Increased number of excitatory synapsis and decreased number of inhibitory synapsis in the prefrontal cortex in autism.

Previous studies in autism spectrum disorder demonstrated an increased number of excitatory pyramidal cells and a decreased number of inhibitory parvalbumin+ chandelier interneurons in the prefrontal cortex of postmortem brains. How these changes in cellular composition affect the overall abundance of excitatory and inhibitory synapses in the cortex is not known. Herein, we quantified the number of excitatory and inhibitory synapses in the prefrontal cortex of 10 postmortem autism spectrum disorder brains and 10 control cases. To identify excitatory synapses, we used VGlut1 as a marker of the presynaptic component and postsynaptic density protein-95 as marker of the postsynaptic component. To identify inhibitory synapses, we used the vesicular gamma-aminobutyric acid transporter as a marker of the presynaptic component and gephyrin as a marker of the postsynaptic component. We used Puncta Analyzer to quantify the number of co-localized pre- and postsynaptic synaptic components in each area of interest. We found an increase in the number of excitatory synapses in upper cortical layers and a decrease in inhibitory synapses in all cortical layers in autism spectrum disorder brains compared with control cases. The alteration in the number of excitatory and inhibitory synapses could lead to neuronal dysfunction and disturbed network connectivity in the prefrontal cortex in autism spectrum disorder.

The distribution, number, and certain neurochemical identities of infracortical white matter neurons in the brains of a southern lesser galago, a black-capped squirrel monkey, and a crested macaque.

In the current study, we examined the number, distribution, and aspects of the neurochemical identities of infracortical white matter neurons, also termed white matter interstitial cells (WMICs), in the brains of a southern lesser galago (Galago moholi), a black-capped squirrel monkey (Saimiri boliviensis boliviensis), and a crested macaque (Macaca nigra). Staining for neuronal nuclear marker (NeuN) revealed WMICs throughout the infracortical white matter, these cells being most dense close to inner cortical border, decreasing in density with depth in the white matter. Stereological analysis of NeuN-immunopositive cells revealed estimates of approximately 1.1, 10.8, and 37.7 million WMICs within the infracortical white matter of the galago, squirrel monkey, and crested macaque, respectively. The total numbers of WMICs form a distinct negative allometric relationship with brain mass and white matter volume when examined in a larger sample of primates where similar measures have been obtained. In all three primates studied, the highest densities of WMICs were in the white matter of the frontal lobe, with the occipital lobe having the lowest. Immunostaining revealed significant subpopulations of WMICs containing neuronal nitric oxide synthase (nNOS) and calretinin, with very few WMICs containing parvalbumin, and none containing calbindin. The nNOS and calretinin immunopositive WMICs represent approximately 21% of the total WMIC population; however, variances in the proportions of these neurochemical phenotypes were noted. Our results indicate that both the squirrel monkey and crested macaque might be informative animal models for the study of WMICs in neurodegenerative and psychiatric disorders in humans.

The distribution, number, and certain neurochemical identities of infracortical white matter neurons in a chimpanzee (Pan troglodytes) brain.

We examined the number, distribution, and immunoreactivity of the infracortical white matter neuronal population, also termed white matter interstitial cells (WMICs), throughout the telencephalic white matter of an adult female chimpanzee. Staining for neuronal nuclear marker (NeuN) revealed WMICs throughout the infracortical white matter, these cells being most numerous and dense close to the inner border of cortical layer VI, decreasing significantly in density with depth in the white matter. Stereological analysis of NeuN-immunopositive cells revealed an estimate of approximately 137.2 million WMICs within the infracortical white matter of the chimpanzee brain studied. Immunostaining revealed subpopulations of WMICs containing neuronal nitric oxide synthase (nNOS, approximately 14.4 million in number), calretinin (CR, approximately 16.7 million), very few WMICs containing parvalbumin (PV), and no calbindin-immunopositive neurons. The nNOS, CR, and PV immunopositive WMICs, possibly all inhibitory neurons, represent approximately 22.6% of the total WMIC population. As the white matter is affected in many cognitive conditions, such as schizophrenia, autism, epilepsy, and also in neurodegenerative diseases, understanding these neurons across species is important for the translation of findings of neural dysfunction in animal models to humans. Furthermore, studies of WMICs in species such as apes provide a crucial phylogenetic context for understanding the evolution of these cell types in the human brain.

