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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, elevated 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 flavor enhancer in many processed foods. Previous studies have shown that MSG administration during the early postnatal period results in neurodegenerative changes in several forebrain regions, characterized by neuronal loss and neuroendocrine abnormalities. Systemic delivery of MSG during the neonatal period and induction of glutamate neurotoxicity in the cochlea have both been shown to result in fewer neurons in the spiral ganglion. We hypothesized that an MSG-induced loss of neurons in the spiral ganglion would have a significant impact on the number of neurons in the cochlear nuclei and superior olivary complex (SOC). Indeed, we found that exposure to MSG from postnatal days 4 through 10 resulted in significantly fewer neurons in the cochlear nuclei and SOC and significant dysmorphology in surviving neurons. Moreover, we found that neonatal MSG exposure resulted in a significant decrease in the expression of both calretinin and calbindin. These results suggest that neonatal exposure to MSG interferes with early development of the auditory brainstem and impacts expression of calcium binding proteins, both of which may lead to diminished auditory function., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

It is unclear if habituation of hindbrain A2 metabolo‑sensory neurons to recurrent insulin-induced hypoglycemia (RIIH) correlates with estradiol-dependent adjustments in energy metabolism that favor positive energy balance. Laser-microdissected A2 cells from estradiolor oil-implanted ovariectomized female rats were analyzed by Western blot to assess effects of three prior daily insulin injections on basal and hypoglycemic patterns of catecholamine biosynthetic enzyme dopamine-beta-hydroxylase (DβH) and rate-limiting energy pathway enzyme protein expression. Precedent hypoglycemia respectively decreased or increased baseline DβH expression in estradiol- (E) vs. oil (O)-treated rats; this protein profile was further suppressed or augmented in those animals at 2 hr after re-induction of hypoglycemia. These data suggest that estradiol may curtail A2 noradrenergic‑controlled functions both in the midst of and between hypoglycemic bouts. Results also show that prior hypoglycemia exposure upregulated A2 neuron glycolytic enzyme protein levels when E was present, and exerted differential effects on basal and hypoglycemia-associated respiratory chain and fatty acid synthetic pathway enzyme expression. E may thus accordingly amplify glycolysis-derived metabolites/energy, coupled with reduced reliance on oxidative phosphorylation, and activate the fatty acid synthetic pathway during RIIH. E may also be of benefit by preventing maladaptive reductions in A2 neuron Krebs cycle/electron transport enzyme expression during re-exposure to hypoglycemia. Augmentation of negative energy balance during this recurring metabolic stress in the absence of E is a likely impetus for augmented vs. decreased A2 signaling of energy imbalance by DβH in O vs. E rats during RIIH.

Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPA receptor-related proteins, known as TARPs. Little is known about the role of TARPs during development, or about their function in non-mammalian organisms. Here we report the presence of TARPs, specifically the prototypical TARP, stargazin, in developing zebrafish. We find that zebrafish express two forms of stargazin, Cacng2a and Cacng2b from as early as 12-h post fertilization (hpf). Knockdown of Cacng2a and Cacng2b via splice-blocking morpholinos resulted in embryos that exhibited deficits in C-start escape responses, showing reduced C-bend angles, smaller tail velocities and aberrant C-bend turning directions. Injection of the morphants with Cacng2a or 2b mRNA rescued the morphological phenotype and the synaptic deficits. To investigate the effect of reduced Cacng2a and 2b levels on synaptic physiology, we performed whole cell patch clamp recordings of AMPA mEPSCs from zebrafish Mauthner cells. Knockdown of Cacng2a results in reduced AMPA currents and lower mEPSC frequencies, whereas knockdown of Cacng2b displayed no significant change in mEPSC amplitude or frequency. Non-stationary fluctuation analysis confirmed a reduction in the number of active synaptic receptors in the Cacng2a but not in the Cacng2b morphants. Together, these results suggest that Cacng2a is required for normal trafficking and function of synaptic AMPARs, while Cacng2b is largely non-functional with respect to the development of AMPA synaptic transmission., (© 2015 Wiley Periodicals, Inc.)

