Neuroanatomy of autism: what is the role of the cerebellum?

Autism (or autism spectrum disorder) was initially defined as a psychiatric disorder, with the likely cause maternal behavior (the very destructive "refrigerator mother" theory). It took several decades for research into brain mechanisms to become established. Both neuropathological and imaging studies found differences in the cerebellum in autism spectrum disorder, the most widely documented being a decreased density of Purkinje cells in the cerebellar cortex. The popular interpretation of these results is that cerebellar neuropathology is a critical cause of autism spectrum disorder. We challenge that view by arguing that if fewer Purkinje cells are critical for autism spectrum disorder, then any condition that causes the loss of Purkinje cells should also cause autism spectrum disorder. We will review data on damage to the cerebellum from cerebellar lesions, tumors, and several syndromes (Joubert syndrome, Fragile X, and tuberous sclerosis). Collectively, these studies raise the question of whether the cerebellum really has a role in autism spectrum disorder. Autism spectrum disorder is now recognized as a genetically caused developmental disorder. A better understanding of the genes that underlie the differences in brain development that result in autism spectrum disorder is likely to show that these genes affect the development of the cerebellum in parallel with the development of the structures that do underlie autism spectrum disorder.

Glycine is a transmitter in the human and chimpanzee cochlear nuclei.

Introduction: Auditory information is relayed from the cochlea via the eighth cranial nerve to the dorsal and ventral cochlear nuclei (DCN, VCN). The organization, neurochemistry and circuitry of the cochlear nuclei (CN) have been studied in many species. It is well-established that glycine is an inhibitory transmitter in the CN of rodents and cats, with glycinergic cells in the DCN and VCN. There are, however, major differences in the laminar and cellular organization of the DCN between humans (and other primates) and rodents and cats. We therefore asked whether there might also be differences in glycinergic neurotransmission in the CN. Methods: We studied brainstem sections from humans, chimpanzees, and cats. We used antibodies to glycine receptors (GLYR) to identify neurons receiving glycinergic input, and antibodies to the neuronal glycine transporter (GLYT2) to immunolabel glycinergic axons and terminals. We also examined archival sections immunostained for calretinin (CR) and nonphosphorylated neurofilament protein (NPNFP) to try to locate the octopus cell area (OCA), a region in the VCN that rodents has minimal glycinergic input. Results: In humans and chimpanzees we found widespread immunolabel for glycine receptors in DCN and in the posterior (PVCN) and anterior (AVCN) divisions of the VCN. We found a parallel distribution of GLYT2-immunolabeled fibers and puncta. The data also suggest that, as in rodents, a region containing octopus cells in cats, humans and chimpanzees has little glycinergic input. Discussion: Our results show that glycine is a major transmitter in the human and chimpanzee CN, despite the species differences in DCN organization. The sources of the glycinergic input to the CN in humans and chimpanzees are not known.

Comparative analysis of four nuclei in the human brainstem: Individual differences, left-right asymmetry, species differences.

Introduction: It is commonly thought that while the organization of the cerebral cortex changes dramatically over evolution, the organization of the brainstem is conserved across species. It is further assumed that, as in other species, brainstem organization is similar from one human to the next. We will review our data on four human brainstem nuclei that suggest that both ideas may need modification. Methods: We have studied the neuroanatomical and neurochemical organization of the nucleus paramedianus dorsalis (PMD), the principal nucleus of the inferior olive (IOpr), the arcuate nucleus of the medulla (Arc) and the dorsal cochlear nucleus (DC). We compared these human brainstem nuclei to nuclei in other mammals including chimpanzees, monkeys, cats and rodents. We studied human cases from the Witelson Normal Brain collection using Nissl and immunostained sections, and examined archival Nissl and immunostained sections from other species. Results: We found significant individual variability in the size and shape of brainstem structures among humans. There is left-right asymmetry in the size and appearance of nuclei, dramatically so in the IOpr and Arc. In humans there are nuclei, e.g., the PMD and the Arc, not seen in several other species. In addition, there are brainstem structures that are conserved across species but show major expansion in humans, e.g., the IOpr. Finally, there are nuclei, e.g. the DC, that show major differences in structure among species. Discussion: Overall, the results suggest several principles of human brainstem organization that distinguish humans from other species. Studying the functional correlates of, and the genetic contributions to, these brainstem characteristics are important future research directions.

