Notochord grafts do not suppress formation of neural crest cells or commissural neurons.

Grafting experiments previously have established that the notochord affects dorsoventral polarity of the neural tube by inducing the formation of ventral structures such as motor neurons and the floor plate. Here, we examine if the notochord inhibits formation of dorsal structures by grafting a notochord within or adjacent to the dorsal neural tube prior to or shortly after tube closure. In all cases, neural crest cells emigrated from the neural tube adjacent to the ectopic notochord. When analyzed at stages after ganglion formation, the dorsal root ganglia appeared reduced in size and shifted in position in embryos receiving grafts. Another dorsal cell type, commissural neurons, identified by CRABP and neurofilament immunoreactivity, differentiated in the vicinity of the ectopic notochord. Numerous neuronal cell bodies and axonal processes were observed within the induced, but not endogenous, floor plate 1 to 2 days after implantation but appeared to be cleared with time. These results suggest that dorsally implanted notochords cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest-derived dorsal root ganglia.     

GAD and GABA in an enriched population of cultured GABAergic neurons from rat cerebral cortex

To study various aspects of GABAergic metabolism in an easily accessible system, dissociated cells from postnatal rat cerebral cortex were cultured in a serum-based medium and characterized morphologically and biochemically. The majority (70–90%) of the neurons were GABAergic as determined by three double-labeling procedures. The specific activity of glutamine synthetase in the cultures was 4–5% of the levels in rat astrocyte cultures and intact rat brain, indicating that glia were a minor component. The developmental increase of GABA levels preceded the increase of GAD activity in both immunocytochemical and biochemical experiments. GABA turnover rates also increased with culture age and were 20–30% of GAD activity. Four anti-GAD antibodies, which recognize GAD subunits with differing molecular masses to varying degrees, were used to stain cultured neurons and make immunoblots. Immunoblots showed that the neurons contained two major subunits of GAD which differed in mass by 2 kDa. All four antibodies immunostained both neuronal perikarya and neurites but one antibody, which on the immunoblots predominantly labeled the GAD protein with the lower molecular weight, showed a somewhat more pronounced punctate staining, possibly indicating a principal localization to neurites.     

BCL-2 and BAX protect adult mice from lethal Sindbis virus infection but do not protect spinal cord motor neurons or prevent paralysis

Cellular proteins that regulate apoptotic cell death can modulate the outcome of Sindbis virus (SV) encephalitis in mice. Both endogenous and overexpressed BCL-2 and BAX proteins protect newborn mice from fatal SV infection by blocking apoptosis in infected neurons. To determine the effects of these cellular factors on the course of infection in older animals, a more neurovirulent SV vector (dsNSV) was constructed from a viral strain that causes both prominent spinal cord infection with hind-limb paralysis and death in weanling mice. This vector has allowed assessment of the effects of BCL-2 and BAX on both mortality and paralysis in these hosts. Similar to newborn hosts, weanling mice infected with dsNSV encoding BCL-2 or BAX survived better than animals infected with control viruses. This finding indicates that BCL-2 and BAX both protect neurons that mediate host survival. Neither cellular factor, however, could suppress the development of hind-limb paralysis or prevent the degeneration of motor neurons in the lumbar spinal cord. Infection of BAX knockout mice with dsNSV demonstrated that endogenous BAX also enhances the survival of animals but has no effect on paralysis. These findings for the spinal cord are consistent with earlier data showing that dying lumbar motor neurons do not exhibit an apoptotic morphology. Thus, divergent cell death pathways are activated in different target populations of neurons during neurovirulent SV infection of weanling mice.     

Human spinal GABA neurons alleviate spasticity and improve locomotion in rats with spinal cord injury

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Vasopressin and oxytocin excite MCH neurons, but not other lateral hypothalamic GABA neurons

