Brainstem motor neuron dysmorphology and excitatory/inhibitory imbalance in an animal model of autism.

BACKGROUND: Autism spectrum disorder (ASD) is a developmental disorder associated with in utero exposure to the antiepileptic valproic acid (VPA) in humans, and similar exposure serves as a validated animal model. Animals exposed to VPA in utero have a number of structural, function and behavioural deficits associated with ASD. Furthermore, VPA-exposed animals have shorter body lengths, lower body and brain weights. This difference in body weight may result from impaired caloric intake due to impaired oropharyngeal function. MATERIALS AND METHODS: Specifically, it is hypothesized that in utero VPA exposure results in fewer lower motor neurons associated with feeding behaviours, that surviving neurons will exhibit dysmorphology and altered balance of excitatory and inhibitory inputs. Further, it is hypothesized that VPA exposure will result in altered oropharyngeal musculature that will impact skull morphology. RESULTS: These hypotheses were investigated using quantitative morphometrics and immunofluorescence. CONCLUSIONS: Results support dysmorphology and excitatory/inhibitory imbalance and these alterations may contribute to dysphagia and poor weight gain in VPA-exposed animals.

Premature termination of the sympathetic chain.

The sympathetic chain serves to distribute visceral efferents and afferents over the entire body. The sympathetic chain courses from the base of the skull to the coccyx and sends branches to distribute along spinal nerves and a number of visceral nerves that distribute to cardiac muscle, smooth muscle and glands. During dissection of the posterior abdominal wall, we identified a rare variation of the sympathetic chain. In this subject, the sympathetic chain failed to send grey rami to the L2-4 spinal nerves and terminated by joining the S1 anterior ramus. Such a variation has only been reported once in the literature in 1895. We provide both schematic and photographic documentation of this variation and propose a number of possible circuits whereby visceral axons can reach their target despite these anatomical barriers.

A tale of two arteries: dual posterior cerebral arteries with vascular bridges. A possible protective pattern?

Stroke is a common morbidity and a frequent cause of disability and even death. The impact of cerebrovascular events is dictated by the brain region involved and can be complicated by anatomical variations. One of the most common variations impacting the cerebral vasculature is the presence of a foetal posterior cerebral artery. This vessel arises from the internal carotid artery instead of the basilar artery and is often associated with more extensive injury in cerebrovascular events. Herein, we report the case of a 60-year-old male who had numerous arterial abnormalities, including a kink and a coil of the left internal carotid, two posterior communicating arteries on the right and two posterior cerebral arteries (PCA) on the left, one arising from the internal carotid (foetal PCA) and one from the basilar. The foetal PCA supplied the thalamus, splenium of the corpus callosum and primary visual cortex. The basilar PCA supplied the midbrain and parts of the occipital lobe. These PCA were connected to each other by a vascular bridge and the foetal PCA was connected to the middle cerebral artery by an additional vascular bridge. This vascular pattern would appear to provide collateral support around blockages in the internal carotid and main stem middle and PCA.

Neonatal exposure to monosodium glutamate results in dysmorphology of orofacial lower motor neurons.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and is stored and released by both neurons and astrocytes. Despite the important role of glutamate as a neurotransmitter, high levels of extracellular glutamate can result in excitotoxicity and apoptosis. Monosodium glutamate (MSG) is a naturally occurring sodium salt of glutamic acid that is used as a flavour enhancer in many processed foods. Neonatal exposure to MSG has been shown to result in neurodegeneration in several forebrain regions, characterised by neuronal loss and neuroendocrine abnormalities. However, the brainstem effects of neonatal MSG exposure have not been investigated. It is therefore hypothesized that MSG exposure during the early postnatal period would impact brainstem lower motor neurons involved in feeding behaviour. The effect of neonatal MSG exposure on brainstem lower motor neurons was investigated by exposing rat pups to either 4 mg/g MSG or saline from postnatal day (P) 4 through 10. On P28, brains were preserved by vascular perfusion with fixative, frozen sectioned and stained for Nïssl substance. The number, size and shape of brainstem motor neurons were compared between MSG and saline-exposed animals. MSG exposure had no impact on the total number of neurons in the nuclei examined. However, MSG exposure was associated with a significant increase in the number of round somata in both the trigeminal and facial nuclei. Furthermore, MSG exposure resulted in significantly smaller neurons in all motor nuclei examined. These results suggest that neonatal exposure to MSG impacts the development of brainstem lower motor neurons which may impact feeding and swallowing behaviours in young animals.