Characterization of the superior olivary complex of chimpanzees (Pan troglodytes) in comparison to humans.

The superior olivary complex (SOC) is a collection of nuclei in the hindbrain of mammals with numerous roles in hearing, including localization of sound sources in the environment, encoding temporal and spectral elements of sound, and descending modulation of the cochlea. While there have been several investigations of the SOC in primates, there are discrepancies in the descriptions of nuclear borders and even the presence of certain cell groups among studies and species. Herein, we aimed to clarify some of these issues by characterizing the SOC from chimpanzees using Nissl staining, quantitative morphometry and immunohistochemistry. We found the medial superior olive (MSO) to be the largest of the SOC nuclei and the arrangement of its neurons and peri-MSO to be very similar to humans. Additionally, we found neurons in the medial nucleus of the trapezoid body (MNTB) to be immunopositive for the calcium binding protein calbindin. Further, most neurons in the MNTB, and some neurons in the lateral nucleus of the trapezoid body were associated with large, calretinin-immunoreactive calyx terminals. Together, these findings indicate the organization of the SOC of chimpanzees is organized very similar to the SOC in humans and suggests modifications to this region among species consistent with differences in head/body size, restricted hearing range and sensitivity to low frequency sounds.

Individual variability in the size and organization of the human arcuate nucleus of the medulla.

The arcuate nucleus (Arc) of the medulla is found in almost all human brains and in a small percentage of chimpanzee brains. It is absent in the brains of other mammalian species including mice, rats, cats, and macaque monkeys. The Arc is classically considered a precerebellar relay nucleus, receiving input from the cerebral cortex and projecting to the cerebellum via the inferior cerebellar peduncle. However, several studies have found aplasia of the Arc in babies who died of SIDS (Sudden Infant Death Syndrome), and it was suggested that the Arc is the locus of chemosensory neurons critical for brainstem control of respiration. Aplasia of the Arc, however, has also been reported in adults, suggesting that it is not critical for survival. We have examined the Arc in closely spaced Nissl-stained sections in thirteen adult human cases to acquire a better understanding of the degree of variability of its size and location in adults. We have also examined immunostained sections to look for neurochemical compartments in this nucleus. Caudally, neurons of the Arc are ventrolateral to the pyramidal tracts (py); rostrally, they are ventro-medial to the py and extend up along the midline. In some cases, the Arc is discontinuous, with a gap between sections with the ventrolaterally located and the ventromedially located neurons. In all cases, there is some degree of left-right asymmetry in Arc position, size, and shape at all rostro-caudal levels. Somata of neurons in the Arc express calretinin (CR), neuronal nitric oxide synthase (nNOS), and nonphosphorylated neurofilament protein (NPNFP). Calbindin (CB) is expressed in puncta whereas there is no expression of parvalbumin (PV) in somata or puncta. There is also immunostaining for GAD and GABA receptors suggesting inhibitory input to Arc neurons. These properties were consistent among cases. Our data show differences in location of caudal and rostral Arc neurons and considerable variability among cases in the size and shape of the Arc. The variability in size suggests that "hypoplasia" of the Arc is difficult to define. The discontinuity of the Arc in many cases suggests that establishing aplasia of the Arc requires examination of many closely spaced sections through the brainstem.

Functional and Neuropathological Evidence for a Role of the Brainstem in Autism.

