Neuronal cell types in the anterior ventral thalamic nucleus of the camel

The anterior ventral nucleus neurons in of the camel brain were morphologically studied by Golgi impregnation method. Two neuronal types of were found in the camel anterior ventral thalamic nucleus, namely, Golgi‐type I neurons and Golgi‐type II neurons. Those neurons were generally similar to their counterparts in the human thalamus. The Golgi‐type I neurons exhibited medium to large cell body (mean diameter = 25 μm) which was either multipolar or triangular in shape. They had from 3 to 10 primary dendrites with many branches but with no spines or appendages. The Golgi‐type II neurons had small to medium size (somatic mean diameter = 17.5 μm), their cell bodies were variable in shape, some were round, and others were multipolar or fusiform. These cells bodies had two to six primary dendrites with few branches that may have spines and/or grape‐like appendages. Our findings shed some light on the anterior ventral thalamic nucleus structure of the camel as one of the strongest adaptive mammals to the hard climatic conditions.

No small neurons (Golgi-type II) were found in one thalamic nucleus namely the dorsomedial nucleus (Ma et al., 1998). The same study reported that all of the neurons in the dorsomedial nucleus were large and considered as thalamocortical projection neurons. Another thalamic nucleus namely the anteroventral nucleus had been studied in both human (Hani et al., 2007) and cat (Somogyi, Tömböl, & Hajdu, 1973). In the human AV, two morphologically different types of neurons were found, one corresponded to Golgi-type I with large body and dense dendritic trees while the other type corresponded to Golgi-type II with small body, few branches and grape-like dendritic appendages. Although long segments of the axons of these small neurons were impregnated in one study (Hani et al., 2007), no local axonal branches were found. This may indicate that these neurons could be in fact thalamocortical projecting neurons rather than interneurons which come in agreement with the result of the other study (Somogyi et al., 1973). Finally, in another group of thalamic nuclei namely the intralaminar nuclei, denser dendritic trees were found in Golgi-type II neurons compared to Golgi-type I neurons (Hazlett, Dutta, & Fox, 1976;Tömböl, Bentivoglio, & Macchi, 1990;Tseng & Royce, 1986).
The aim of this study is to describe the microscopic features of different neuronal types in the camel thalamic AV nucleus and compare these neurons with their counterparts previously described in different thalamic nuclei in human and some other species. Hopefully, these results will contribute to our understanding of the comparative neuroanatomy.

| MATERIALS AND METHODS
The Golgi-Kopsch method of silver impregnation method was used. This method usually stains between 1 and 5% of neurons without overlapping between the stained neurons. This enabled us to study the different parts of neurons including their spines and appendages.
Camel brains were bought from local butchery stores. No animals were sacrificed in this study, therefore Institutional Animal Care and Use Committee (IACUC) approval was waived. Two modifications of Golgi-Kopsch impregnation were carried out on seven well-fixed camel brains (fixed in 10% formalin for 1-3 months). Coronal blocks containing the AV thalamic nucleus were cut and processed according to:

| Fox, et al. (1951) modification
Block (4 mm thick) containing the AV thalamic nucleus was cut, dried, and kept in a mixture of 3% Zink chromate and 2% formic acid for 7 days. Then the block was removed from the chromate solution, dried without washing, and immersed in a 0.75% silver nitrate solution for another 7 days. This method gave the best results regarding the impregnation of axons of Golgi-type II neurons, impregnation of very few neurons with a very clear background.

| Braitenberg et al. (1967) modification
Blocks (about 2 mm thick) containing the anterior ventral nucleus of the thalamus were cut, dried and placed in a mixture of 3% potassium dichromate, 0.5% formaldehyde, and 12.5% sucrose for 12 days. Then they were incubated in a 0.75% silver nitrate solution for 7 days.

