer (Fig. 3a). The axons of basket cells make a meshwork around the cell bodies of PCs (Fig. 3a), while their dendrites extend perpendicular to the dendretic tree of PC (Fig. 3b). The axons of granular cells traversing the molecular layer to variable depth then bifurcated at right angles to give rise to parallel fibers (Fig. 3c). Whereas, both silver and Luxol fast blue could not visualize the climbing fiber nor the mossy fibers.
The PC layer made up of one row of cell bodies was located at the border between the molecular and granular layers. The PCs in camel were characterized by their large, flask-shaped cell bodies. Each cell contained a vesicular nucleus with a prominent circular nucleolus (Fig. 3a,c) and we observed some Nissles granules arranged  Table 1 .
The inner most granular layer is extremely cellular and densely populated by small spheroid somata with dark-stained nuclei and scanty cytoplasm (Fig. 4a,b). The granular cells arranged in clumps, rosettes or cords, and many spaces exist within the granular layer, most of them are lightly stained with silver stain (Fig. 4b,c). The granule cell measured 7.1 × 6.2 µm with CSA 35.7 ± 2 µm 2 . Granule cell density was 80.6 ± 9.26 cells/0.01 mm 2 CSA. The calculated density was 1289 ± 148 cells/0.001 mm 3 volume.
Luxol fast blue staining showed that white matter and many myelinated axons traverse through the granular layer from the Purkinje cell layer and the molecular layer either individually or in bundles (Fig. 4a,b). Additionally, many luxol fast blue stained endings surround the somata of the PCs, which are likely from axons of basket cells (Fig. 4b). Occasionally, some bundles run immediately beneath and parallel to the arranged PC somata (Fig. 4d,e), which are likely axons from Lugaro cells, basket cells as well as collaterals from PC axons. Moreover, we were able to detect non-traditional large neurons, such as Lugaro (Fig. 4e) and synaromatic neurons (Fig. 4f), with cresyl violet and silver staining.
Molecular characterization of the CaBP in camel cerebellum. Immunohistochemical staining against different molecules of the CaBP in camel cerebella such as CB, PV and CR were performed. However, immunopositive structures were detected throughout the cerebellar cortex, with layer-specific patterns of expression. The immunoreactivity was visualized in neuronal bodies, and in some instances, in dendrites and axons. Negative control sections incubated without the primary antibodies revealed no staining for neurons or fibers (See supplementary file). www.nature.com/scientificreports/

Calbindin-D 28 k (CB) expression in the camel cerebellum.
The Purkinje neurons showed the highest CB immunoreactivity, which demarcates the characteristic morphology of the Purkinje neurons throughout its extent (Fig. 5a). Moreover, the dendrites extend the whole length of the molecular layer and showing obvious dendritic arborization including the primary and secondary trunks and the spiny branchlets (Fig. 5a). However, seldom low CB-immunopositive PC bodies were observed (Fig. 5b). The ratio of CB-immunopositive cells were about 99 percent of the PC. The nature of the strong immunoreactivity was seen in densely packed homogeneous deposits in their cytoplasm, and in some cases, a similar immunoreactivity was seen within the nuclei (Fig. 5c,d).
Strong CB-ir fibers were seen beneath and surrounding the PC, which are likely from basket and/or Lugaro cells (Fig. 5c). Also, some coarse CB-immunopositive fibers were traversing through the granular layer and were oriented obliquely or vertically till reaching the white matter. They are characterized by varicosities along with their profile and some of them could be traced originated from PC (Fig. 5d). Interestingly, the immunoreactivity was www.nature.com/scientificreports/ observed in the spiny branchlets with many irregularities on their surface indicating dendritic spines (Fig. 5e).