The brainstem includes many nuclei and fiber tracts that mediate a wide range of functions. Data from two parallel approaches to the study of autistic spectrum disorder (ASD) implicate many brainstem structures. The first approach is to identify the functions affected in ASD and then trace the neural systems mediating those functions. While not included as core symptoms, three areas of function are frequently impaired in ASD: (1) Motor control both of the limbs and body and the control of eye movements; (2) Sensory information processing in vestibular and auditory systems; (3) Control of affect. There are critical brainstem nuclei mediating each of those functions. There are many nuclei critical for eye movement control including the superior colliculus. Vestibular information is first processed in the four nuclei of the vestibular nuclear complex. Auditory information is relayed to the dorsal and ventral cochlear nuclei and subsequently processed in multiple other brainstem nuclei. Critical structures in affect regulation are the brainstem sources of serotonin and norepinephrine, the raphe nuclei and the locus ceruleus. The second approach is the analysis of abnormalities from direct study of ASD brains. The structure most commonly identified as abnormal in neuropathological studies is the cerebellum. It is classically a major component of the motor system, critical for coordination. It has also been implicated in cognitive and language functions, among the core symptoms of ASD. This structure works very closely with the cerebral cortex; the cortex and the cerebellum show parallel enlargement over evolution. The cerebellum receives input from cortex via relays in the pontine nuclei. In addition, climbing fiber input to cerebellum comes from the inferior olive of the medulla. Mossy fiber input comes from the arcuate nucleus of the medulla as well as the pontine nuclei. The cerebellum projects to several brainstem nuclei including the vestibular nuclear complex and the red nucleus. There are thus multiple brainstem nuclei distributed at all levels of the brainstem, medulla, pons, and midbrain, that participate in functions affected in ASD. There is direct evidence that the cerebellum may be abnormal in ASD. The evidence strongly indicates that analysis of these structures could add to our understanding of the neural basis of ASD.

The Claustrum in the Squirrel Monkey.

The claustrum (CLA) is a subcortical structure that is reciprocally and topographically connected with the cerebral cortex. The complexity of the cerebral cortex varies dramatically across mammals, raising the question of whether there might also be differences in CLA organization, circuitry, and function. Species variations in the shape of the CLA are well documented. Studies in multiple species have identified subsets of neurochemically distinct interneurons; some data suggest species variations in the nature, distribution, and numbers of different neurochemically identified neuronal types. We have studied the CLA in a smooth-brained primate, the squirrel monkey, using Nissl-stained sections and immunohistochemistry. We found that the shape of the CLA is different from that in other primates. We found several different neurochemically defined populations of neurons equally distributed throughout the CLA. Immunoreactivity to GAD65/67 and GABAA receptors suggest that GABAergic interneurons provide widespread inhibitory input to CLA neurons. Immunoreactivity to glutamate transporters suggests widespread and overlapping excitatory input from cortical and possibly subcortical sources. Comparison of CLA organization in different species suggests that there may be major species differences both in the organization and in the functions of the CLA. Anat Rec, 303:1439-1454, 2020. © 2019 American Association for Anatomy.

The transcription factor Nfix is essential for normal brain development.

BACKGROUND: The Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nfic-deficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice. RESULTS: Here we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant Pax6- and doublecortin-positive cells in the lateral ventricles of Nfix-/- mice, increased brain weight, expansion of the cingulate cortex and entire brain along the dorsal ventral axis, and aberrant formation of the hippocampus. On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22. If their food is supplemented with a soft dough chow from P10, Nfix-/- animals show a lag in weight gain from P8 to P20 but then increase their growth rate. A fraction of the animals survive to adulthood and are fertile. The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals. CONCLUSION: These data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects.

Individual variability in the structural properties of neurons in the human inferior olive.

