Nuclear organization of cholinergic,catecholaminergic, serotonergic and orexinergic systems in the brain of the Tasmanian devil (Sarcophilus harrisii)

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brains of three male Tasmanian devils (Sarcophilus harrisii), which had a mean brain mass of 11.6 g. We found that the nuclei generally observed for these systems in other mammalian brains were present in the brain of the Tasmanian devil. Despite this, specific differences in the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems appear to carry a phylogenetic signal. In the cholinergic system, only the dorsal hypothalamic cholinergic nucleus could be observed, while an extra dorsal subdivision of the laterodorsal tegmental nucleus and cholinergic neurons within the gelatinous layer of the caudal spinal trigeminal nucleus were observed. Within the catecholaminergic system the A4 nucleus of the locus coeruleus complex was absent, as was the caudal ventrolateral serotonergic group of the serotonergic system. The organization of the orexinergic system was similar to that seen in many mammals previously studied. Overall, while showing strong similarities to the organization of these systems in other mammals, the specific differences observed in the Tasmanian devil reveal either order specific, or class specific, features of these systems. Further studies will reveal the extent of change in the nuclear organization of these systems in marsupials and how these potential changes may affect functionality.     

Nuclear organization of cholinergic,putative catecholaminergic and serotonergic nuclei in the brain of the eastern rock elephant shrew,Elephantulus myurus

The organization of the nuclear subdivisions of the cholinergic, putative catecholaminergic and serotonergic systems of the brain of the elephant shrew (Elephantulus myurus) were determined following immunohistochemistry for choline acetyltransferase, tyrosine hydroxylase and serotonin, respectively. This was done in order to determine if differences in the nuclear organization of these systems in comparison to other mammals were evident and how any noted differences may relate to specialized behaviours of the elephant shrew. The elephant shrew belongs to the order Macroscelidea, and forms part of the Afrotherian mammalian cohort. In general, the organization of the nuclei of these systems resembled that described in other mammalian species. The cholinergic system showed many features in common with that seen in the rock hyrax, rodents and primates; however, specific differences include: (1) cholinergic neurons were observed in the superior and inferior colliculi, as well as the cochlear nuclei; (2) cholinergic neurons were not observed in the anterior nuclei of the dorsal thalamus as seen in the rock hyrax; and (3) cholinergic parvocellular nerve cells forming subdivisions of the laterodorsal and pedunculopontine tegmental nuclei were not observed at the midbrain/pons interface as seen in the rock hyrax. The organization of the putative catecholaminergic system was very similar to that seen in the rock hyrax and rodents except for the lack of the rodent specific C3 nucleus, the dorsal division of the anterior hypothalamic group (A15d) and the compact division of the locus coeruleus (A6c). The nuclear organization of the serotonergic system was identical to that seen in all eutherian mammals studied to date. The additional cholinergic neurons found in the cochlear nucleus and colliculi may relate to a specific acoustic signalling system observed in elephant shrews expressed when the animals are under stress or detect a predator. These neurons may then function to increase attention to this type of acoustic signal termed foot drumming.     

Distribution and morphology of cholinergic,catecholaminergic and serotonergic neurons in the brain of Schreiber's long-fingered bat,Miniopterus schreibersii

The current study describes the nuclear parcellation and neuronal morphology of the cholinergic, catecholaminergic and serotonergic systems within the brain of a representative species of microbat. While these systems have been investigated in detail in the laboratory rat, and examined in several other mammalian species, no chiropterans, to the author's knowledge, have been examined. Using immunohistochemical stains for choline-acetyltransferase, tyrosine hydroxylase and serotonin, we were able to observe and document these systems in relation to the cytoarchitecture. The majority of cholinergic nuclei typically found in mammals were evident in the microbat, however we could not find evidence for choline-acetyltransferase immunopositive neurons in the Edinger–Westphal nucleus, parabigeminal nucleus, and the medullary tegmental field, as seen in several other mammalian species. A typically mammalian appearance of the catecholaminergic nuclei was observed, however, the anterior hypothalamic groups (A15 dorsal and ventral), the dorsal and dorsal caudal subdivisions of the ventral tegmental area (A10d and A10dc), and the ventral (pars reticulata) substantia nigra (A9v) were not present. The serotonergic nuclei were similar to that reported in all eutherian mammalian species studied to date. The overall complement of nuclei of these systems in the microbat, while different to the species examined in other orders of mammals, resembles most closely the complement seen in earlier studies of insectivore species, and is clearly distinguished from that seen in rodents, carnivores and primates. This data is discussed in terms of the phylogenetic relationships of the chiropterans.     