There are also abundant immunopositive punctate elements (puncta) distributed in the neuropil between neurons of the molecular layer (Fig. 5e). All of the abovementioned immunopositive structures impart an intense coloration to the. In the molecular layer, CB-ir cells showed various degrees of intensity. The immunoreactivity was observed within the cytoplasm of the bodies and extended into the initial segments of the processes. Basket cells were recognized by their shape, orientation and position as well as their elongated somata whose long axes were parallel www.nature.com/scientificreports/ to the PC layer and localized in the deeper region of the molecular layer (Fig. 6a). Notably, there was CB-ir ring surrounding the somata of the PC, which originated from the axon terminals of the basket cells (Fig. 6b). The stellate neurons were characterized by their rounded/polymorphic somata and their settlement in the outer www.nature.com/scientificreports/ zone of the molecular layer. The dense CB-ir was also observed within the cytoplasm of PC dendritic tree which extend along the distal ramifications, just underneath the pial surface (Fig. 6c).
In the granular layer, moderate CB-ir was observed in a fraction of the granular cells, however, the expression was heterogeneous. The immunopositive cells displayed moderate, finely granular cytoplasmic immunoreactivity (Fig. 6d,e). The large Golgi neurons showed positive CB-ir (Fig. 6d). There were islands of CB-immunopositive a cellular spots in the granular layer among granule cells (islands of Held) (Fig. 6d,e). The heterogeneous CB expression was also obvious in the medulla, where fibers reacted differently with CB. Moreover, the perivascular large non-traditional neurons showed positive CB-ir (Fig. 6f).
Parvalbumin (PV) expression in the camel cerebellum. PV-ir was obvious in the PCs and molecular layer cells (Fig. 7a). Parvalbumin was expressed by the PCs which appeared as a diffuse granular reaction within the cytoplasm of the cell bodies and the dendrites, whereas, the nuclei showed less immunoreactivity. Notably, PV-ir was less intense compared to CB (Fig. 7b) and the ratio of PV-immunopositive cells were about 92 percent of the PC. Molecular layer cells showed higher PV-ir compared to CB. Moreover, the molecular layer cells were heterogeneously expressing PV. Its expression was also diffused within the cells with strong expression by the nuclear membrane. The processes of basket cells could be visualized clearly using this marker. These processes extending from basket cells towards the PCs and form a network surrounding the PC bodies (Fig. 7c,d). The stellate cells were expressing PV, with varying degrees of expression in their nuclei. In contrast to PC and basket cells, we could not detect the processes of stellate cells (Fig. 8a,b). We could detect solitary fibers traversing the granular layer, which are likely the axons of PCs (Fig. 8c). Furthermore, some, but not all, fibers of the white matter were expressing PV (Fig. 8d).

Calretinin (CR) expression in the camel cerebellum. CR-ir was obvious in almost all cell layers with
varying degrees (Fig. 9a). The molecular layer cells showed strong immunoreactivity for CR (Fig. 9a). The density of CR immune positive cells of the molecular layer was 129 cells per 50,000 μm 3 . The basket neurons were recognized by their elongated body whose long axis parallel to the cerebellar surface and localized in the inner region of the molecular layer ( Fig. 9 b-d). In addition to the well-known arrangement of the basket cell fibers surrounding the PC somata, we noticed also the dendrites of the basket cells form synapses with dendrites of the Purkinje cells (Fig. 9c,d). The stellate neurons were characterized by their polymorphic body and their localization in the outer zone of the molecular layer (Fig. 9e). PC showed any or very weak CR-ir (Fig. 9c,d) with a ratio of CR-immunopositive cells were about 4 percent of the PC. The granular layer showed positive CR-ir and is composed of two main groups of neurons: the granule neurons (granules) and the large neurons (Fig. 10a). The granule cells have a small, spheroid body and they aggregated in cords or clumps leaving large spaces in between. Large neurons showed stronger CR-ir and have voluminous polygonal or ovoid body. They involve the neuron of Golgi, one of the five traditional corticocerebellar neurons (Fig. 10b). Besides, several other large neuron types, generically indicated as non-traditional neurons were demonstrated. The expression of different CaBP in different cerebellar cell components are summarized in Table 2.

Non-traditional large neurons.
