A dual role for the RhoGEF Ephexin5 in regulation of dendritic spine outgrowth.

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines.

Activity-Dependent Degradation of Synaptic Vesicle Proteins Requires Rab35 and the ESCRT Pathway.

UNLABELLED: Synaptic vesicle (SV) pools must maintain a functional repertoire of proteins to efficiently release neurotransmitter. The accumulation of old or damaged proteins on SV membranes is linked to synaptic dysfunction and neurodegeneration. However, despite the importance of SV protein turnover for neuronal health, the molecular mechanisms underlying this process are largely unknown. Here, we have used dissociated rat hippocampal neurons to investigate the pathway for SV protein degradation. We find that neuronal activity drives the degradation of a subset of SV proteins and that the endosomal sorting complex required for transport (ESCRT) machinery and SV-associated GTPase Rab35 are key elements of this use-dependent degradative pathway. Specifically, neuronal activity induces Rab35 activation and binding to the ESCRT-0 protein Hrs, which we have identified as a novel Rab35 effector. These actions recruit the downstream ESCRT machinery to SV pools, thereby initiating SV protein degradation via the ESCRT pathway. Our findings show that the Rab35/ESCRT pathway facilitates the activity-dependent removal of specific proteins from SV pools, thereby maintaining presynaptic protein homeostasis. SIGNIFICANCE STATEMENT: Synaptic transmission is mediated by the release of chemical neurotransmitters from synaptic vesicles (SVs). This tightly regulated process requires a functional pool of SVs, necessitating cellular mechanisms for removing old or damaged proteins that could impair SV cycling. Here, we show that a subset of SV proteins is degraded in an activity-dependent manner and that key steps in this degradative pathway are the activation of the small GTPase Rab35 and the subsequent recruitment of the endosomal sorting complex required for transport (ESCRT) machinery to SV pools. Further, we demonstrate that ESCRT-0 component Hrs is an effector of Rab35, thus providing novel mechanistic insight into the coupling of neuronal activity with SV protein degradation and the maintenance of functional SV pools.

The α2ß2 isoform combination dominates the astrocytic Na+ /K+ -ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation.

Synaptic activity results in transient elevations in extracellular K+ , clearance of which is critical for sustained function of the nervous system. The K+ clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na+ /K+ -ATPase. The Na+ /K+ -ATPase consists of an α and a ß subunit, each with several isoforms present in the central nervous system, of which the α2ß2 and α2ß1 isoform combinations are kinetically geared for astrocytic K+ clearance. While transcript analysis data designate α2ß2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and ß isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na+ /K+ -ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both ß1 and ß2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na+ /K+ -ATPase is dominated by α2, paired with ß1 or ß2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics.

Excitation-induced ataxin-3 aggregation in neurons from patients with Machado-Joseph disease.

Machado-Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that L-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca(2+)-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na(+) and K(+) channels as well as ionotropic and voltage-gated Ca(2+) channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.

Cortactin-binding protein 2 modulates the mobility of cortactin and regulates dendritic spine formation and maintenance.

Dendritic spines, the actin-rich protrusions emerging from dendrites, are the locations of excitatory synapses in mammalian brains. Many molecules that regulate actin dynamics also influence the morphology and/or density of dendritic spines. Since dendritic spines are neuron-specific subcellular structures, neuron-specific proteins or signals are expected to control spinogenesis. In this report, we characterize the distribution and function of neuron-predominant cortactin-binding protein 2 (CTTNBP2) in rodents. An analysis of an Expressed Sequence Tag database revealed three splice variants of mouse CTTNBP2: short, long, and intron. Immunoblotting indicated that the short form is the dominant CTTNBP2 variant in the brain. CTTNBP2 proteins were highly concentrated at dendritic spines in cultured rat hippocampal neurons as well as in the mouse brain. Knockdown of CTTNBP2 in neurons reduced the density and size of dendritic spines. Consistent with these morphological changes, the frequencies of miniature EPSCs in CTTNBP2 knockdown neurons were lower than those in control neurons. Cortactin acts downstream of CTTNBP2 in spinogenesis, as the defects caused by CTTNBP2 knockdown were rescued by overexpression of cortactin but not expression of a CTTNBP2 mutant protein lacking the cortactin interaction. Finally, immunofluorescence staining demonstrated that, unlike cortactin, CTTNBP2 stably resided at dendritic spines even after glutamate stimulation. Fluorescence recovery after photobleaching further suggested that CTTNBP2 modulates the mobility of cortactin in neurons. CTTNBP2 may thus help to immobilize cortactin in dendritic spines and control the density of dendritic spines.