| Sectioning and mounting
The blocks of the two modifications were removed from the silver nitrate, and then cut into 100 μm thick sections using a sliding microtome. The sections were kept in absolute alcohol in their original serial order for 2 hr before transferring them to xylene for clearing where they were kept for 5 min. Sections were mounted on slides using DPX mounting medium and then slides were covered with cover slips.

| Drawing and photographs
Selected well-impregnated neurons were studied under Nikon light microscope equipped with camera Lucida (drawing tube), oculometer, and imaging system.

| RESULTS
Two types of neurons, namely Golgi-type I (TCPN) and Golgi-type II neurons (LCN) were found in the camel AV nucleus. These cell classes were distinguished by the soma size, density of the dendritic tree, and the dendritic appendages.

| Golgi-type I neurons (TCPN)
These neurons (Figure 1) had medium to large cell body with diameter range from 18 to 34 μm with mean diameter = 25 μm ± 4.5 (n = 20). Their cell bodies exhibited primarily multipolar shape and sometimes they may be triangular with no spines or appendages. They had from 3 to 10 primary dendrites. These dendrites had no spines or appendages. The dendrites of these cells showed rich arborizations with two branching patterns (radiating and tufted). The radiating branching pattern was more common than the tufted one. The tufted pattern was seen more often in the proximal dendrites comparing to the distal dendrites, which exhibited more radiating pattern.

| Golgi-type II neurons (LCN)
Golgi-type II neurons (Figures 2-4) represented the most frequent impregnated cell type in this study. These cells had small to medium size with somatic diameter range from 8 to 24 μm with mean diameter = 17.5 μm ± 4.7 (n = 20). Their cell bodies may be round, multipolar, or fusiform in shape. These neurons had few (2 to 6) primary dendrites with less dense dendritic trees comparing to Golgi-type I neurons. In this study, Golgi-type II neurons were divided in two subtypes: Golgi-type IIa (Figure 2) had round cell body and scarce primary dendrites (one to three). These dendrites were sparsely branching and had no spines or appendages. Golgi-type IIb (Figures 3 and 4) neurons had round, multipolar or fusiform cell body. They possessed dendritic tree of moderate density consisting of three to six primary dendrites.
The dendrites of Golgi-type IIb neurons may have spines and/or grape-like appendages (Figures 3b and 4). Moreover, these dendrites had two branching patterns, the radiating and the tufted. The radiating branching pattern was clearly more common than the tufted. The tufted pattern was less common in Golgi-type IIb than Golgi-type I neurons. Proximal parts of the axons of Golgi-type IIb neurons were impregnated with no branches for these parts of the axons.