The distribution, number, and certain neurochemical identities of infracortical white matter neurons in a lar gibbon (Hylobates lar) brain.

We examined the number, distribution, and immunoreactivity of the infracortical white matter neuronal population, also termed white matter interstitial cells (WMICs), in the brain of a lesser ape, the lar gibbon. Staining for neuronal nuclear marker (NeuN) revealed WMICs throughout the infracortical white matter, these cells being most numerous and dense close to cortical layer VI, decreasing significantly in density with depth in the white matter. Stereological analysis of NeuN-immunopositive cells revealed a global estimate of ~67.5 million WMICs within the infracortical white matter of the gibbon brain, indicating that the WMICs are a numerically significant population, ~2.5% of the total cortical gray matter neurons that would be estimated for a primate brain the mass of that of the lar gibbon. Immunostaining revealed subpopulations of WMICs containing neuronal nitric oxide synthase (nNOS, ~7 million in number, with both small and large soma volumes), calretinin (~8.6 million in number, all of similar soma volume), very few WMICs containing parvalbumin, and no calbindin-immunopositive neurons. These nNOS, calretinin, and parvalbumin immunopositive WMICs, presumably all inhibitory neurons, represent ~23.1% of the total WMIC population. As the white matter is affected in many cognitive conditions, such as schizophrenia, autism and also in neurodegenerative diseases, understanding these neurons across species is important for the translation of findings of neural dysfunction in animal models to humans. Furthermore, studies of WMICs in species such as apes provide a crucial phylogenetic context for understanding the evolution of these cell types in the human brain.

Nuclear organization of the African elephant (Loxodonta africana) amygdaloid complex: an unusual mammalian amygdala.

Here we describe the nuclear organization of the African elephant amygdaloid complex using Nissl, myelin, and a range of immunohistochemical stains. The African elephant is thought to exhibit many affect-laden and social-empathic behaviours; however, to date the amygdaloid complex, which is the generator of emotional states of the brain is yet to be fully explored in the elephants. For the most part, the amygdaloid complex of the African elephant is similar to that observed in other mammals in terms of the presence of nuclei and their topological relationships; however, we did observe several specific differences in amygdaloid organization. The elephant amygdala has undergone rotation in both the coronal and sagittal planes, seemingly associated with the expansion of the temporal lobe. Numerous scalloped cell clusters, termed glomeruli, forming the intermediate nuclei of the basal, accessory basal and central nuclear groups, were occupied by structures immunopositive to doublecortin. The nuclei typically associated with the accessory olfactory system (posterior cortical nucleus and medial nuclear complex) were absent from the elephant amygdala. The anterior cortical nucleus is very large and appears to be comprised of two subdivisions. The lateral nuclear complex is expanded and has two novel subdivisions. The amygdalohippocampal area appears relatively enlarged. The numerous shared and derived characters make the elephant amygdaloid complex very unusual and unique amongst mammals, but the derived characters appear to relate to observed elephant affect-laden behaviours.

Nuclear organisation of cholinergic, catecholaminergic, serotonergic and orexinergic neurons in two relatively large-brained rodent species-The springhare (Pedetes capensis) and Beecroft's scaly-tailed squirrel (Anomalurus beecrofti).