Distribution and morphology of cholinergic, catecholaminergic and serotonergic neurons in the brain of Schreiber's long-fingered bat, Miniopterus schreibersii

The current study describes the nuclear parcellation and neuronal morphology of the cholinergic, catecholaminergic and serotonergic systems within the brain of a representative species of microbat. While these systems have been investigated in detail in the laboratory rat, and examined in several other mammalian species, no chiropterans, to the author's knowledge, have been examined. Using immunohistochemical stains for choline-acetyltransferase, tyrosine hydroxylase and serotonin, we were able to observe and document these systems in relation to the cytoarchitecture. The majority of cholinergic nuclei typically found in mammals were evident in the microbat, however we could not find evidence for choline-acetyltransferase immunopositive neurons in the Edinger–Westphal nucleus, parabigeminal nucleus, and the medullary tegmental field, as seen in several other mammalian species. A typically mammalian appearance of the catecholaminergic nuclei was observed, however, the anterior hypothalamic groups (A15 dorsal and ventral), the dorsal and dorsal caudal subdivisions of the ventral tegmental area (A10d and A10dc), and the ventral (pars reticulata) substantia nigra (A9v) were not present. The serotonergic nuclei were similar to that reported in all eutherian mammalian species studied to date. The overall complement of nuclei of these systems in the microbat, while different to the species examined in other orders of mammals, resembles most closely the complement seen in earlier studies of insectivore species, and is clearly distinguished from that seen in rodents, carnivores and primates. This data is discussed in terms of the phylogenetic relationships of the chiropterans.     

Distribution and morphology of putative catecholaminergic and serotonergic neurons in the medulla oblongata of a sub-adult giraffe,Giraffa camelopardalis

The current study details the nuclear parcellation and appearance of putative catecholaminergic and serotonergic neurons within the medulla oblongata of a sub-adult giraffe, using immunohistochemistry for tyrosine hydroxylase and serotonin. We hypothesized that the unusual phenotype of the giraffe, this being the long neck and potential axonal lengthening of these neurons, may pose specific problems in terms of the efficient functioning of these systems, as several groups of catecholaminergic and serotonergic neurons, especially of the medulla, are known to project to the entire spinal cord. This specific challenge may lead to observable differences in the nuclear parcellation and morphology of these systems in the giraffe. Our personal observations in the giraffe reveal that, as with other Artiodactyls, the spinal cord extends to the caudal end of the sacral vertebrae. Within the giraffe medulla we found evidence for five putative catecholaminergic (neurons containing tyrosine hydroxylase) and five serotonergic nuclei. In terms of both morphological appearance of the neurons and nuclear parcellation we did not find any evidence for features that may be considered affected by the phenotype of the giraffe. The nuclear parcellation and appearance of both the putative catecholaminergic and serotonergic systems in the medulla of the giraffe studied are strikingly similar to that seen in previous studies of other Artiodactyls. We interpret these findings in terms of a growing literature detailing order specific phylogenetic constraints in the evolution of these neuromodulatory systems.     

Distribution and morphology of putative catecholaminergic and serotonergic neurons in the medulla oblongata of a sub-adult giraffe, Giraffa camelopardalis