A case of a single intracranial vertebral artery and cerebral infarct.

The vertebral arteries are commonly affected by anatomical variation. This variation ranges from slight asymmetry in arterial diameter between the right and left sides to complete absence of a vertebral artery on one side. Asymmetry in diameter is a common observation, although complete absence of the artery is rare. Herein, we report on a 79-year-old male anatomical donor who, upon brain removal, was found to have a single intracranial vertebral artery which was the sole source of the basilar artery. During dissection of the neck, both right and left vertebral arteries were identified arising from the subclavian arteries. The vertebral arteries were dissected from the transverse foramina and followed into the skull. The right vertebral artery terminated by supplying the spinal cord, consistent with the distribution of the posterior spinal artery. Such vascular anomalies are clinically significant, as they may lead to abnormal patterns of sensory-motor deficiencies in stroke and are at risk of iatrogenic injury during surgical procedures.

Prenatal valproic acid exposure disrupts tonotopic c-Fos expression in the rat brainstem.

Autism spectrum disorder (ASD) is a group of neurodevelopmental conditions characterized by difficulties in communication and social interactions, restricted, repetitive behaviors and sensory abnormalities. Notably, the vast majority of individuals with ASD experience some degree of auditory dysfunction and we have recently reported consistent hypoplasia and dysmorphology in auditory brainstem centers in individuals with ASD. Prenatal exposure to the antiepileptic drug valproic acid (VPA) is associated with an increased risk of ASD. In rodents, prenatal exposure to VPA is employed as an animal model of ASD and is associated with a number of anatomical, physiological and behavioral deficits, including hypoplasia and dysmorphology of auditory brainstem centers. Based on these observations, we hypothesized that such dysmorphology in VPA-exposed animals would translate into abnormal neuronal activity in brainstem circuits and irregular tonotopic maps. Herein, we have subjected control and VPA-exposed animals to 4- or 16-kHz tones and examined neuronal activation with immunohistochemistry for c-Fos. After these exposures, we identified significantly more c-Fos-positive neurons in the auditory brainstem of VPA-exposed animals. Additionally, we observed a larger dispersion of c-Fos-positive neurons and shifted tonotopic bands in VPA-exposed rats. We interpret these findings to suggest hyper-responsiveness to sounds and disrupted mapping of sound frequencies after prenatal VPA exposure. Based on these findings, we suggest that such abnormal patterns of activation may play a role in auditory processing deficits in ASD.

Prenatal valproic acid exposure disrupts tonotopic c-Fos expression in the rat brainstem.

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by difficulties with communication and social interactions, restricted, repetitive behaviors and sensory abnormalities. Additionally, the vast majority of subjects with ASD suffer some degree of auditory dysfunction and we have previously identified significant hypoplasia and dysmorphology in auditory brainstem centers in individuals with ASD. Prenatal exposure to the antiepileptic drug valproic acid (VPA) is associated with an increased risk of ASD. In rodents, prenatal exposure to VPA is utilized as an animal model of ASD and is associated with a number of anatomical, physiological and behavioral deficits, including hypoplasia and dysmorphology in the auditory brainstem. Based on these observations, we hypothesized that such dysmorphology in VPA-exposed animals would translate into abnormal activity in brainstem circuits and irregular tonotopic maps. Herein, we have subjected control and VPA-exposed animals to 4 or 16 kHz tones and examined neuronal activation with immunohistochemistry for c-Fos. After these sound exposures, we found significantly more c-Fos-positive neurons in the auditory brainstem of VPA-exposed animals. Further, we found a larger dispersion of c-Fos-positive neurons and shifted tonotopic bands in VPA-exposed rats. We interpret these findings to suggest hyper-responsiveness to sounds and disrupted mapping of sound frequencies after prenatal VPA exposure. Based on these findings, we suggest that such abnormal patterns of activation may play a role in auditory processing deficits in ASD.

Abnormal neuronal morphology and neurochemistry in the auditory brainstem of Fmr1 knockout rats.