The proper development and maturation of neuronal circuits require precise migration of component neurons from their birthplace (germinal zone) to their final positions. Little is known about the effects of aberrant neuronal position on the functioning of organized neuronal groups, especially in mammals. Here, we investigated the formation and properties of brainstem respiratory neurons in looptail (Lp) mutant mice in which facial motor neurons closely apposed to some respiratory neurons fail to migrate due to loss of function of the Wnt/Planar Cell Polarity (PCP) protein Vangl2. Using calcium imaging and immunostaining on embryonic hindbrain preparations, we found that respiratory neurons constituting the embryonic parafacial oscillator (e-pF) settled at the ventral surface of the medulla in Vangl2(Lp/+) and Vangl2(Lp/Lp) embryos despite the failure of tangential migration of its normally adjacent facial motor nucleus. Anatomically, the e-pF neurons were displaced medially in Lp/+ embryos and rostro-medially Lp/Lp embryos. Pharmacological treatments showed that the e-pF oscillator exhibited characteristic network properties in both Lp/+ and Lp/Lp embryos. Furthermore, using hindbrain slices, we found that the other respiratory oscillator, the preBötzinger complex, was also anatomically and functionally established in Lp mutants. Importantly, the displaced e-pF oscillator established functional connections with the preBötC oscillator in Lp/+ mutants. Our data highlight the robustness of the developmental processes that assemble the neuronal networks mediating an essential physiological function.

Evidence suggests that release of oxytocin in the nucleus tractus solitarius (NTS) of the hindbrain from descending projections that originate in the paraventricular nucleus can inhibit food intake by amplifying the satiety response to cholecystokinin (CCK). To further evaluate this mechanism in rats, we used a novel cytotoxin, saporin conjugated to oxytocin (OXY-SAP), a compound designed to destroy cells that express oxytocin receptors (OXYr). OXY-SAP was injected directly into the NTS to lesion neurons that express OXYr and that are implicated in potentiating CCK's satiety effects. The control consisted of injection of saporin conjugated to a nonsense peptide. We found that OXY-SAP was cytotoxic to human uterine smooth muscle cells in vitro, demonstrating that OXY-SAP can lesion cells that express OXYr. Using laser capture microdissection and real-time quantitative PCR, we demonstrated that OXYr mRNA levels were reduced in the NTS after OXY-SAP administration. Moreover, we found that OXY-SAP attenuated the efficacy of CCK-8 to reduce food intake and blocked the actions of an OXYr antagonist to stimulate food intake. The findings suggest that OXY-SAP is an effective neurotoxin for in vivo elimination of cells that express OXYr and is potentially useful for studies to analyze central nervous system mechanisms that involve the action of oxytocin on food intake and other physiological processes.

It is well known that ethanol damages the developing nervous system by augmenting apoptosis. Previously, this laboratory reported that ethanol augments apoptosis in fetal rhombencephalic neurons, and that the increased apoptosis is associated with reduced activity of the phosphatidylinositol 3-kinase pathway and downstream expression of pro-survival genes. Other laboratories have shown that another mechanism by which ethanol induces apoptosis in developing neurons is through the generation of reactive oxygen species (ROS) and the associated oxidative stress. The present study used an in vitro model to investigate the potential neuroprotective effects of several antioxidants against ethanol-associated apoptosis in fetal rhombencephalic neurons. The investigated antioxidants included three phenolics: (-)-epigallocatechin-3-gallate (EGCG), a flavanoid polyphenol found in green tea; curcumin, found in tumeric; and resveratrol (3,5,4'-trihydroxystilbene), a component of red wine. Additional antioxidants, including melatonin, a naturally occurring indole, and alpha-lipoic acid, a naturally occurring dithiol, were also investigated. These studies demonstrated that a 24-hour treatment of fetal rhombencephalic neurons with 75 mM ethanol caused a 3-fold increase in the percentage of apoptotic neurons. However, co-treatment of these cultures with any of the five different antioxidants prevented ethanol-associated apoptosis. Antioxidant treatment did not alter the extent of apoptosis in control neurons, i.e., those cultured in the absence of ethanol. These studies showed that several classes of antioxidants can exert neuroprotection against ethanol-associated apoptosis in fetal rhombencephalic neurons.