The current study details the nuclear parcellation and appearance of putative catecholaminergic and serotonergic neurons within the medulla oblongata of a sub-adult giraffe, using immunohistochemistry for tyrosine hydroxylase and serotonin. We hypothesized that the unusual phenotype of the giraffe, this being the long neck and potential axonal lengthening of these neurons, may pose specific problems in terms of the efficient functioning of these systems, as several groups of catecholaminergic and serotonergic neurons, especially of the medulla, are known to project to the entire spinal cord. This specific challenge may lead to observable differences in the nuclear parcellation and morphology of these systems in the giraffe. Our personal observations in the giraffe reveal that, as with other Artiodactyls, the spinal cord extends to the caudal end of the sacral vertebrae. Within the giraffe medulla we found evidence for five putative catecholaminergic (neurons containing tyrosine hydroxylase) and five serotonergic nuclei. In terms of both morphological appearance of the neurons and nuclear parcellation we did not find any evidence for features that may be considered affected by the phenotype of the giraffe. The nuclear parcellation and appearance of both the putative catecholaminergic and serotonergic systems in the medulla of the giraffe studied are strikingly similar to that seen in previous studies of other Artiodactyls. We interpret these findings in terms of a growing literature detailing order specific phylogenetic constraints in the evolution of these neuromodulatory systems.     

Nuclear organization of some immunohistochemically identifiable neural systems in two species of the Euarchontoglires: A Lagomorph,Lepus capensis,and a Scandentia,Tupaia belangeri

The present study describes the organization of the nuclei of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of two members of Euarchontoglires, Lepus capensis and Tupaia belangeri. The aim of the present study was to investigate the nuclear complement of these neural systems in comparison to previous studies on Euarchontoglires and generally with other mammalian species. Brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The majority of nuclei revealed in the current study were similar between the species investigated and to mammals generally, but certain differences in the nuclear complement highlight potential phylogenetic interrelationships within the Euarchontoglires and across mammals. In the northern tree shrew the nucleus of the trapezoid body contained neurons immunoreactive to the choline acetyltransferase antibody with some of these neurons extending into the lamellae within the superior olivary nuclear complex (SON). The cholinergic nature of the neurons of this nucleus, and the extension of cholinergic neurons into the SON, has not been noted in any mammal studied to date. In addition, cholinergic neurons forming the medullary tegmental field were also present in the northern tree shrew. Regarding the catecholaminergic system, the cape hare presented with the rodent specific rostral dorsal midline medullary nucleus (C3), and the northern tree shrew lacked both the ventral and dorsal divisions of the anterior hypothalamic group (A15v and A15d). Both species were lacking the primate/megachiropteran specific compact portion of the locus coeruleus complex (A6c). The nuclei of the serotonergic and orexinergic systems of both species were similar to those seen across most Eutherian mammals. Our results lend support to the monophyly of the Glires, and more broadly suggest that the megachiropterans are more closely related to the primates than are any other members of Euarchontoglires studied to date.     

Distribution of orexin-A immunoreactive neurons and their terminal networks in the brain of the rock hyrax, Procavia capensis

The present study describes the distribution of orexin-A immunoreactive neurons and terminal networks in relation to the previously described catecholaminergic, cholinergic and serotonergic systems within the brain of the rock hyrax, Procavia capensis. Adult female rock hyrax brains were sectioned and immunohistochemically stained with an antibody to orexin-A. The staining revealed that the neurons were mainly located within the hypothalamus as with other mammals. The orexinergic terminal network distribution also resembled the typical mammalian plan. High-density orexinergic terminal networks were located within regions of the diencephalon (e.g. paraventricular nuclei), midbrain (e.g. serotonergic nuclei) and pons (locus coeruleus), while medium density orexinergic terminal networks were evident in the telencephalic (e.g. basal forebrain), diencephalic (e.g. hypothalamus), midbrain (e.g. periaqueductal gray matter), pontine (e.g. serotonergic nuclei) and medullary regions (e.g. serotonergic and catecholaminergic nuclei). Although the distribution of the orexinergic terminal networks was typically mammalian, the rock hyrax did show one atypical feature, the presence of a high-density orexinergic terminal network within the anterodorsal nucleus of the dorsal thalamus (AD). The dense orexinergic innervation of the AD nucleus has only been reported previously in the Nile grass rat, Arvicanthis niloticus and Syrian hamster, Mesocricetus auratus, both diurnal mammals. It is possible that orexinergic innervation of the AD nucleus might be a unique feature associated with diurnal mammals. It was also noted that the dense orexinergic innervation of the AD nucleus coincided with previously identified cholinergic neurons and terminal networks in this particular nucleus of the rock hyrax brain. It is possible that this dense orexinergic innervation of the AD nucleus in the brain of the rock hyrax may act in concert with the cholinergic neurons and/or the cholinergic axonal terminals, which in turn may influence arousal states and motivational processing.     