The inferior olive (IO) is the sole source of the climbing fibers innervating the cerebellar cortex. We have previously shown both individual differences in the size and folding pattern of the principal nucleus (IOpr) in humans as well as in the expression of different proteins in IOpr neurons. This high degree of variability was not present in chimpanzee samples. The neurochemical differences might reflect static differences among individuals, but might also reflect age-related processes resulting in alterations of protein synthesis. Several observations support the latter idea. First, accumulation of lipofuscin, the "age pigment" is well documented in IOpr neurons. Second, there are silver- and abnormal tau-immunostained intraneuronal granules in IOpr neurons (Ikeda et al. Neurosci Lett 258:113-116, 1998). Finally, Olszewski and Baxter (Cytoarchitecture of the human brain stem, Second edn. Karger, Basel, 1954) observed an apparent loss of IOpr neurons in older individuals. We have further investigated the possibility of age-related changes in IOpr neurons using silver- and immunostained sections. We found silver-labeled intraneuronal granules in neurons of the IOpr in all human cases studied (n = 17, ages 25-71). We did not, however, confirm immunostaining with antibodies to abnormal tau. There was individual variability in the density of neurons as well as in the expression of the calcium-binding protein calretinin. In the chimpanzee, there were neither silver-stained intraneuronal granules nor irregularities in immunostaining. Overall, the data support the hypothesis that in some, but not all, humans there are functional changes in IOpr neurons and ultimately cell death. Neurochemical changes of IOpr neurons may contribute to age-related changes in motor and cognitive skills mediated by the cerebellum.

Species Differences in the Organization of the Ventral Cochlear Nucleus.

The mammalian cochlear nuclei (CN) consist of two major subdivisions, the dorsal (DCN) and ventral (VCN) nuclei. We previously reported differences in the structural and neurochemical organization of the human DCN from that in several other species. Here we extend this analysis to the VCN, considering both the organization of subdivisions and the types and distributions of neurons. Classically, the VCN in mammals is composed of two subdivisions, the anteroventral (VCA) and posteroventral cochlear nuclei (VCP). Anatomical and electrophysiological data in several species have defined distinct neuronal types with different distributions in the VCA and VCP. We asked if VCN subdivisions and anatomically defined neuronal types might be distinguished by patterns of protein expression in humans. We also asked if the neurochemical characteristics of the VCN are the same in humans as in other mammalian species, analyzing data from chimpanzees, macaque monkeys, cats, rats and chinchillas. We examined Nissl- and immunostained sections, using antibodies that had labeled neurons in other brainstem nuclei in humans. Nissl-stained sections supported the presence of both VCP and VCA in humans and chimpanzees. However, patterns of protein expression did not differentiate classes of neurons in humans; neurons of different soma shapes and dendritic configurations all expressed the same proteins. The patterns of immunostaining in macaque monkey, cat, rat, and chinchilla were different from those in humans and chimpanzees and from each other. The results may correlate with species differences in auditory function and plasticity. Anat Rec, 301:862-886, 2018. © 2017 Wiley Periodicals, Inc.

Neurochemically defined cell types in the claustrum of the cat.

The claustrum is a subcortical structure reciprocally and topographically connected with all sensory and motor domains of the cerebral cortex. Previous anatomical and electrophysiological data suggested that most cells in the claustrum are large neurons that both receive cortical input and project back to cortex, forming excitatory connections with their cortical targets. These data have been interpreted to imply a relay function for the claustrum, with information from different functional cortical domains remaining segregated. The possibility that the claustrum might mediate a more "global" function has been recently been developed by Crick and Koch [Crick, F. C., Koch, C., 2005. What is the function of the claustrum? Philos. Trans. R. Soc. Lond., B Biol. Sci. 360, 1271-1279]. We have reexamined the anatomical substrate for information processing in the claustrum of the cat by analyzing the patterns of immunoreactivity to calcium-binding proteins, GAD, serotonin, nNOS and the glutamate transporter EAAC1. We found multiple neurochemically defined cell types, suggesting multiple classes of projection neurons and interneurons. Each class was found throughout the entire claustrum, in all functionally defined subdivisions. Many neurons in the claustrum were surrounded by parvalbumin, calretinin, GAD or nNOS immunoreactive terminals, suggesting that many neurons of the claustrum make extensive intraclaustral connections. The entire claustrum also receives a serotonergic input. The identification of multiple neurochemical cell classes, their distribution and the extent of their dendritic arborizations relative to functional compartments suggest a substrate for information processing in the claustrum that may allow integration of information across functional subdivisions.