Microarray analysis of rat hippocampus exposed to excitotoxicity: reversal Na(+)/Ca(2+) exchanger NCX3 is overexpressed in glial cells.

Multiple factors are involved in the glutamate-induced excitotoxicity phenomenon, such as overload of ionotropic and metabotropic receptors, excess Ca(2+) influx, nitric oxide synthase activation, oxidative damage due to increase in free radicals, and release of endogenous polyamine, among others. In order to attempt a more integrated approach to address this issue, we established, by microarray analysis, the hippocampus gene expression profiles under glutamate-induced excitotoxicity conditions. Increased gene expression is mainly related to excitotoxicity (CaMKII, glypican 2, GFAP, NCX3, IL-2, and Gmeb2) or with cell damage response (dynactin and Ecel1). Several genes that augmented their expression are related to glutamatergic system modulation, in particular with NMDA receptor modulation and calcium homeostasis (IL-2, CaMKII, acrosin, Gmeb2, hAChE, Slc83a, and SP1 factor). Conversely, among genes that diminished their expression, we found the Syngap 1, which is downregulated by CaMKII, and the MHC II, which is downregulated by glutamate. Changes observed in gene expression induced by monosodium glutamate (MSG) neonatal treatment in the hippocampus are consistent with the activation of the mechanisms that modulate NMDA receptor function as well as with the implementation of plastic response to cell damage and intracellular calcium homeostasis. Regarding this aspect, we report here that NCX3/Slc8a3, a Na(+)/Ca(2+) membrane exchanger, is highly expressed in astrocytes, both in vitro and in vivo, in response to glutamate-induced excitotoxicity. Hence, the results of this analysis present a broad view of the expression profile elicited by MSG neonatal treatment, and lead us to suggest the possible molecular pathways of action and reaction involved under this experimental model of excitotoxicity.

Presynaptic inhibition of glutamate transmission by α2 receptors in the VTA.

The ventral tegmental area (VTA) forms part of the mesocorticolimbic system and plays a pivotal role in reward and reinforcing actions of drugs of abuse. Glutamate transmission within the VTA controls important aspects of goal-directed behavior and motivation. Noradrenergic receptors also present in the VTA have important functions in the modulation of neuronal activity. Here we studied the effects of α2 noradrenergic receptor activation in the alteration of glutamate neurotransmission in VTA dopaminergic neurons from male Sprague-Dawley rats. We used whole-cell patch-clamp recordings from putative VTA dopaminergic neurons and measured excitatory postsynaptic currents. Clonidine (40 µm) and UK 14,304 (40 µm), both α2 receptor agonists, reduced (approximately 40%) the amplitude of glutamate-induced excitatory postsynaptic currents. After clonidine administration, there was a dose-dependent reduction over the concentration range of 15-40 µm. Using yohimbine (20 µm) and two other α2 adrenergic receptor antagonists, idaxozan (40 µm) and atipemazole (20 µm), we demonstrated that the inhibitory action is specifically mediated by α2 receptors. Moreover, by inhibiting protein kinases with H-7 (75 µm), Rp-adenosine 3',5'-cyclic (11 µm) and chelerythrine (1 µm) it was shown that the clonidine-induced inhibition seems to involve a selective activation of the protein kinase C intracellular pathway. Increased paired-pulse ratios and changes in spontaneous and miniature excitatory postsynaptic current frequencies but not amplitudes indicated that the effect of the α2 agonist was presynaptically mediated. It is suggested that the suppression of glutamate excitatory inputs onto VTA dopaminergic neurons might be relevant in the regulation of reward and drug-seeking behaviors.

Physiological evidence that D-aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica neurons.

D-Aspartate (D-Asp) activates an excitatory current in neurons of Aplysia californica. Although D-Asp is presumed to activate a subset of L-glutamate (L-Glu) channels, the identities of putative d-Asp receptors and channels are unclear. Whole cell voltage- and current-clamp studies using primary cultures of Aplysia buccal S cluster (BSC) neurons were executed to characterize D-Asp-activated ion channels. Both D-Asp and L-Glu evoked currents with similar current-voltage relationships, amplitudes, and relatively slow time courses of activation and inactivation when agonists were pressure applied. D-Asp-induced currents, however, were faster and desensitized longer, requiring 40 s to return to full amplitude. Of cells exposed to both agonists, 25% had D-Asp- but not L-Glu-induced currents, suggesting a receptor for D-Asp that was independent of l-Glu receptors. D-Asp channels were permeable to Na(+) and K(+), but not Ca²âº, and were vulnerable to voltage-dependent Mg²âº block similarly to vertebrate NMDA receptor (NMDAR) channels. d-Asp may activate both NMDARs and non-l-Glu receptors in the nervous system of Aplysia.