Mechanothermal nociceptors in the scaly skin of the chicken leg

Corticotropin-releasing hormone (CRH) interacts with noradrenergic, dopaminergic and cholinergic systems of the brain, and these interactions are thought to be of relevance for the stress response, anxiety-related behavior, and cognitive function. CRH mediates its central effects through two high-affinity membrane receptors, CRH receptor subtypes 1 and 2. It is however unclear at present whether cholinergic or catecholaminergic cells express these receptors themselves or whether the effects of CRH are indirectly mediated through interaction with other neurotransmitter systems. Therefore, this study investigated whether choline acetyltransferase immunoreactive neurons of the murine basal forebrain and brainstem nuclei, and tyrosine hydroxylase immunoreactive neurons located within the locus coeruleus, ventral tegmental area and substantia nigra co-express CRH receptor 1, employing a double-immunocytochemical procedure. Using an antibody against the C-terminus of the CRH type 1 receptor (CRH-R1), CRH-R1-like immunoreactivity was found in all cholinergic basal forebrain nuclei except the nucleus basalis magnocellularis. In particular, the diagonal band of Broca (vertical and horizontal limbs) showed a high degree of co-localization of CRH-R1 immunoreactivity and choline acetyltransferase immunoreactivity (both limbs >90%). A less intense immunoreactivity but still high rate of co-localization was detected in the cholinergic neurons of the medial septum (80%), while lowest co-localization was observed in choline acetyltransferase immunoreactive neurons of the substantia innominata (58%). An intermediate degree of co-localization (75%) was seen in the brainstem pedunculopontine tegmental nucleus, while the other major brainstem cholinergic nucleus, the laterodorsal tegmental nucleus, showed an even higher degree of choline acetyltransferase immunoreactivity-positive cells also immunoreactive for CRH-R1 (92%). All catecholaminergic structures studied displayed a pattern of CRH-R1 immunoreactivity strongly overlapping the pattern of tyrosine hydroxylase immunoreactivity. The intensity of the CRH-R1 signal was relatively low within the ventral tegmental area and the substantia nigra pars compacta, while the CRH-R1 signal was very intense and detected in almost all of the neurons of the locus coeruleus.These results clearly demonstrate that the cholinergic and catecholaminergic systems provide direct anatomical substrates for CRH action through the CRH-R1. These findings are of particular relevance for understanding the action of recently developed CRH-R1 antagonistic drugs which may offer a new therapeutic approach to treat stress-related disorders such as anxiety and depression and their concomitant alterations in arousal and cognitive functions.     

The organization of the brainstem and spinal cord of the mouse: relationships between monoaminergic, cholinergic, and spinal projection systems

Information regarding the organization of the CNS in terms of neurotransmitter systems and spinal connections in the mouse is sparse, especially at the level of the brainstem. An overview is presented of monoaminergic and cholinergic systems in the brainstem and spinal cord that were visualized immunohistochemically in inbred C57BL/6 and outbred CD-1 mice. This information is complemented with data on spinal cord-projecting systems that were characterized using retrograde tracing, spinal hemisections, and double labeling techniques. Attention is given to differences in these systems related to spinal levels. The data are discussed with reference to studies in the rat, and to standardized information as provided in the atlas of the mouse brain. Although the overall organization of these systems in the mouse is largely similar to those in the rat, species differences are present in relative location, size and/or connectivity of cell groups. For example, catecholaminergic neurons in the (ventro)lateral pons (A5 and A7 cell groups) in the mouse project to the spinal cord mainly via contralateral, and not ipsilateral, pathways. The data further supplement information as provided in standardized brainstem sections of the C57BL/6 mouse [Paxinos, G., Franklin, K.B.J., 2001. The mouse brain in stereotaxic coordinates. Academic Press, San Diego], especially with respect to the size and/or location of the catecholaminergic retrorubral field (A8 group), A5, A1, and C1 cell groups, and the serotonergic B4 group, reticulotegmental nucleus (B9 group), lateral paragigantocellular nucleus and raphe magnus nucleus (B3 group). Altogether this study provides a comprehensive overview of the spatial relationships of neurochemically and anatomically defined neuronal systems in the mouse brainstem and spinal cord.     