Neurons that synthesize melanin-concentrating hormone (MCH) colocalize GABA, regulate energy homeostasis, modulate water intake, and influence anxiety, stress, and social interaction. Similarly, vasopressin and oxytocin can influence the same behaviors and states, suggesting that these neuropeptides may exert part of their effect by modulating MCH neurons. Using whole cell recording in MCH-green fluorescent protein (GFP) transgenic mouse hypothalamic brain slices, we found that both vasopressin and oxytocin evoked a substantial excitatory effect. Both peptides reversibly increased spike frequency and depolarized the membrane potential in a concentration-dependent and tetrodotoxin-resistant manner, indicating a direct effect. Substitution of lithium for extracellular sodium, Na(+)/Ca(2+) exchanger blockers KB-R7943 and SN-6, and intracellular calcium chelator BAPTA, all substantially reduced the vasopressin-mediated depolarization, suggesting activation of the Na(+)/Ca(2+) exchanger. Vasopressin reduced input resistance, and the vasopressin-mediated depolarization was attenuated by SKF-96265, suggesting a second mechanism based on opening nonselective cation channels. Neither vasopressin nor oxytocin showed substantial excitatory actions on lateral hypothalamic inhibitory neurons identified in a glutamate decarboxylase 67 (GAD67)-GFP mouse. The primary vasopressin receptor was vasopressin receptor 1a (V1aR), as suggested by the excitation by V1aR agonist [Arg(8)]vasotocin, the selective V1aR agonist [Phe(2)]OVT and by the presence of V1aR mRNA in MCH cells, but not in other nearby GABA cells, as detected with single-cell RT-PCR. Oxytocin receptor mRNA was also detected in MCH neurons. Together, these data suggest that vasopressin or oxytocin exert a minimal effect on most GABA neurons in the lateral hypothalamus but exert a robust excitatory effect on presumptive GABA cells that contain MCH. Thus, some of the central actions of vasopressin and oxytocin may be mediated through MCH cells.     

Avian pancreatic polypeptide (APP) immunoreactive neurons in the spinal cord and spinal trigeminal nucleus

Using indirect immunofluorescence technique, avian pancreatic polypeptide (APP) immunoreactive cell bodies and fibres have been observed in the superficial laminae of the dorsal horn of the spinal cord and of the spinal trigeminal nucleus. Fibres were also seen in the ventral horns, in low numbers at the cervical and thoracic levels and in high numbers at the lower lumbar and upper sacral levels. Neither total cord transection, nor dorsal rhizotomy, nor capsaicin treatment seemed to affect the APP systems described above. The present findings suggest that an APP-like peptide may be involved in processing of sensory information at the level of the first relay station.     

Cerebrospinal fluid cyclic nucleotides and GABA do not change in alcohol withdrawal

Gamma-aminobutyric acid (GABA) and cyclic adenosine monophosphate (AMP) and guanosine monophosphate (GMP) were compared in the cerebrospinal fluid (CSF) of 13 patients suffering from acute alcohol withdrawal, and the same patients during convalescence. No significant changes were found, but CSF norepinephrine (NE) levels were elevated in acute alcohol withdrawal and decreased toward normal during convalescence.     

Development of a high-affinity GABA uptake system in embryonic amphibian spinal neurons

High-affinity uptake systems for amino acid neurotransmitter precursors have been highly correlated with the use of the particular amino acid or its derivative as a transmitter. We have found interneurons in the Xenopus embryo spinal cord which accumulate GABA by a high-affinity uptake system. They originate near the end of gastrulation and their ability to accumulate GABA first appears at the early tail bud stage. By position and appearance they are comparable to some of the embryonic interneurons described by A. Roberts and J. D. W. Clarke (1982, Phil. Trans. R. Soc. London Ser. B 296, 195-212). GABA-accumulating neurons also develop in dissociated cell cultures made from the presumptive spinal cord of neural plate stage Xenopus embryos. GABA accumulation in cultured neurons, as in cells in vivo, occurs via a high-affinity uptake system; GABA-accumulating cells have the same time of origin as the cells in vivo, and the ability to accumulate GABA in the population of cultured neurons appears at a time equivalent to that observed in intact sibling embryos. Thus it seems likely that the population of GABA-accumulating neurons developing in cell culture corresponds to the GABA-accumulating interneurons in vivo. The development of these neurons in dissociated cell cultures permits perturbation experiments that would be difficult to perform in vivo. We have examined the development of high-affinity GABA uptake in conditions that permit no electrical impulse activity in the cultures. The onset and extent of development of GABA accumulation in the neuronal population are normal under these conditions.     

Some monoclonal anti-human lymphocyte and class II MHC antigen antibodies do not stain snap frozen Macaca fascicularis lymphocytes or tissue sections.

Experiments were designed to stain snap frozen and fixed Macaca fascicularis peripheral blood mononuclear cell (PBMC) preparations and colon sections assuming that monoclonal mouse anti-human lymphocyte and class II major histocompatibility complex antigen antibodies would cross-react with the monkey counterparts to the human antigens. Most of the monoclonals used in this study did not stain the monkey frozen and fixed tissues in immunoenzymatic protocols despite adequate staining of control human tissues. The fact that snap frozen Macaca fascicularis PBMCs and tissue sections were not always stained is of practical importance for experimental design in Macaca fascicularis using anti-human reagents.     