Neurochemically defined cell columns in the nucleus prepositus hypoglossi of the cat and monkey.

Many studies have shown that the nucleus prepositus hypoglossi (PH) participates with the vestibular nuclear complex, the cerebellum and the oculomotor nuclei in the control of eye movements. We have looked at the neurochemical organization of PH in the cat and monkey using a recently developed antibody, 8B3, that recognizes a chondroitin sulfate proteoglycan. In the cat, immunoreactivity to 8B3 labels a set of cells in PH. On frontal sections, these cells form a cluster that is seen over the entire anterior-posterior (A-P) extent of PH, but the number of cells in the cluster changes with A-P level. Earlier studies have identified an A-P cell column in PH of the cat whose neurons synthesize nitric oxide. We have used both single- and double-label protocols to investigate the relation between the two cell groups. Single-label studies show spatial overlap but that the cells immunoreactive to nitric oxide synthase (nNOS) are more numerous than cells immunoreactive to 8B3. Double-label studies show that all cells immunoreactive to 8B3 were also immunoreactive to nNOS, but, as suggested by the single-label data, there are many nNOS-immunoreactive cells not immunoreactive to 8B3. Populations of 8B3 and nNOS-immunoreactive cells are also found in PH of squirrel and macaque monkeys. The results suggest that nNOS-immunoreactive cells in PH may consist of two functionally different populations.

Variations in the structure of the prelunate gyrus in Old World monkeys.

Anatomical and electrophysiological studies have revealed a complex organization in the macaque prelunate gyrus. We investigated the morphology and architecture of the prelunate gyrus in Old World monkeys. In Macaca nemestrina, we observed a sulcus crossing the prelunate gyrus within 2 mm of the vertical meridian representation. In other macaque species and other cercopithecines, we observed substantial variations in sulcal morphology across the prelunate gyrus. We did not find a sulcus in all species, and the location and depth of that indentation on the gyrus varied among species. A deep sulcus was observed in all species that emerged earlier in evolution than macaques, such as guenons, baboons, and colobines. We analyzed the regional and parcellation features of the prelunate gyrus in three macaque species, M. maura, M. mulatta, and M. radiata, and in Erythrocebus patas, with emphasis on the relation of structure to the distribution of prelunate visual areas. Nonphosphorylated neurofilament protein immunoreactivity permitted the delineation of a novel area in the prelunate gyrus of Old World monkeys, located around the prelunate sulcus. Species-specific patterns were also observed in the prelunate gyrus of the patas monkey compared to macaques. These observations, as well as a cladistic analysis of the data, suggest an expanded and diversified organization of the prelunate gyrus in some cercopithecoids that may reflect adaptation to specific ecological environments. It was, however, progressively lost in most macaques, being retained only in species that diverged early in the evolution of the genus Macaca, such as M. nemestrina and M. maura.

Unique features of the human brainstem and cerebellum.

The cerebral cortex is greatly expanded in the human brain. There is a parallel expansion of the cerebellum, which is interconnected with the cerebral cortex. We have asked if there are accompanying changes in the organization of pre-cerebellar brainstem structures. We have examined the cytoarchitectonic and neurochemical organization of the human medulla and pons. We studied human cases from the Witelson Normal Brain Collection, analyzing Nissl sections and sections processed for immunohistochemistry for multiple markers including the calcium-binding proteins calbindin, calretinin, and parvalbumin, non-phosphorylated neurofilament protein, and the synthetic enzyme for nitric oxide, nitric oxide synthase. We have also compared the neurochemical organization of the human brainstem to that of several other species including the chimpanzee, macaque and squirrel monkey, cat, and rodent, again using Nissl staining and immunohistochemistry. We found that there are major differences in the human brainstem, ranging from relatively subtle differences in the neurochemical organization of structures found in each of the species studied to the emergence of altogether new structures in the human brainstem. Two aspects of human cortical organization, individual differences and left-right asymmetry, are also seen in the brainstem (principal nucleus of the inferior olive) and the cerebellum (the dentate nucleus). We suggest that uniquely human motor and cognitive abilities derive from changes at all levels of the central nervous system, including the cerebellum and brainstem, and not just the cerebral cortex.