Nuclear organization and morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brain of the Cape porcupine (Hystrix africaeaustralis): increased brain size does not lead to increased organizational complexity

The distribution, morphology and nuclear organization of the cholinergic, putative catecholaminergic and serotonergic systems within the brain of the Cape porcupine (Hystrix africaeaustralis) were identified following immunohistochemistry for choline acetyltransferase, tyrosine hydroxylase and serotonin. The aim of the present study was to investigate possible differences in the complement of nuclear subdivisions of these systems in the Cape porcupine in comparison with previous studies of these systems in other rodents. The Cape porcupine is the largest rodent in which these systems have been examined and has an adult body mass of 10-24kg and an average brain mass of approximately 37g, around 15 times larger than the laboratory rat. The Cape porcupines were taken from the wild and while these differences, especially that of mass, may lead to the prediction of a significant difference in the nuclear organization or number within these systems, all the nuclei observed in all three systems in the laboratory rat and in other rodents had direct homologues in the brain of the Cape porcupine. Moreover, there were no additional nuclei in the brain of the Cape porcupine that are not found in the laboratory rat or other rodents studied and vice versa. It is noted that the medial septal nucleus of the Cape porcupine appeared qualitatively to have a reduced number of neurons in comparison to the laboratory rat and other rodents. The locus coeruleus of the laboratory rat differs in location to that observed for the Cape porcupine and several other rodent species. The Cape porcupine is distantly related to the laboratory rat, but still a member of the order Rodentia; thus, changes in the organization of these systems appears to demonstrate a form of constraint related to the phylogenetic level of the order.     

Low expression of extracellular matrix components in rat brain stem regions containing modulatory aminergic neurons

Extracellular matrix proteoglycans, particularly those accumulated in perineuronal nets (PNs), have been shown to form characteristic distribution patterns in cortical and subcortical regions of adult mammals. Their involvement in sustaining mechanisms that are especially related to fast activities of neurons has been discussed as one of the possible functions. The present study deals with the spatial organization of extracellular matrix proteoglycans in brain stem regions that contain aminergic neurons, such as substantia nigra, ventral tegmental area (VTA), raphe nuclei and locus coeruleus (LC). As these nuclei are known to influence brain activity by modulatory functions exerting patterns of slow electric activity, it could be expected that PNs would be absent around aminergic cells. The staining of PNs with Wisteria floribunda agglutinin (WFA) was combined with the detection of catecholaminergic neurons by tyrosine hydroxylase immunoreactivity and of serotonergic neurons by tryptophan hydroxylase (TH) immunoreactivity using double fluorescence microscopy. It was found that the catecholaminergic and serotonergic neurons in the nuclear accumulations, as well as those scattered in adjacent regions, were not ensheathed by PNs. In contrast, several non-aminergic neurons intermingled with aminergic neurons in the raphe nuclei, in the substantia nigra pars compacta (SNC) and in the VTA, as well as many cells in the reticular part of the substantia nigra, were found to be surrounded by PNs. It can be concluded from these results that the absence of PNs around aminergic brain stem neurons, also previously shown for cholinergic basal forebrain neurons, appears as a characteristic feature common to cells that exert slow modulatory functions.     

Nuclear organization of cholinergic, putative catecholaminergic, serotonergic and orexinergic systems in the brain of the African pygmy mouse (Mus minutoides): organizational complexity is preserved in small brains

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brain of the African pygmy mouse (Mus minutoides). The African pygmy mice studied had a brain mass of around 275 mg, making these the smallest rodent brains to date in which these neural systems have been investigated. In contrast to the assumption that in this small brain there would be fewer subdivisions of these neural systems, we found that all nuclei generally observed for these systems in other rodent brains were also present in the brain of the African pygmy mouse. As with other rodents previously studied in the subfamily Murinae, we observed the presence of cortical cholinergic neurons and a compactly organized locus coeruleus. These two features of these systems have not been observed in the non-Murinae rodents studied to date. Thus, the African pygmy mouse displays what might be considered a typical Murinae brain organization, and despite its small size, the brain does not appear to be any less complexly organized than other rodent brains, even those that are over 100 times larger such as the Cape porcupine brain. The results are consistent with the notion that changes in brain size do not affect the evolution of nuclear organization of complex neural systems. Thus, species belonging to the same order generally have the same number and complement of the subdivisions, or nuclei, of specific neural systems despite differences in brain size, phenotype or time since evolutionary divergence.     