The present study describes the nuclear organization of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of the springhare and Beecroft's scaly-tailed squirrel following immunohistochemical labelling. We aimed to investigate any differences in the nuclear organization of these neural systems when compared to previous data on other species of rodents, as these two rodent species have relatively large brains - 1.2 to 1.4 times larger than would be expected for mammals of their body mass and 1.7-1.9 times larger than would be expected for rodents of their body mass. A series of coronal sections were taken through two brains of each species and immunohistochemically labelled with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. Generally, the nuclear complement of these systems revealed extensive similarities between both species and to previously studied rodents. While no differences were observed in the nuclear complement of the serotonergic and orexinergic systems, some differences were observed in the nuclear complement of the cholinergic and catecholaminergic systems. These include the presence of cholinergic neurons in the cerebral cortex and nucleus of the trapezoid body in the springhare; while the Beecroft's scaly-tailed squirrel exhibited cholinergic neurons in the pretectal area of the midbrain. For the catecholaminergic system it was observed that Beecroft's scaly-tailed squirrel possessed immunoreactive neurons in the accessory olfactory bulb. Despite these four differences, most not previously observed in rodents, the remaining complement of cholinergic and catecholaminergic nuclei were identical to that observed in other rodents, including the presence of the rodent specific catecholaminergic rostral dorsal midline medullary (C3) nucleus in the medulla oblongata. Thus, even with a significant increase in relative brain size, the overall complement of nuclei forming these systems shows minimal changes in complexity within a specific mammalian order.

Nuclear organization of the rock hyrax (Procavia capensis) amygdaloid complex.

The current study details the nuclear organization of the rock hyrax amygdaloid complex using both Nissl and myelin stains, along with a range of immunohistochemical stains. The rock hyrax appears to be the least derived of the Afrotherians, a group with a huge range of body phenotypes, life histories and specialized behaviours, brain sizes, and ecological niches. In this sense, the rock hyrax represents a species where the organization of the amygdaloid complex may be reflective of that in stem Eutherian mammals. Our analysis indicates that the nuclear organization of the rock hyrax amygdaloid complex is indeed very similar to that in other mammals studied, with four major nuclear groupings (the deep or basolateral group; the superficial or cortical-like or corticomedial group; the centromedial group; and the other amygdaloid nuclei) being observed, which is typical of Eutherian mammals. Moreover, each of these groupings is composed of several nuclei, the vast majority of which were readily identified in the rock hyrax. Small nuclei identified in rodents and primates were absent in the superficial and centromedial groups, seemingly involved with olfaction. A novel shell-like nucleus of the accessory basal nuclear cluster was observed in the rock hyrax, again, likely to be involved in olfaction. The current study underlines the conserved nature of nuclear parcellation in the Eutherian mammal amygdaloid complex and indicates that across most species, the flow of information processing related to species-specific affective-laden stimuli and the resultant physiological and behavioural outcomes are likely to be similar across species.

The mummified brain of a pleistocene woolly mammoth (Mammuthus primigenius) compared with the brain of the extant African elephant (Loxodonta africana).

This study presents the results of an examination of the mummified brain of a pleistocene woolly mammoth (Mammuthus primigenius) recovered from the Yakutian permafrost in Siberia, Russia. This unique specimen (from 39,440-38,850 years BP) provides the rare opportunity to compare the brain morphology of this extinct species with a related extant species, the African elephant (Loxodonta africana). An anatomical description of the preserved brain of the woolly mammoth is provided, along with a series of quantitative analyses of various brain structures. These descriptions are based on visual inspection of the actual specimen as well as qualitative and quantitative comparison of computed tomography imaging data obtained for the woolly mammoth in comparison with magnetic resonance imaging data from three African elephant brains. In general, the brain of the woolly mammoth specimen examined, estimated to weigh between 4,230 and 4,340 g, showed the typical shape, size, and gross structures observed in extant elephants. Quantitative comparative analyses of various features of the brain, such as the amygdala, corpus callosum, cerebellum, and gyrnecephalic index, all indicate that the brain of the woolly mammoth specimen examined has many similarities with that of modern African elephants. The analysis provided here indicates that a specific brain type representative of the Elephantidae is likely to be a feature of this mammalian family. In addition, the extensive similarities between the woolly mammoth brain and the African elephant brain indicate that the specializations observed in the extant elephant brain are likely to have been present in the woolly mammoth.