Comparative organization of the claustrum: what does structure tell us about function?

The claustrum is a subcortical nucleus present in all placental mammals. Many anatomical studies have shown that its inputs are predominantly from the cerebral cortex and its outputs are back to the cortex. This connectivity thus suggests that the claustrum serves to amplify or facilitate information processing in the cerebral cortex. The size and the complexity of the cerebral cortex varies dramatically across species. Some species have lissencephalic brains, with few cortical areas, while others have a greatly expanded cortex and many cortical areas. This evolutionary diversity in the cerebral cortex raises several questions about the claustrum. Does its volume expand in coordination with the expansion of cortex and does it acquire new functions related to the new cortical functions? Here we survey the organization of the claustrum in animals with large brains, including great apes and cetaceans. Our data suggest that the claustrum is not always a continuous structure. In monkeys and gorillas there are a few isolated islands of cells near the main body of the nucleus. In cetaceans, however, there are many isolated cell islands. These data suggest constraints on the possible function of the claustrum. Some authors propose that the claustrum has a more global role in perception or consciousness that requires intraclaustral integration of information. These theories postulate mechanisms like gap junctions between claustral cells or a "syncytium" to mediate intraclaustral processing. The presence of discontinuities in the structure of the claustrum, present but minimal in some primates, but dramatically clear in cetaceans, argues against the proposed mechanisms of intraclaustral processing of information. The best interpretation of function, then, is that each functional subdivision of the claustrum simply contributes to the function of its cortical partner.

Laminar and neurochemical organization of the dorsal cochlear nucleus of the human, monkey, cat, and rodents.

The dorsal cochlear nucleus (DCN) is a brainstem structure that receives input from the auditory nerve. Many studies in a diversity of species have shown that the DCN has a laminar organization and identifiable neuron types with predictable synaptic relations to each other. In contrast, studies on the human DCN have found a less distinct laminar organization and fewer cell types, although there has been disagreement among studies in how to characterize laminar organization and which of the cell types identified in other animals are also present in humans. We have reexamined DCN organization in the human using immunohistochemistry to analyze the expression of several proteins that have been useful in delineating the neurochemical organization of other brainstem structures in humans: nonphosphorylated neurofilament protein (NPNFP), nitric oxide synthase (nNOS), and three calcium-binding proteins. The results for humans suggest a laminar organization with only two layers, and the presence of large projection neurons that are enriched in NPNFP. We did not observe evidence in humans of the inhibitory interneurons that have been described in the cat and rodent DCN. To compare humans and other animals directly we used immunohistochemistry to examine the DCN in the macaque monkey, the cat, and three rodents. We found similarities between macaque monkey and human in the expression of NPNFP and nNOS, and unexpected differences among species in the patterns of expression of the calcium-binding proteins.

Neurochemical organization of the vestibular brainstem in the common chimpanzee (Pan troglodytes).