Fragile X syndrome (FXS) is an inherited neurodevelopmental disorder affecting nearly one in 5000 newborn males and is a leading genetic cause of autism spectrum disorder. In addition to developmental delays and intellectual impairment, FXS is characterized by seizures, attention deficit, and hypersensitivity to visual, tactile and auditory stimuli. The Fmr1 gene encodes Fragile X mental retardation protein (FMRP), which is abundant in neurons, binds select mRNAs and functions as a negative regulator of mRNA translation. A deficiency in FMRP, as in FXS and Fmr1 knockout (KO) animals, results in neuronal dysmorphology and altered synaptic function. Additionally, there is evidence for disruption of GABAergic circuits in subjects lacking FMRP. Our previous studies demonstrated widespread expression of FMRP in human auditory brainstem neurons. Given this observation, we hypothesized that FMRP is highly expressed in rat auditory brainstem neurons and that the auditory hypersensitivity characteristic of FXS results from dysfunction of brainstem networks secondary to decreased expression of FMRP. In our investigation of postnatal day 50 (P50) control rats, we found that FMRP was widely expressed in neurons of the superior olivary complex (SOC). In P50 Fmr1 KO rats, many SOC neurons had a smaller soma when compared to controls, indicative of abnormal neuronal morphology. Additionally, neurons in the medial superior olive (MSO) were more round in Fmr1 KO rats. There was also reduced expression of glutamic acid decarboxylase (GAD67) in neurons of the superior paraolivary nucleus (SPON) and a reduction in the number of calretinin-immunoreactive terminals associated with neurons of the medial nucleus of the trapezoid body (MNTB). Together, these findings support the conclusion that the auditory dysfunction characteristic of FXS arises, at least in part, from defective brainstem networks.

Organization of the human superior olivary complex in 15q duplication syndromes and autism spectrum disorders.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by a number of behavioral and social features. Although the etiology of most cases of ASD is idiopathic, a significant number of cases can be attributed to genetic causes, such as chromosome 15q duplications [dup(15q)]. Recent neuropathological investigations have provided evidence for distinct patterns of heterotopias and dysplasias in ASD and subjects with both ASD and dup(15q). Individuals with ASD characteristically have hearing difficulties and we have previously demonstrated significant and consistent hypoplasia in a number of auditory brainstem nuclei in subjects with ASD. Herein, we compare results from a morphometric investigation of auditory brainstem nuclei in subjects with ASD, dup(15q) and controls. Our observations in subjects with ASD support our previous reports. However, in subjects with dup(15q), we find significantly fewer neurons and in many nuclei, neurons were significantly smaller than in ASD subjects. Finally, we find a notably higher incidence of ectopic neurons in dup(15q). These results suggest that in the brainstem, these neuropathological conditions may evolve from some of the same developmental errors but are distinguished on microscopic features.

Anatomical dissection of a cadaver with congenital scoliosis.

Congenital scoliosis is a developmental anomaly involving poorly formed or fused vertebral segments resulting in an abnormal lateral curvature of the vertebral column and is often accompanied by significant rotational defects. Despite abundant literature on causes, diagnosis and treatment of scoliosis, little attention has been given to impacts of this condition on the musculoskeletal system beyond the bony defects. This report describes the detailed, layer-by-layer dissection of the superficial and deep back musculature and examination of the axial skeleton of a 47-year-old male with severe congenital scoliosis. The subject presented with both cervico-thoracic and thoraco-lumbar scoliotic curves. Dissection of the back muscles revealed notable asymmetry in the superficial muscles and marked atrophy of the deep back muscles on the left side. Examination of the axial skeleton revealed numerous bones which were abnormally porous, reduced thoracic volume, attenuated intercostal spaces on the left side and 2 separate fusion deficits, including an unsegmented bar spanning 6 vertebral segments.

Distribution of fragile X mental retardation protein in the human auditory brainstem.