Neurons of Lugaro (NL or horizontal neurons). They are located in the external zone of the granular layer and in more/less close contact with PC layer. Their bodies are fusiform and horizontally oriented, parallel to the surface of the folium. The processes extend from the 2 poles of the body and are oriented horizontally along the boundary between the granular layer and PC and forms synapses with PC. They showed positive immunoreactivity for CB and CR ( Fig. 5b and Fig. 10c).
Synarmotic neurons (SNs). They are located in the inner zone of the granular layer near the subcortical white matter. Their bodies are ovoid and horizontally oriented. They showed positive reactivity with silver impregnation, CB and CR (Figs. 4f, 6e and 10d).  (UBN; monodendritic neurons). These neurons have round, or ovoid and vertical cell bodies. They are localized throughout the granular layer. A single thick dendrite trunk originates from the external body pole, extends for a short distance and gives rise to a push-like end. These neurons could be visulaized only by its positive CR-ir (Fig. 10 f-h).

Discussion
The current study aimed to extend the knowledge about the camel cerebellum that helps to learn more about the cerebellum. We demonstrated the detailed characterization of the architecture, both of cellular and fiber components, of the cerebellar cortex in the camel cerebellum. To this end, different histochemical stains were applied. Furthermore, numerous markers were used to visualize the different CaBPs by immunohistochemical analysis. One of the characteristic features in the results of histological stains of the camel cerebellum was that the granular cells were arranged in cords or rosettes 21,22 . According to our observation the cells in the granular layer were not crowded and many spaces were observed.
The relative weight of camel's cerebellum is 13.86% which is larger than that of donkey 10.8% 23   www.nature.com/scientificreports/ and 1254 ± 116, respectively). The density was lesser than that of bull, horse and man (1416 ± 141, 1503 ± 165 and 1609 ± 171 cells/0.001 mm 3 , respectively). The density was far lesser than that of rat, mouse and squirrel monkey (3216 ± 287, 3111 ± 341 and 2990 ± 301 cells/0.001 mm 3 , respectively). These results run in parallel to notion that the cell density (Number of cells) decreases with higher brain weight animals 25 . PC dimension in camel measured 43.76 × 31 µm, with a CSA of 797.73 ± 90 µm 2 . It is larger than that of rat (21 × 25 µm), mice (CSA 188 µm 2 ) and cat (width 29 µm), whereas, it is comparable to that of man (30-35 × 50-70) (reviewed in 26 ). There three different methods to analyze the PC density; to be PC number with respect to volume in µm 3 , to area in µm 2 or in certain length (linear). We preferred the later because it is the simplest and not subjected to calculations; however, we presented all three values. Due to the ambiguity inherent in measuring the volume of the PC layer we took the PC density to be number with respect to the area of the PC layer interface rather than to a volume. The units were accordingly neurons/mm 227 . PC linear density was 7.4 ± 1.1 cells/1 mm and the calculated density was 140.8 ± 2 cells/1 mm 2 or 139.4 ± 12.8 cells/0.001 mm 3 volume. We thought that the PC densities to be high because of the large body and long extrimites of camel; however, they had the lowest densities among several previously studied species. PC linear density in camel was much less than that of mice (38.5 ± 1.2 cells/mm) 28 . The calculated PC density in camel was also much less than that of rat (930 cells per mm 2 in the posterior lobe), cat (567 per mm 2 ) and monkey (510 cells per mm 2 ). PC density in camel was the nearest to that of man (300 cells per mm 2 ) among different species; however, it still far less than that of man, about half the densities (reviewed in 26 ). These low PC and granule cell densities were recorded also in the African elephant 29 . Together with the abundant cerebellar islands, this might indicate an abundant synapses in the neuropil to control the complex motor patterns and to process the sophisticated neural information which increases during phylogenesis 29 .