Development of sensitivity to glycine, GABA and beta-alanine in cultured spinal neurons

Whole cell patch-clamp recording of cultured chick spinal neurons, presumed to be motoneurons, revealed currents elicited in these cells by GABA, glycine and beta-alanine, corresponding to the opening of chloride channels. During maturation, sensitivity to all three transmitters were first detected on the day 6 of culture, and appeared in most neurons by day 8. However, at all stages of development, a fraction of the cells were sensitive only to GABA; this observation supports the notion that the GABA and the glycine receptors are distinct. On the other hand, separate activation by glycine and beta-alanine was never observed, in agreement with the postulate that these amino acids bind to the same receptor.     

Effect of fenibut on the GABA B receptors of the spinal motor neurons

It has been established in experiments on the isolated spinal cord of 7-14-day-old rats that the GABAB-mimetic phenibut (10(-5)--10(-4) M) elicits a slow-developing depolarization of motoneurons, suppression of spontaneous activity and polysynaptic reflex discharges of motoneurons, recorded from the ventral roots. Administered under the same conditions GABA produces de- and hyperpolarization of motoneurons. The depolarization of motoneurons elicited by phenibut and GABA is not reversed by picrotoxin in contradistinction to the GABA-induced hyperpolarization of motoneurons, being associated with a direct action of the GABA-mimetics on postsynaptic GABAB receptors of motoneurons. Diazepam (10(-9)--10(-6) M) potentiates the effects of phenibut supposedly via benzodiazepine receptors bound with GABAA receptors (an independent interaction).     

Immunoreactivity for GABA,GAD65, GAD67 and Bestrophin-1 in the Meninges and the Choroid Plexus: Implications for Non-Neuronal Sources for GABA in the Developing Mouse Brain

Neural progenitors in the developing neocortex, neuroepithelial cells and radial glial cells, have a bipolar shape with a basal process contacting the basal membrane of the meninge and an apical plasma membrane facing the lateral ventricle, which the cerebrospinal fluid is filled with. Recent studies revealed that the meninges and the cerebrospinal fluid have certain roles to regulate brain development. γ-aminobutyric acid (GABA) is a neurotransmitter which appears first during development and works as a diffusible factor to regulate the properties of neural progenitors. In this study, we examined whether GABA can be released from the meninges and the choroid plexus in the developing mouse brain. Immunohistochemical analyses showed that glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67), both of which are GABA-synthesizing enzymes, are expressed in the meninges. The epithelial cells in the choroid plexus express GAD65. GABA immunoreactivity could be observed beneath the basal membrane of the meninge and in the epithelial cells of the choroid plexus. Expression analyses on Bestrophin-1, which is known as a GABA-permeable channel in differentiated glial cells, suggested that the cells in the meninges and the epithelial cells in the choroid plexus have the channels able to permeate non-synaptic GABA into the extracellular space. Further studies showed that GAD65/67-expressing meningeal cells appear in a manner with rostral to caudal and lateral to dorsal gradient to cover the entire neocortex by E14.5 during development, while the cells in the choroid plexus in the lateral ventricle start to express GAD65 on E11–E12, the time when the choroid plexus starts to develop in the developing brain. These results totally suggest that the meninges and the choroid plexus can work as non-neuronal sources for ambient GABA which can modulate the properties of neural progenitors during neocortical development.     

Malaria parasites do not contain or synthesize sialic acids

The capacity of Plasmodia to synthesize sialic acids was investigated by adding radioactive acetate to short-term in vitro cultures of the intraerythrocytic asexual forms of three malaria parasites (the human malaria Plasmodium falciparum in Aotus trivirgatus erythrocytes; the simian malaria P. knowlesi in rhesus monkey erythrocytes; the rodent malaria P. berghei in mouse erythrocytes) and to cultures of extracellular zygotes of the avian malaria P. gallinaceum. Radioactive acetate was added to normal rhesus monkey erythrocytes and to cells of the murine myeloma NS-1 for comparison. Although [1-14C]-acetate labeled many proteins with each malaria parasite and the NS-1 cells, analysis of purified sialic acids revealed that only with the NS-1 cells was radioactivity incorporated into sialic acids. Furthermore, N-acetyl[6-3H]mannosamine was not incorporated into sialic acids or malarial glycoproteins when added to P. knowlesi cultures. All of the malaria parasites underwent growth or differentiation during these experiments as measured by [35S]methionine uptake into protein and by light microscopy. Extracellular parasites largely free of erythrocyte membranes were prepared to determine whether Plasmodia contain sialic acids that are not labeled by exogenous precursors. Purified merozoites of P. knowlesi and zygotes of P. gallinaceum did not contain detectable amounts of sialic acids on chemical analysis. Thus, although we could show that Plasmodia can incorporate radioactive sugars such as glucosamine, galactose and mannose into proteins, presumably glycoproteins, they do not synthesize sialic acids or sialo-glycoproteins, nor do they contain sialo-glycoconjugates of host origin.     