Organization of cholinergic,catecholaminergic, serotonergic and orexinergic nuclei in three strepsirrhine primates: Galago demidoff,Perodicticus potto and Lemur catta

The nuclear organization of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of three species of strepsirrhine primates is presented. We aimed to investigate the nuclear complement of these neural systems in comparison to those of simian primates, megachiropterans and other mammalian species. The brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The nuclei identified were identical among the strepsirrhine species investigated and identical to previous reports in simian primates. Moreover, a general similarity to other mammals was found, but specific differences in the nuclear complement highlighted potential phylogenetic interrelationships. The central feature of interest was the structure of the locus coeruleus complex in the primates, where a central compactly packed core (A6c) of tyrosine hydroxylase immunopositive neurons was surrounded by a shell of less densely packed (A6d) tyrosine hydroxylase immunopositive neurons. This combination of compact and diffuse divisions of the locus coeruleus complex is only found in primates and megachiropterans of all the mammalian species studied to date. This neural character, along with variances in a range of other neural characters, supports the phylogenetic grouping of primates with megachiropterans as a sister group.     

Nuclear organization of catecholaminergic neurons in the brains of a lar gibbon and a chimpanzee

Using tyrosine hydroxylase immunohistochemistry, we describe the nuclear parcellation of the catecholaminergic system in the brains of a lar gibbon (Hylobates lar) and a chimpanzee (Pan troglodytes). The parcellation of catecholaminergic nuclei in the brains of both apes is virtually identical to that observed in humans and shows very strong similarities to that observed in mammals more generally, particularly other primates. Specific variations of this system in the apes studied include an unusual high-density cluster of A10dc neurons, an enlarged retrorubral nucleus (A8), and an expanded distribution of the neurons forming the dorsolateral division of the locus coeruleus (A4). The additional A10dc neurons may improve dopaminergic modulation of the extended amygdala, the enlarged A8 nucleus may be related to the increased use of communicative facial expressions in the hominoids compared to other primates, while the expansion of the A4 nucleus appears to be related to accelerated evolution of the cerebellum in the hominoids compared to other primates. In addition, we report the presence of a compact division of the locus coeruleus proper (A6c), as seen in other primates, that is not present in other mammals apart from megachiropteran bats. The presence of this nucleus in primates and megachiropteran bats may reflect homology or homoplasy, depending on the evolutionary scenario adopted. The fact that the complement of homologous catecholaminergic nuclei is mostly consistent across mammals, including primates, is advantageous for the selection of model animals for the study of specific dysfunctions of the catecholaminergic system in humans.     

Distribution and morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brain of the Egyptian rousette flying fox, Rousettus aegyptiacus

Over the past decade much controversy has surrounded the hypothesis that the megachiroptera, or megabats, share unique neural characteristics with the primates. These observations, which include similarities in visual pathways, have suggested that the megabats are more closely related to the primates than to the other group of the Chiropteran order, the microbats, and suggests a diphyletic origin of the Chiroptera. To contribute data relevant to this debate, we used immunohistochemical techniques to reveal the architecture of the neuromodulatory systems of the Egyptian rousette (Rousettus aegypticus), an echolocating megabat. Our findings revealed many similarities in the nuclear parcellation of the cholinergic, putative catecholaminergic and serotonergic systems with that seen in other mammals including the microbat. However, there were 11 discrete nuclei forming part of these systems in the brain of the megabat studied that were not evident in an earlier study of a microbat. The occurrence of these nuclei align the megabat studied more closely with primates than any other mammalian group and clearly distinguishes them from the microbat, which aligns with the insectivores. The neural systems investigated are not related to such Chiropteran specializations as echolocation, flight, vision or olfaction. If neural characteristics are considered strong indicators of phylogenetic relationships, then the data of the current study strongly supports the diphyletic origin of Chiroptera and aligns the megabat most closely with primates in agreement with studies of other neural characters.     