The CaBPs are widely expressed throughout the nervous system however they are among the most abundant proteins in the cerebellum 8 . These proteins expressed in the peripheral tissues including the expression of CR in the thymus, CB in the kidney and PV in the skeletal muscles and endocrine tissues 15,30 . In fact, no data on the expression of the CaBP within the camel cerebellum are available to date. Therefore, the results of the current study provide for the first time the detailed characterization of cellular, together with fiber, components in the www.nature.com/scientificreports/ camel cerebellar cortex. To this end, different immunohistochemical analyses were applied using markers for CB, CR and PV. We observed that the expression of each molecule was differentially expressed among different cerebellar populations. As a ubiquitous second messenger, Ca 2+ has been shown to regulate membrane excitability 31 , dendrite development 32 , synaptogenesis 33 , and many other processes contributing to the neuronal primary functions of the information processing and memory storage 34 .
CB was detected in the nervous system 35 , after its discovery in the chicken intestine 36 . Its expression is started early in cerebellar development in fetuses and newborn kittens during neuronal migration and is affected in aged dogs 37 . CB expression was intensely and widely distributed in different compartments; neuronal bodies, their main processes and the axon and dendritic terminal compartments, thus indicating its importance for normal cerebellar function. This some studies of various mammalian species stated that CB expression was predominant in Purkinje neurons 38,39 , the present study demonstrated that the CB was expressed in almost all cortical neuron types, however, with different intensity. The PCs showed the highest CB immunoreactivity, which distributed throughout the cytoplasm of cell bodies, axons and dendritic tree. Therefore, CB demarcates the characteristic morphology of the Purkinje neurons throughout its extent. However, a few low CB-immunopositive PC bodies were observed. The appearance of the detailed morphology of PC, including its very distal tiny branchlets and spines, using these antibodies, made it a marker of choice to study PCs 40 . The cytoplasmic abundance of CB is consistent with the previous studies that demonstrated the interaction between CB and cytoplasm proteins, such as myoinositol mono-phosphatase and protein M 41 . Additionally, the nuclear localization of CB in the PC supports the hypothesis that is plays a role in the regulation of gene expressions 42 . This homogeneous, intense expression of CB along the entire cytoplasm of the PC indicates the involvement of CB in many neuronal functions, especially in this large neurons which receive huge numbers of synapses from numerous types of cells. Furthermore, the experimental data demonstrated that CB plays a neuroprotective role against the oxidative stress or toxic effect of prolonged stimulation by excitatory amino acids (i.e.: glutamate; aspartate) 43 . Therefore, the absence or alteration in the expression of calcium-buffer proteins by any way will result in marked abnormalities in cell firing, with alterations in simple and complex spikes 37 . This will be accompanied by many neurological disorders and neurodegenerative conditions concerning motor coordination and sensory functions 44 . It has been reported that PC loss CB-ir in some viral encephalitides from HIV encephalitis in humans 45 , rabies-infected cattle 37 , neonatal Borna disease infection in rats 46 , and experimental infection of in mice 47 , or aging in dogs 48 . These notions postulate a relationship between loss of CB-ir in PC, altered calcium homeostasis and calcium  SN), and we assumed that CB is mainly associated with the neurotransmission of inhibitory type 42 . In line with this notion, the current study demonstrated the expression of CB in the GABAergic inhibitory neurons including stellate, basket, and Lugaro neurons 51,52 .On the other hand, we found also that CB expression was also demonstrated in some glutamatergic excitatory neuron including a large subpopulation of the granule neurons. Taken together, our results indicate the expression of CB by neurons is implicated in both the projective (extrinsic) and regulatory (intrinsic) circuits of the camel cerebellar cortex and there is no correlation between CB expression and type of neurotransmitter 42,52 . PV was expressed by PC, however, its intensity in the soma and dendrites was lower when compared to CB. Moreover, PV showed variations in the staining intensity within the PC cells. The same results were demonstrated in the cerebellum of rat, primates and human 8,35 . Two subpopulations within the molecular layer were also expressing PV, the stellate and basket cells 53 . These inhibitory GABAergic interneurons receive excitatory inputs from the granule cells and climbing fibers