Fragile X mental retardation protein (FMRP) binds select mRNAs, functions in intracellular transport of these mRNAs and represses their translation. FMRP is highly expressed in neurons and lack of FMRP has been shown to result in dendritic dysmorphology and altered synaptic function. FMRP is known to interact with mRNAs for the Kv3.1b potassium channel which is required for neurons to fire action potentials at high rates with remarkable temporal precision. Auditory brainstem neurons are known for remarkably high spike rates and expression of Kv3.1b potassium channels. Fragile X syndrome (FXS) is a genetic disorder caused by a mutation in the fragile X mental retardation 1 gene (Fmr1) resulting in decreased expression of FMRP and subsequent intellectual disability, seizures, attention deficit and hypersensitivity to auditory and other sensory stimuli. We therefore hypothesize that the auditory difficulties in FXS result, at least in part, from dysfunction of auditory brainstem neurons. To examine this hypothesis, we have studied normal human brainstem tissue with immunohistochemical techniques and confocal microscopy. Our results demonstrate that FMRP is widely expressed in cell bodies and dendritic arbors of neurons in the human cochlear nucleus and superior olivary complex and also that coincidence detector neurons of the medial superior olive colocalization of FMRP and Kv3.1b. We interpret these observations to suggest that the lower auditory brainstem is a potential site of dysfunction in FXS.

Characterization of human auditory brainstem circuits by calcium-binding protein immunohistochemistry.

The cochlear nucleus (CN) and superior olivary complex are auditory brainstem centers with essential roles in encoding temporal features of vocalizations, localization of sound sources and descending modulation of the cochlea. Numerous neuronal populations, across a multitude of laboratory mammals, have been characterized within these brainstem centers based on cell body morphology, dendritic architecture, afferent/efferent connections and neurochemistry. However, scant details are available for these neuronal populations in humans. The objective of this study is to further characterize human auditory hindbrain nuclei and examine the axonal connections between these structures. To this end, we have used immunohistochemistry and morphometric techniques to characterize neuronal populations and axonal projections in the human brainstem. Herein, we provide evidence for calretinin immunoreactive neurons and synaptic boutons in the ventral CN, axons in the trapezoid body, peridendritic boutons in the medial superior olive and calyceal endings in the medial nucleus of the trapezoid body (MNTB). Further, we demonstrate that the majority of neurons in the human MNTB are calbindin and Kv3.1b immunoreactive and that perisomatic profiles in this nucleus are vesicular glutamate transporter and Rab3a positive, suggesting that such profiles are in fact synaptic terminals.

Vascular anomalies in a case of situs inversus.

Situs inversus is a developmental condition in which the thoracic and abdominal organs fail to negotiate their normal migration patterns and the result is a mirror-image arrangement of these viscera. The literature provides evidence that individuals with this condition have a higher incidence of other congenital malformations (e.g. heart anomalies). Here we describe the dissection of a 71 year-old female cadaver with situs inversus, in which we discovered multiple anomalous vessels associated with the coeliac trunk directed toward the liver. In addition, we identified the inferior vena cava on the left side and a persistent supracardinal vein on the right, constituting a double inferior vena cava. Finally, we identified multiple abnormal venous channels associated with the sub-renal inferior vena cava. These vascular patterns are indeed a rare finding and have surgical implications but may indicate a higher incidence of vascular anomalies in cases of situs inversus.

Superior paraolivary nucleus of the rat is a GABAergic nucleus.

The presence of the inhibitory neurotransmitters glycine and GABA (gamma-amino butyric acid) and GAD (glutamic acid decarboxylase), the synthesizing enzyme for GABA, was examined by immunocytochemistry in the superior paraolivary nucleus (SPON) of the rat. Only rarely were SPON neurons observed to be glycine-immunoreactive, but the majority were GABA-immunoreactive. Using unbiased stereological counting methods, we estimated that this nucleus contains approximately 2500 neurons. Moreover, 90% of SPON neurons were immunolabeled by antisera directed against either the 65- or 67-kD isoform of GAD, or a third antiserum that recognizes both GAD isoforms. Morphometric analysis of GAD-immunolabeled neurons indicated that SPON neurons possess cell bodies and dendritic arbors that are elongated rostrocaudally and relatively flattened parasagittally. Abundant glycine-, GABA-, and GAD-immunoreactive punctate profiles-presumed to represent, for the most part, presynaptic axon terminals-were observed in apposition to SPON neurons. We conclude that the rat SPON contains a homogeneous population of multipolar GABAergic neurons that receive abundant GABAergic and glycinergic innervation. The vast majority of glycinergic inputs to SPON are presumed to originate in the ipsilateral medial nucleus of the trapezoid body, but the source(s) of its GABAergic innervation remains to be determined.