A quantitative study of the brainstem cholinergic projections to the ventral part of the oral pontine reticular nucleus (REM sleep induction site) in the cat

The ventral part of the cat oral pontine reticular nucleus (vRPO) is the site in which microinjections of small dose and volume of cholinergic agonists produce long-lasting rapid eye movement sleep with short latency. The present study determined the precise location and proportions of the cholinergic brainstem neuronal population that projects to the vRPO using a double-labeling method that combines the neuronal tracer horseradish peroxidase–wheat germ agglutinin with choline acetyltransferase immunocytochemistry in cats. Our results show that 88.9% of the double-labeled neurons in the brainstem were located, noticeably bilaterally, in the cholinergic structures of the pontine tegmentum. These neurons occupied not only the pedunculopontine and laterodorsal tegmental nuclei, which have been described to project to other pontine tegmentum structures, but also the locus ceruleus complex principally the locus ceruleus and peri-, and the parabrachial nuclei. Most double-labeled neurons were found in the pedunculopontine tegmental nucleus and locus ceruleus complex and, much less abundantly, in the laterodorsal tegmental nucleus and the parabrachial nuclei. The proportions of these neurons among all choline acetyltransferase positive neurons within each structure were highest in the locus ceruleus complex, followed in descending order by the pedunculopontine and laterodorsal tegmental nuclei and then, the parabrachial nuclei. The remaining 11.1% of double-labeled neurons were found bilaterally in other cholinergic brainstem structures: around the oculomotor, facial and masticatory nuclei, the caudal pontine tegmentum and the praepositus hypoglossi nucleus. The disperse origins of the cholinergic neurons projecting to the vRPO, in addition to the abundant noncholinergic afferents to this nucleus may indicate that cholinergic stimulation is not the only or even the most decisive event in the generation of REM sleep.     

Localisation of GABA(A) receptor epsilon-subunit in cholinergic and aminergic neurones and evidence for co-distribution with the theta-subunit in rat brain

In situ hybridisation and immunohistochemical methodologies suggest the existence of a large diversity of GABA(A) receptor subtypes in the brain. These are hetero-oligomeric proteins modulated by a number of clinically important drugs, depending on their subunit composition. We recently cloned and localised the rat GABA(A) receptor epsilon-subunit by in situ hybridisation and immunohistochemical procedures. Here, in a dual-labelling immunohistochemical study in the rat brain, we used our affinity-purified antiserum to epsilon with antisera to markers of cholinergic, catecholaminergic, and serotonergic neurones. As far as cholinergic systems were concerned, epsilon-immunoreactivity was expressed in all forebrain cell-groups, as well as in the caudal lateral pontine tegmentum and dorsal motor nucleus of the vagus nerve. As far as dopaminergic systems were concerned, epsilon-immunoreactivity was found to be expressed in a great number of hypothalamic cell-groups (A15, A14 and A12) and in the substantia nigra pars compacta. The noradrenergic, and to a lesser extent, adrenergic cell-groups were all epsilon-immunoreactive. Also, epsilon-immunoreactivity was detected in all serotonergic cell-groups. We also revealed by in situ hybridisation in a monkey brain that epsilon mRNA was expressed in the locus coeruleus, as previously observed in rats. Finally, by using in situ hybridisation in rat brains, we compared the distribution of the mRNA of epsilon with that of the recently cloned theta-subunit of the GABA(A) receptor. Both subunits showed strikingly overlapping expression patterns throughout the brain, especially in the septum, preoptic areas, various hypothalamic nuclei, amygdala, and thalamus, as well as the aforementioned monoaminergic cell-groups. No theta-mRNA signals were detected in cholinergic cell-groups.Taken together with previously published evidence of the presence of the alpha3-subunit in monoamine- or acetylcholine-containing systems, our data suggest the existence of novel GABA(A) receptors comprising alpha3/epsilon in cholinergic and alpha3/theta/epsilon in monoaminergic cell-groups.