Chimpanzees are one of the closest living relatives of humans. However, the cognitive and motor abilities of chimpanzees and humans are quite different. The fact that humans are habitually bipedal and chimpanzees are not implies different uses of vestibular information in the control of posture and balance. Furthermore, bipedal locomotion permits the development of fine motor skills of the hand and tool use in humans, suggesting differences between species in the structures and circuitry for manual control. Much motor behavior is mediated via cerebro-cerebellar circuits that depend on brainstem relays. In this study, we investigated the organization of the vestibular brainstem in chimpanzees to gain insight into whether these structures differ in their anatomy from humans. We identified the four nuclei of vestibular nuclear complex in the chimpanzee and also looked at several other precerebellar structures. The size and arrangement of some of these nuclei differed between chimpanzees and humans, and also displayed considerable inter-individual variation. We identified regions within the cytoarchitectonically defined medial vestibular nucleus visualized by immunoreactivity to the calcium-binding proteins calretinin and calbindin as previously shown in other species including human. We have found that the nucleus paramedianus dorsalis, which is identified in the human but not in macaque monkeys, is present in the chimpanzee brainstem. However, the arcuate nucleus, which is present in humans, was not found in chimpanzees. The present study reveals major differences in the organization of the vestibular brainstem among Old World anthropoid primate species. Furthermore, in chimpanzees, as well as humans, there is individual variability in the organization of brainstem nuclei.

The nucleus pararaphales in the human, chimpanzee, and macaque monkey.

The human cerebral cortex and cerebellum are greatly expanded compared to those of other mammals, including the great apes. This expansion is reflected in differences in the size and organization of precerebellar brainstem structures, such as the inferior olive. In addition, there are cell groups unique to the human brainstem. One such group may be the nucleus pararaphales (PRa); however, there is disagreement among authors about the size and location of this nucleus in the human brainstem. The name "pararaphales" has also been used for neurons in the medulla shown to project to the flocculus in the macaque monkey. We have re-examined the existence and status of the PRa in eight humans, three chimpanzees, and four macaque monkeys using Nissl-stained sections as well as immunohistochemistry. In the human we found a cell group along the midline of the medulla in all cases; it had the form of interrupted cell columns and was variable among cases in rostrocaudal and dorsoventral extent. Cells and processes were highly immunoreactive for non-phosphorylated neurofilament protein (NPNFP); somata were immunoreactive to the synthetic enzyme for nitric oxide, nitric oxide synthase, and for calretinin. In macaque monkey, there was a much smaller oval cell group with NPNFP immunoreactivity. In the chimpanzee, we found a region of NPNFP-immunoreactive cells and fibers similar to what was observed in macaques. These results suggest that the "PRa" in the human may not be the same structure as the flocculus-projecting cell group described in the macaque. The PRa, like the arcuate nucleus, therefore may be unique to humans.

Tinnitus, unipolar brush cells, and cerebellar glutamatergic function in an animal model.

Unipolar brush cells (UBCs) are excitatory interneurons found in the dorsal cochlear nucleus (DCN) and the granule cell layer of cerebellar cortex, being particularly evident in the paraflocculus (PFL) and flocculus (FL). UBCs receive glutamatergic inputs and make glutamatergic synapses with granule cells and other UBCs. It has been hypothesized that UBCs comprise local networks of tunable feed-forward amplifiers. In the DCN they might also participate in feed-back amplification of signals from higher auditory centers. Recently it has been shown that UBCs, in the vestibulocerebellum and DCN of adult rats, express doublecortin (DCX), previously considered a marker of newborn and migrating neurons. In an animal model, both the DCN, and more recently the PFL, have been implicated in contributing to the sensation of acoustic-exposure-induced tinnitus. These studies support the working hypothesis that tinnitus emerges after loss of peripheral sensitivity because inhibitory processes homeostatically down regulate, and excitatory processes up regulate. Here we report the results of two sequential experiments that examine the potential role of DCN and cerebellar UBCs in tinnitus, and the contribution of glutamatergic transmission in the PFL. In Experiment 1 it was shown that adult rats with psychophysical evidence of tinnitus induced by a single unilateral high-level noise exposure, had elevated DCX in the DCN and ventral PFL. In Experiment 2 it was shown that micro-quantities of glutamatergic antagonists, delivered directly to the PFL, reversibly reduced chronically established tinnitus, while similarly applied glutamatergic agonists induced tinnitus-like behavior in non-tinnitus controls. These results are consistent with the hypothesis that UBC up regulation and enhanced glutamatergic transmission in the cerebellum contribute to the pathophysiology of tinnitus.