and exert inhibitory signals to the PC 54 . Both populations have relatively comparable morphologies with short dendritic trees, which are organized in an almost right angle www.nature.com/scientificreports/ to the dendrites of the PC cells. The axons of basket cells extend and, as their name implies, organized around the PC somata as a basket 47 The dendrites of both cells and the axons of the stellate cells form synapses on the dendritic tree of Purkinje cells. In contrast to the human cerebellum, we found that PV was not expressed by the GABAergic Golgi cells within the granular layer of the camel cerebellum 54 . Although the PCs have both CB and PV, the autism spectrum disorder (ASD) was linked to PV reduction. The cerebellum neuropathology in ASD was first detected over 35 years ago 55 , where the significant reduction in PV-expressing PC in autistic individuals is considered a histopathological feature of autism 56 . Therefore, altered calcium metabolism plays a key role in ASD pathophysiology, where it might, in turn, impact GABAergic signaling 57 . PV expressing neurons are more subjected to degeneration. In contrast, CR and/or CB expressing neurons are more likely resistant reviewed in 58 . CR is structurally related to CB and was first described in 1987 in embryonic chick retina, hence its name is driven from CB and retina 59,60 . According to the speed of Ca 2+ buffering, CB is the fastest, PV is the slowest  The authors claimed the great variability in CR-immunopositivity cannot be associated to methodological variables instead, this notion confirms the idea of a heterogeneous functional organization for the cerebellar cortex within an apparent homogeneous anatomical and histological structure. In camels, The majority of granule cells were expressing CR, however, was relatively weaker than other cells. This was in line with that reported in rodents [63][64][65] . Moreover, CR-ir was detected also in the axons of granule cells, the parallel fibers, suggesting an important role in Ca +2 -dependent synaptic plasticity 65,66 . CR was also expressed by large non-traditional neurons of the granular layer: unipolar brush cells (UBC), Lugaro cells, synaromatic cells and candelabrum cells. These populations were more intensely stained with CR antibody than the surrounding granule cells, supporting the notion that they contain higher concentrations of CR. Schwaller, et al. 54 and colleagues reported only that CR is expressed by unipolar brush cells (UBC) 67 and Lugaro cells in rodents 68 . The authors did not mention whether other large non-traditional neurons expressed CR. 68 . Lugaro cells and their processes are usually located just below the PC layer, which we could detect CB and even using the Luxol fast blue staining. They are believed to exert an indirect inhibitory influence on PC via a glycinergic inhibition of Golgi cells 69 . CR-ir by Lugaro cells is not conserved in all vertebrates, because Lugaro cells of Pigeon are not expressing it, instead they express secretagogin 70 . We found UBC in the granule cell layer of the posterior cerebellar lobe in camel, similar to that described in rodents 65 and sheep 71 . They have a single, thick process which forms a bush-like tip which is reported to function as excitatory glutamatergic neurons 67 . We found distinct large CR-ir globular cells deep in the granular layers suggestive to be Golgi cells. However, it has been reported that Golgi cells in all species except for guinea pig are immunonegative for calretinin 72 . It has been suggested recently that these cells might be a subtype of Lugaro cells in pigeon, where there are both globular and fusiform Lugaro cells 70 . These results might be indicate that CR expression is not conserved in certain cells among all vertebrates.
The neuroprotective role of CR against calcium-stimulated cytotoxicity has been suggested 60 . Because of the crucial role played by Ca +2 in neuronal physiology, it is not surprising that even modest impairments of Ca +2 homeostasis result in profound functional alterations. Despite the heterogeneous etiology of neurodegenerative disorders, as well as the physiological aging process, are all characterized by disruption of Ca +2 homeostasis and signaling.

Conclusion
In summary, the results of the current study achieved a complete map for the neurochemical organization of CaBP expression and distribution by different populations in the camel cerebellum. Understanding of these data under normal conditions represents a prerequisite for studying the cerebellar pathophysiology. PCs are expressing high CB, low CR and moderate PV. Molecular layer cells share the same expression profile; low CB and high CR and PV. Granule cells are expressing heterogeneous (low/moderate) CB, moderate CR and no PV, whereas this phenotype is the reverse in Lugaro cells. Golgi, UBC and synaromatic share the high CR-ir and no PV-ir and low CB-ir, however, UBC were not expressing CB.