Recent studies implicate the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), in septopreoptic (SPO) mechanisms that suppress preovulatory pituitary luteinizing hormone (LH) secretion during neuroglucopenia. Since Fos immunolabeling of the SPO of rats treated by caudal fourth ventricular (CV4) administration of the glucose antimetabolite, 5-thioglucose (5TG), parallels the distribution of GABA neuronal perikarya, the current studies investigated the genomic responsiveness of neuroanatomically-defined populations of glutamate decarboxylase (GAD)-immunoreactive (-ir) neurons in this region of the brain to hindbrain glucoprivation. In lieu of reports that CV4 5TG enhances SPO GABA turnover via mu opioid receptor (mu-R)-dependent mechanisms and evidence that GAD- and mu-R-ir are codistributed within the SPO, patterns of cellular colocalization of these antigens were also evaluated here. Neural tissue was obtained from groups of steroid-primed ovariectomized female rats 2 h after CV4 injection of vehicle or 5TG. Neuronal cell bodies in the lateral and medial septum, medial (MPN) and median preoptic nuclei (MEPO), and rostral medial preoptic area (rMPO) were immunostained for cytoplasmic GAD-ir, but only GAD-reactive neurons in the rMPO and MEPO exhibited robust nuclear colabeling for Fos in response to 5TG. SPO GABA neurons in the vehicle-treated controls were uniformly Fos-ir-negative. Dual immunolabeling for GAD- and mu-R revealed approximately 52% and 36% colabeling of this phenotype in the MEPO and MPN, and colocalization of lesser magnitude (18%) in the rMPO. These results demonstrate site-specific genomic activation of GABAergic neurons in the female rat SPO by CV4 glucose antimetabolite administration, and implicate MEPO and rMPO GABA cell populations in neural pathways that mediate regulatory effects of hindbrain glucoprivic signaling on CNS functions, including inhibition of the steroid positive feedback-activated gonadotropin-releasing hormone/LH neuroendocrine axis. The current studies also support the view that a proportion of neuroglucoprivic-sensitive GABA neurons in the MEPO and rMPO may be direct substrates for mu-R ligand modulatory actions during this state of central substrate imbalance., ((c) 2007 S. Karger AG, Basel)

We report here the characterization of a mutant mouse line with a specific gene trap event in the Mdm4 locus. Absence of Mdm4 expression results in embryonic lethality (10.5 days postcoitum [dpc]), which was rescued by transferring the Mdm4 mutation into a Trp53-null background. Mutant embryos were characterized by overall growth deficiency, anemia, improper neural tube closure, and dilation of lateral ventricles. In situ analysis demonstrated increased levels of p21(CIP1/Waf1) and lower levels of Cyclin E and proliferating cell nuclear antigen expression. Consistent with lack of 5-bromo-2'-deoxyuridine incorporation, these data suggest a block of mutant embryo cells in the G(1) phase of the cell cycle. Accordingly, Mdm4-deficient mouse embryonic fibroblasts manifested a greatly reduced proliferative capacity in culture. Moreover, extensive p53-dependent cell death was specifically detected in the developing central nervous system of the Mdm4 mutant embryos. These findings unambiguously assign a critical role for Mdm4 as a negative regulator of p53 and suggest that Mdm4 could contribute to neoplasias retaining wild-type Trp53. Finally, we provide evidence indicating that Mdm4 plays no role on cell proliferation or cell cycle control that is distinct from its ability to modulate p53 function.

Glutamate is involved in the degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). However, the aetiology of ALS appears heterogeneous, leading to the possibility that patient subgroups with different pathophysiology may exist. The concentration of glutamate in cerebrospinal fluid (CSF) is measured using a new HPLC method with coulometric detection in a large cohort of ALS patients and controls: 377 ALS patients, 88 neurological patients and 18 normal controls. In ALS patients, and only in these subjects, the existence of two groups was observed, one with normal glutamate concentrations and one (40.8% of ALS patients) with high glutamate concentrations. High glutamate concentrations were correlated with a spinal onset of the disease, more impaired limb function and a higher rate of muscle deterioration. These results suggest that elevations of CSF glutamate concentrations could reflect the intensity of cell insult in the spinal cord. It remains to be determined if the group of patients with high CSF glutamate concentrations represents a specific subgroup of patients in terms of mechanism of disease, or only in terms of the spatial extent of motor neuron insult.