Comparative neuronal morphology of the cerebellar cortex in afrotherians, carnivores, cetartiodactyls, and primates.

Although the basic morphological characteristics of neurons in the cerebellar cortex have been documented in several species, virtually nothing is known about the quantitative morphological characteristics of these neurons across different taxa. To that end, the present study investigated cerebellar neuronal morphology among eight different, large-brained mammalian species comprising a broad phylogenetic range: afrotherians (African elephant, Florida manatee), carnivores (Siberian tiger, clouded leopard), cetartiodactyls (humpback whale, giraffe) and primates (human, common chimpanzee). Specifically, several neuron types (e.g., stellate, basket, Lugaro, Golgi, and granule neurons; N = 317) of the cerebellar cortex were stained with a modified rapid Golgi technique and quantified on a computer-assisted microscopy system. There was a 64-fold variation in brain mass across species in our sample (from clouded leopard to the elephant) and a 103-fold variation in cerebellar volume. Most dendritic measures tended to increase with cerebellar volume. The cerebellar cortex in these species exhibited the trilaminate pattern common to all mammals. Morphologically, neuron types in the cerebellar cortex were generally consistent with those described in primates (Fox et al., 1967) and rodents (Palay and Chan-Palay, 1974), although there was substantial quantitative variation across species. In particular, Lugaro neurons in the elephant appeared to be disproportionately larger than those in other species. To explore potential quantitative differences in dendritic measures across species, MARSplines analyses were used to evaluate whether species could be differentiated from each other based on dendritic characteristics alone. Results of these analyses indicated that there were significant differences among all species in dendritic measures.

Architectural organization of the african elephant diencephalon and brainstem.

The current study examined the organization of the diencephalon and brainstem of the African elephant (Loxodonta africana) - a region of the elephant brain that has not been examined for at least 50 years. The current description, employing material amenable for use with modern neuroanatomical methods, shows that, for the most part, the elephant diencephalon and brainstem are what could be considered typically mammalian, with subtle differences in proportions and topology. The variations from these previous descriptions, where they occurred, were related to four specific aspects of neural information processing: (1) the motor systems, (2) the auditory and vocalization systems, (3) the orexinergic satiety/wakefulness centre of the hypothalamus and the locus coeruleus, and (4) the potential neurogenic lining of the brainstem. For the motor systems, three specific structures exhibited interesting differences in organization - the pars compacta of the substantia nigra, the facial motor nerve nucleus, and the inferior olivary nuclear complex, all related to the timing and learning of movements and likely related to the control of the trunk. The dopaminergic neurons of the substantia nigra appear to form distinct islands separated from each other by large fibre pathways, an appearance unique to the elephant. Each island may send topologically organized projections to the striatum forming a dopaminergic innervation mosaic that may relate to trunk movements. At least five regions of the combined vocalization production and auditory/seismic reception system were specialized, including the large and distinct nucleus ellipticus of the periaqueductal grey matter, the enlarged lateral superior olivary nucleus, the novel transverse infrageniculate nucleus of the dorsal thalamus, the enlarged dorsal column nuclei and the ventral posterior inferior nucleus of the dorsal thalamus. These specializations, related to production and reception of infrasound, allow the proposal of a novel concept regarding the reception and localization of infrasonic sources. The orexinergic system of the hypothalamus displayed a medial hypothalamic parvocellular cluster of neurons in addition to the magnocellular clusters typical of mammals located in the lateral hypothalamus, and a novel medial division of the locus coeruleus was observed in the pons. These systems are related to appetitive drive and promotion of wakefulness, two aspects of elephant behaviour that appear to be inextricably linked. Lastly, we observed an extensive potential neurogenic lining of the ventricles throughout the brainstem that is present in even quite old elephants, although the function of these cells remains elusive. These observations combined demonstrate that, while much of the elephant brainstem is typically mammalian, certain aspects of the anatomy related to specialized behaviour of elephants are present and instructive in understanding elephant behaviour.