| DISCUSSION
In this study, two main types of nerve cells were described in the camel AV thalamic nucleus and compared with their counterparts in other thalamic nuclei described in previous similar Golgi studies in human and other animals.
Golgi-type I nerve cells found in this study were found to be generally similar to their counterparts previously described in other thalamic nuclei including the human anterior ventral, ventral anterior, ventral posterior, ventral lateral nuclei as well as medial geniculate, and lateral geniculate bodies (Al-Hussain Bani Hani et al., 2008;Braak & Braak, 1984;Grodd et al., 2020;Hajdu, Hassler, & Somogyi, 1982;Hani et al., 2007;Hazlett et al., 1976;Pearson & Haines, 1980;Schaltenbrand & Wahen, 1977;Tseng & Royce, 1986). In all these studies, Golgi-type I nerve cells were found to have medium to large cell bodies and dense dendritic trees. In this study, Golgi-type I neuronal cell bodies were not seen to have any spines or appendages. These results are in line with similar findings reported in many previous studies (Al-Hussain, 1992;Babb, 1980;Pearson & Haines, 1980) but contradicted with the results reported in the centromedian-parafascicular complex of the monkey (Hazlett et al., 1976). The authors of the last study reported cell bodies of Golgi-type I neurons with spines and appendages. Like their counterparts in the human AV nucleus (Al-Hussain Bani Hani et al., 2008;Hani et al., 2007), the dendrites of these neurons have both radiating-tufted dendritic branching patterns but with no dendritic spines or hair-like appendages. A combined horseradish peroxidase (HRP) staining and electrophysiological study on the AV thalamic nucleus in cats identified relay, projection and interneurons (Yamamoto et al., 1985). The study distinguished three types of dendritic patterns (radial, tufted, and linear-oriented) in the relay and projection neurons. The first two patterns are in line with dendritic pattern in Golgi-type I neurons described in our present study and in other study conducted in cats (Kiss & Tömböl, 1972). Electrophysiological experiments confirmed that these neurons are relay neurons in the thalamus (Yamamoto et al., 1985).
Golgi-type II nerve cells were found to have both similarities and differences comparing with their counterparts already reported in other thalamic nuclei. Like Golgi-type II neurons described in other studies (Al-Hussain, 1992;Tömböl et al., 1969), these neurons were found in this study to have small to medium cell body. No somatic spines or appendages. This was in line with similar results reported in several species but contradicted with results reported in two previous studies (Al-Hussain, 1992;Hazlett et al., 1976) who reported such somatic spines and appendages. Golgi-type II nerve cells showed grape-like appendages. The grape-like appendage was found to be a part of complicated synaptic structures known as glomeruli that were mentioned in previous study (Morest, 1975). In those glomeruli, dendrites receive synapses from afferent fibers and make synapses on dendrites of Golgi-type I cells (Al-Hussain, 1992). The grape-like appendages in this nucleus were detected not only along the shaft of dendrites like those reported for the Golgi-type II cells in the motor thalamic nuclei of the cat (Somogyi et al., 1973;Tömböl et al., 1990;Tseng & Royce, 1986) and humans (Al-Hussain, 1992) but also at dendritic tips. Concerning the axons of Golgi-type II cells, F I G U R E 3 (a) Photomicrograph and (b) drawing of Golgi-type IIb neuron with grape-like appendages (long arrows) and spines (short arrows; X for drawing = 529). The arrows shown in the photomicrograph (a) indicate to two grape-like appendages shown on higher magnification in Figure 4 F I G U R E 4 Two photomicrographs of two grape-like appendages (arrows) of Golgi-type II neurons that shown in Figure 3a there were no branches found for the impregnated part. Whether or not these axons had any branches like the axons of Golgi-type II cell in the motor thalamic nuclei of the cat (Somogyi et al., 1973;Tömböl et al., 1990;Tseng & Royce, 1986), or those that found in the dorsal lateral geniculate body of the Macaca monkey (Wilson & Hendrickson, 1981) was difficult to determine. If these axons of the camel brain thalamic AV nucleus were really with no branches, this may make them functionally different from their counterparts in other thalamic nuclei mentioned above. Special interest is the absence of these branches in the long segment of the Golgi-type II axons that were impregnated in the human AV nucleus (Al-Hussain Bani Hani et al., 2008;Hani et al., 2007). Therefore, these axons might make synapses with cells away from their native cells within the AV nucleus itself or in other thalamic nuclei or they might also be projecting to areas out of the thalamus such as the cerebral cortex. In fact, some neurons in the lateral geniculate body with morphological features similar to Golgi-type II neurons were found to be projection neurons (Pearson & Haines, 1980). Our results will be an addition to our previous findings on the gracile and cuneate nuclei (Al-Hussain, Al-saffar, Zaqout, Al-Hussain, Al-Saffar, & El-Dwairi, 2012) and shed some light on the neuronal structures in different brain regions of the camel. Collectively, these findings illustrate part of the adaptive mechanisms developed in camel as one of the species living in hard climate conditions. This will open the door for further investigations using immunohistochemistry and electron microscopy techniques to dissect the neuronal subtypes in different brain regions of the camel.

ACKNOWLEDGMENTS
Open Access funding provided by the Qatar National Library

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.