To determine whether neurons lacking huntingtin can participate in development and survive in postnatal brain, we used two approaches in an effort to create mice consisting of wild-type cells and cells without huntingtin. In one approach, chimeras were created by aggregating the 4-8 cell embryos from matings of Hdh (+/-) mice with wild-type 4-8 cell embryos. No chimeric offspring that possessed homozygous Hdh (-/-) cells were obtained thereby, although statistical considerations suggest that such chimeras should have been created. By contrast, Hdh (-/-) ES cells injected into blastocysts yielded offspring that were born and in adulthood were found to have Hdh (-/-) neurons throughout brain. The Hdh (-/-) cells were, however, 5-10 times more common in hypothalamus, midbrain, and hindbrain than in telencephalon and thalamus. Chimeric animals tended to be smaller than wild-type littermates, and chimeric mice rich in Hdh (-/-) cells tended to show motor abnormalities. Nonetheless, no brain malformations or pathologies were evident. The apparent failure of aggregation chimeras possessing Hdh (-/-) cells to survive to birth is likely attributable to the previously demonstrated critical role of huntingtin in extraembryonic membranes. That Hdh (-/-) cells in chimeric mice created by blastocyst injection are under-represented in adult telencephalon and thalamus implies a role for huntingtin in the development of these regions, whereas the neurological dysfunction in brains enriched in Hdh (-/-) cells suggests a role for huntingtin in adult brain. Nonetheless, the lengthy survival of Hdh (-/-) cells in adult chimeric mice indicates that individual neurons in many brain regions do not require huntingtin to participate in normal brain development and to survive.

Advanced silver stains and immunohistochemical reactions against alpha-synuclein were used to detect Parkinson's disease-related cytoskeletal abnormalities in select lower brain stem nuclei. Various types of inclusion bodies including inconspicuous and heretofore unnoted granular particles and thread-like Lewy neurites were visualized. Of the nuclei investigated (gigantocellular reticular nucleus, bulbar raphe nuclei, coeruleus-subcoeruleus area), only lipofuscin- or neuromelanin-laden neuronal types showed a propensity to develop the pathological changes. Neuronal types devoid of pigment deposits remained free of the cytoskeletal abnormalities. Fine, dust-like particles and small globular Lewy bodies were encountered solely within the limits of intraneuronal lipofuscin or neuromelanin deposits.

The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive condition involving craniofacial and central nervous system malformations with occasional holoprosencephaly (HPE). It is caused by a defect in the 7-dehydrocholesterol (7-DHC) reductase, the enzyme catalyzing the last step of cholesterol biosynthesis. Treatment of pregnant rats with inhibitors of 7-DHC reductase, either AY9944 or BM15.766, has provided a valuable model to study the pathogenesis in SLOS. Recently, cholesterol has been shown to be involved in the post-translational activation of the signaling protein Sonic Hedgehog. To identify the early defects associated with HPE in a rat model of SLOS, and to compare the phenotype of the treated embryos with that of the Shh(-/-) mutants, we examined brain morphology and expression of three developmental genes (Shh, Otx2, and Pax6 ) in 23-somite stage embryos from AY9944-treated dams. We report clearly abnormal morphology of the developing brain, concerning primarily the ventral aspect of the neural tube. We observed a reduced or absent expression of Shh and Otx2 in their ventral domain associated with extended ventral expression of Pax6. The results suggest an absence of the midline ventral cell type at all levels of the cranial neural tube. They provide further evidence that cholesterol-deficiency-induced HPE originates from impaired Shh signaling activity in the ventral neural tube., (Copyright 1999 Wiley-Liss, Inc.)