Qualitative and quantitative aspects of the microanatomy of the African elephant cerebellar cortex.

The current study provides a number of novel observations on the organization and structure of the cerebellar cortex of the African elephant by using a combination of basic neuroanatomical and immunohistochemical stains with Golgi and stereologic analysis. While the majority of our observations indicate that the cerebellar cortex of the African elephant is comparable to other mammalian species, several features were unique to the elephant. The three-layered organization of the cerebellar cortex, the neuronal types and some aspects of the expression of calcium-binding proteins were common to a broad range of mammalian species. The Lugaro neurons observed in the elephant were greatly enlarged in comparison to those of other large-brained mammals, suggesting a possible alteration in the processing of neural information in the elephant cerebellar cortex. Analysis of Golgi impregnations indicated that the dendritic complexity of the different interneuron types was higher in elephants than other mammals. Expression of parvalbumin in the parallel fibers and calbindin expressed in the stellate and basket cells also suggested changes in the elephant cerebellar neuronal circuitry. The stereologic analysis confirmed and extended previous observations by demonstrating that neuronal density is low in the elephant cerebellar cortex, providing for a larger volume fraction of the neuropil. With previous results indicating that the elephants have the largest relative cerebellar size amongst mammals, and one of the absolutely largest mammalian cerebella, the current observations suggest that the elephants have a greater volume of a potentially more complexly organized cerebellar cortex compared to other mammals. This quantitatively larger and more complex cerebellar cortex likely represents part of the neural machinery required to control the complex motor patterns involved in movement of the trunk and the production of infrasonic vocalizations.

Elephants have relatively the largest cerebellum size of mammals.

The current study used MR imaging to determine the volume of the cerebellum and its component parts in the brain of three adult male African elephants (Loxodonta africana) and compared this with published data from Asian elephants and other mammalian species including odontocete cetaceans, primates, chiropterans, insectivores, carnivores, and artiodactyls. The cerebellum of the adult elephant has a volume of ∼925 mL (average of both African and Asian species). Allometric analysis indicates that the elephant has the largest relative cerebellum size of all mammals studied to date. In addition, both odontocete cetaceans and microchiropterans appear to have large relative cerebellar sizes. The vermal and hemispheric components of the African elephant cerebellum are both large relative to other mammals of similar brain size, however, for odontocete cetaceans the vermal component is small and the hemispheric component is large. These volumetric observations are related to life-histories and anatomies of the species investigated. The current study provides context for one aspect of the elephant brain in the broader picture of mammalian brain evolution.

Acquisition of brains from the African elephant (Loxodonta africana): perfusion-fixation and dissection.

The current correspondence describes the in situ perfusion-fixation of the brain of the African elephant. Due to both the large size of proboscidean brains and the complex behaviour of these species, the acquisition of good quality material for comparative neuroanatomical analysis from these species is important. Three male African elephants (20-30 years) that were to be culled as part of a larger population management strategy were used. The animals were humanely euthanized and the head removed from the body. Large tubes were inserted into to the carotid arteries and the cranial vasculature flushed with a rapid (20 min) rinse of 100 l of cold saline (4 degrees C). Following the rinse the head was perfusion-fixed with a slower rinse (40 min) of 100 l of cold (4 degrees C) 4% paraformaldehyde in 0.1M phosphate buffer. This procedure resulted in well-fixed neural and other tissue. After perfusion the brains were removed from the skull with the aid of power tools, a procedure taking between 2 and 6h. The brains were immediately post-fixed in the same solution for 72 h at 4 degrees C. The brains were subsequently placed in a sucrose solution and finally an antifreeze solution and are stored in a -20 degrees C freezer. The acquisition of high quality neural material from African elephants that can be used for immunohistochemistry and electron microscopy is of importance in understanding the "hardware" underlying the behaviour of this species. This technique can be used on a variety of large mammals to obtain high quality material for comparative neuroanatomical studies.

Nuclear organization and morphology of serotonergic neurons in the brain of the Nile crocodile, Crocodylus niloticus.