Distribution of glycine/GABA neurons in the ventromedial medulla with descending spinal projections and evidence for an ascending glycine/GABA projection

The ventromedial medulla (VM), subdivided in a rostral (RVM) and a caudal (CVM) part, has a powerful influence on the spinal cord. In this study, we have identified the distribution of glycine and GABA containing neurons in the VM with projections to the cervical spinal cord, the lumbar dorsal horn, and the lumbar ventral horn. For this purpose, we have combined retrograde tracing using fluorescent microspheres with fluorescent in situ hybridization (FISH) for glycine transporter 2 (GlyT2) and GAD67 mRNAs to identify glycinergic and/or GABAergic (Gly/GABA) neurons. Since the results obtained with FISH for GlyT2, GAD67, or GlyT2 + GAD67 mRNAs were not significantly different, we concluded that glycine and GABA coexisted in the various projection neurons. After injections in the cervical cord, we found that 29% ± 1 (SEM) of the retrogradely labeled neurons in the VM were Gly/GABA (RVM: 43%; CVM: 21%). After lumbar dorsal horn injections 31% ± 3 of the VM neurons were Gly/GABA (RVM: 45%; CVM: 12%), and after lumbar ventral horn injections 25% ± 2 were Gly/GABA (RVM: 35%; CVM: 17%). In addition, we have identified a novel ascending Gly/GABA pathway originating from neurons in the area around the central canal (CC) throughout the spinal cord and projecting to the RVM, emphasizing the interaction between the ventromedial medulla and the spinal cord. The present study has now firmly established that GABA and glycine are present in many VM neurons that project to the spinal cord. These neurons strongly influence spinal processing, most notably the inhibition of nociceptive transmission.     

GAD65 is essential for synthesis of GABA destined for tonic inhibition regulating epileptiform activity

GABA is synthesized from glutamate by glutamate decarboxylase (GAD), which exists in two isoforms, that is, GAD65 and GAD67. In line with GAD65 being located in the GABAergic synapse, several studies have demonstrated that this isoform is important during sustained synaptic transmission. In contrast, the functional significance of GAD65 in the maintenance of GABA destined for extrasynaptic tonic inhibition is less well studied. Using GAD65-/- and wild type GAD65+/+ mice, this was examined employing the cortical wedge preparation, a model suitable for investigating extrasynaptic GABA(A) receptor activity. An impaired tonic inhibition in GAD65-/- mice was revealed demonstrating a significant role of GAD65 in the synthesis of GABA acting extrasynaptically. The correlation between an altered tonic inhibition and metabolic events as well as the functional and metabolic role of GABA synthesized by GAD65 was further investigated in vivo. Tonic inhibition and the demand for biosynthesis of GABA were augmented by injection of kainate into GAD65-/- and GAD65+/+ mice. Moreover, [1-(13) C]glucose and [1,2-(13) C]acetate were administered to study neuronal and astrocytic metabolism concomitantly. Subsequently, cortical and hippocampal extracts were analyzed by NMR spectroscopy and mass spectrometry, respectively. Although seizure activity was induced by kainate, neuronal hypometabolism was observed in GAD65+/+ mice. In contrast, kainate evoked hypermetabolism in GAD65-/- mice exhibiting deficiencies in tonic inhibition. These findings underline the importance of GAD65 for synthesis of GABA destined for extrasynaptic tonic inhibition, regulating epileptiform activity.     

Response of the spinal trigeminal nucleus neurons to electric stimulation of the rat dura mater

Aseptic inflammation of tissues surrounding large meningeal blood vessels, e.g. the superior sagittal sinus, underlies pathogenesis of migraine. This inflammation develops due to antidromic activation of sensory trigeminal nerve endings and is followed by changes in responses of the spinal nucleus of the trigeminal nerve neurons to electrical stimulation of the superior sagittal sinus. However, characteristics of these reactions are still unclear. In experiments ou urethane-anesthetized rats, responses of 387 neurons of the spinal nucleus of the trigeminal nerve to electrical stimulation of the superior sagittal sinus, were recorded. It was tial discharge with the latency 7 to 19 ms (11.4 +/- 0.17 ms) and a subsequent long-lasting discharge with the latency 20 to 50 ms (34.2 +/- 0.8 ms). It is presumed that the first phase reflects orthodromic activation of prevascular A delta and C-fibers of the trigeminal nerve while the second phase is connected with activation of meningeal C-fibers which have low conduction velocity, and/or with a secondary activation of perivascular sensory endings of trigeminal nerve by releasing algogenic and vasoactive substances. These changes could be used as an indicator of efficacy of some antimigraine substances in animal experiments.     

Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU⁺ cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU⁺ cells, very few are mash1⁺ or nestin⁺ stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1⁺ microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.