This study tested the hypothesis that systemic stressors in rats activate glucagon-like peptide-1 (GLP-1)-immunoreactive neurons in the caudal brain stem, including those that project to the paraventricular nucleus of the hypothalamus (PVN). Neural tracer was microinjected into the PVN to retrogradely label brain stem neurons. Seven to ten days later, rats were injected with lithium chloride (LiCl; 50 mg/kg). Additional non-tracer-injected rats were treated with lipopolysaccharide (LPS; 100 microgram/kg) or CCK (100 microgram/kg) or were allowed to consume a very large meal. Rats were killed 90-120 min after drug treatment or 30 min after the meal. Brains were processed for immunocytochemical localization of c-Fos (a marker of neuronal activation), GLP-1, and, when appropriate, neural tracer. The majority of GLP-1 neurons were activated to express c-Fos after LiCl, LPS, or CCK treatment, including (in LiCl-treated rats) those projecting to the PVN. In contrast, GLP-1 neurons rarely expressed c-Fos after ingestion of a large meal, despite prominent activation of other brain stem neurons. These results suggest that GLP-1 neurons are uniquely activated in situations of interoceptive stress, and may participate in adaptive hypothalamic stress responses.

The role of zebrafish hedgehog genes in branchiomotor neuron development was analyzed by examining mutations that affect the expression of the hedgehog genes and by overexpressing these genes in embryos. In cyclops mutants, reduction in sonic hedgehog (shh) expression, and elimination of tiggy-winkle hedgehog (twhh) expression, correlated with reductions in branchiomotor neuron populations. Furthermore, branchiomotor neurons were restored in cyclops mutants when shh or twhh was overexpressed. These results suggest that Shh and/or Twhh play an important role in the induction of branchiomotor neurons in vivo. In sonic-you (syu) mutants, where Shh activity was reduced or eliminated due to mutations in shh, branchiomotor neurons were reduced in number in a rhombomere-specific fashion, but never eliminated. Similarly, spinal motor neurons were reduced, but not eliminated, in syu mutants. These results demonstrate that Shh is not solely responsible for inducing branchiomotor and spinal motor neurons, and suggest that Shh and Twhh may function as partially redundant signals for motor neuron induction in zebrafish., (Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved.)

The mouse neurological mutant lurcher (Lc) results from a semidominant mutation. Heterozygous Lc/+ mice are viable but ataxic because Lc/+ Purkinje cells die by apoptosis within the first 3 weeks of life. Lc/Lc mice die shortly after birth. To aid in understanding the function of the lurcher gene product, we have examined the embryonic development of homozygous lurcher animals. The ratio of +/+:Lc/+:Lc/Lc animals did not deviate significantly from the expected 1:2:1. Homozygous lurcher mice at P0 were found to be normal under gross morphological examination. However, these mice weighed less, lacked milk in their stomach, and died within the first day of life. No resorbed embryos were found at embryonic day (E) 17.5, indicating that all homozygous lurchers survived until birth. Histological examination of P0 animals revealed that in homozygous lurcher mice the patterning of the brain is normal but that there has been a massive loss of hindbrain neurons during embryonic development. A particularly conspicuous consequence of the Lc/Lc genotype at birth is the complete absence of large neurons comprising the trigeminal motor nucleus. These neurons arise normally and are maintained until E15.5. However, beginning at E15.5 large numbers of pyknotic cells are evident in the trigeminal motor nucleus, suggesting that these cells die coincident with their terminal differentiation in the developing hindbrain. Because the trigeminal motor nucleus controls muscles required for suckling, these results suggest an explanation for the neonatal death of homozygous Lc animals. These data demonstrate that the severe and dose-dependent developmental consequences of lurcher gene action result from degeneration of distinct neuronal populations on maturation in the developing CNS.

Differential vulnerability in the hindbrain neurons was examined immunohistochemically during hindbrain ischemia in the gerbil. Hindbrain ischemia was produced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Local cerebral blood flow was measured by quantitative autoradiographic technique after 5 min of ischemia and was reduced to less than 5 ml/100 g per min in the cerebellum, the pons, and the medulla, indicating that severe and reproducible hindbrain ischemia was induced immediately after occlusion. For immunohistochemical investigation, four gerbils each were used for each ischemic period of 5, 10, 15, and 30 min. Immunohistochemical lesions, detected by the reaction for microtubule-associated protein 2, were visible in the lateral vestibular nucleus and the cerebellar interpositus nucleus even after 5 min of ischemia. These results suggested that these areas were more vulnerable than others, although blood flow was markedly reduced in various regions of the hindbrain. In contrast, areas related to respiratory or cardiovascular control were rather resistant to ischemia. The present study suggests that selective vulnerability during hindbrain ischemia depends mainly on different metabolic characteristics inherent to various neurons in the hindbrain.