The present study describes the location and nuclear organization of the serotonergic system in a representative of the order Crocodylia, the Nile crocodile (Crocodylus niloticus). We found evidence for serotonergic neurons in three regions of the brain, including the diencephalon, rostral and caudal brainstem, as previously reported in several other species of reptile. Within the diencephalon we found neurons in the periventricular organ of the hypothalamus, but not in the infundibular recess as noted in some other reptilian species. In addition we found serotonergic neurons in the pretectal nucleus, this being the first description of these neurons in any species. Within the rostral brainstem we found medial and lateral divisions of the superior raphe nucleus and a widely dispersed group of neurons in the tegmentum, the superior reticular nucleus. In the caudal brainstem we observed the inferior raphe nucleus and the inferior reticular nucleus. While much of the serotonergic system of the Nile crocodile is similar to that seen in other reptiles the entire suite of features appears to distinguish the crocodile studied from the members of the Squamate (lizards and snakes) and Testudine (turtles, tortoises and terrapins) reptiles previously studied. The observations are suggestive of order-specific patterns of nuclear organization of this system in the reptiles, reflecting potential evolutionary constraints in the mutability of the nuclear organization as seen for similar systems in mammals.

Distribution and morphology of putative catecholaminergic and serotonergic neurons in the brain of the greater canerat, Thryonomys swinderianus.

The distribution, morphology and nuclear subdivisions of the putative catecholaminergic and serotonergic systems within the brain of the greater canerat (sometimes spelt cane rat) were identified following immunohistochemistry for tyrosine hydroxylase and serotonin. The aim of the present study was to investigate possible differences in the complement of nuclear subdivisions of these systems when comparing those of the greater canerat with reports of these systems in other rodents. The greater canerat was chosen for investigation as it is a large rodent (around 2.7kg body mass) and has an average brain mass of 13.75g, more than five times larger than that of the laboratory rat. The greater canerats used in the present study were caught from the wild, which is again another contrast to the laboratory rat. While these differences, especially that of size, may lead to the prediction of significant differences in the nuclear complement of these systems, we found that all nuclei identified in both systems in the laboratory rat and other rodents in several earlier studies had direct homologs in the brain of the greater canerat. Moreover, there were no additional nuclei in the brain of the greater canerat that are not found in the laboratory rat or other rodents. It is noted that the locus coeruleus of the laboratory rat differs in appearance to that reported for several other rodent species. The greater canerat is phylogenetically distant from the laboratory rat, but still a member of the order Rodentia. Thus, changes in the nuclear organization of these systems appears to demonstrate a form of constraint related to the phylogenetic level of the order.

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.

Distribution and morphology of catecholaminergic and serotonergic neurons in the brain of the highveld gerbil, Tatera brantsii.

The distribution, morphology and nuclear subdivisions of the putative catecholaminergic and serotonergic systems within the brain of the highveld gerbil were identified following immunohistochemistry for tyrosine hydroxylase and serotonin. The aim of the present study was to investigate possible differences in the complement of nuclear subdivisions of these systems when comparing those of the highveld gerbil with those of the laboratory rat. The highveld gerbil was chosen as it is relatively closely related to the laboratory rat, but the Gerbillinae and Murinae lineages diverged over 20 million years ago. Moreover, even though brain sizes are similar, the life history and phenotypes between these two species are substantially different. The gerbils used in the present study were caught from the wild, which is again another contrast to the laboratory rat. While these differences may lead to the prediction of significant differences in the nuclear complement of these systems, we found that all nuclei identified in both systems in the laboratory rat in several earlier studies had direct homologs in the brain of the highveld gerbil. Moreover, there were no additional nuclei in the brain of the highveld gerbil that are not found in the laboratory rat. The only discernable difference between the two species was a greater density and number of catecholaminergic neurons in the olfactory bulb of the highveld gerbil. Thus, the evolution of nuclear parcellation in these systems appears to demonstrate a form of phylogenetic constraint related to the order Rodentia.

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.