The capabilities and limitations of conductance-based compartmental neuron models with reduced branched or unbranched morphologies and active dendrites.

Conductance-based neuron models are frequently employed to study the dynamics of biological neural networks. For speed and ease of use, these models are often reduced in morphological complexity. Simplified dendritic branching structures may process inputs differently than full branching structures, however, and could thereby fail to reproduce important aspects of biological neural processing. It is not yet well understood which processing capabilities require detailed branching structures. Therefore, we analyzed the processing capabilities of full or partially branched reduced models. These models were created by collapsing the dendritic tree of a full morphological model of a globus pallidus (GP) neuron while preserving its total surface area and electrotonic length, as well as its passive and active parameters. Dendritic trees were either collapsed into single cables (unbranched models) or the full complement of branch points was preserved (branched models). Both reduction strategies allowed us to compare dynamics between all models using the same channel density settings. Full model responses to somatic inputs were generally preserved by both types of reduced model while dendritic input responses could be more closely preserved by branched than unbranched reduced models. However, features strongly influenced by local dendritic input resistance, such as active dendritic sodium spike generation and propagation, could not be accurately reproduced by any reduced model. Based on our analyses, we suggest that there are intrinsic differences in processing capabilities between unbranched and branched models. We also indicate suitable applications for different levels of reduction, including fast searches of full model parameter space.

21st century excitatory amino acid research: A Q & A with Jeff Watkins and Dick Evans.

In 1981 Jeff Watkins and Dick Evans wrote what was to become a seminal review on excitatory amino acids (EAAs) and their receptors (Watkins and Evans, 1981). Bringing together various lines of evidence dating back over several decades on: the distribution in the nervous system of putative amino acid neurotransmitters; enzymes involved in their production and metabolism; the uptake and release of amino acids; binding of EAAs to membranes; the pharmacological action of endogenous excitatory amino acids and their synthetic analogues, and notably the actions of antagonists for the excitations caused by both nerve stimulation and exogenous agonists, often using pharmacological tools developed by Jeff and his colleagues, they provided a compelling account for EAAs, especially l-glutamate, as a bona fide neurotransmitter in the nervous system. The rest, as they say, is history, but far from being consigned to history, EAA research is in rude health well into the 21st Century as this series of Special Issues of Neuropharmacology exemplifies. With EAAs and their receptors flourishing across a wide range of disciplines and clinical conditions, we enter into a dialogue with two of the most prominent and influential figures in the early days of EAA research: Jeff Watkins and Dick Evans.

Excitatory action of GABA in the terminal nerve gonadotropin-releasing hormone neurons.

The terminal nerve (TN)-gonadotropin-releasing hormone (GnRH) neurons have been suggested to function as a neuromodulatory system that regulates the motivational and arousal state of the animal and have served as a model system for the study of GnRH neuron physiology. To investigate the synaptic control of the TN-GnRH neurons, we analyzed electrophysiologically the effect of GABA on the TN-GnRH neurons. GABA generally hyperpolarizes most of the neurons in the adult brain by activating GABA(A) receptors while the activation of GABA(A) receptors depolarizes some specific neurons in the mature brain. Here we examined the GABA(A) receptor-mediated responses in the TN-GnRH neurons of adult teleost fish, the dwarf gourami, by means of gramicidin-perforated patch-clamp and cell-attached patch-clamp recordings. The reversal potential for the currents through GABA(A) receptors under the voltage clamp was depolarized relative to the resting membrane potential. GABA(A) receptor activation depolarized TN-GnRH neurons under the current clamp and had excitatory effect on their electrical activity, whereas the stronger GABA(A) receptor activation had bidirectional effect (excitatory-inhibitory). This excitatory effect is suggested to arise from high [Cl(-)](i) and was shown to be suppressed by bumetanide, the blocker of Cl(-)-accumulating sodium-potassium-2-chloride co-transporter (NKCC). The present results demonstrate that GABA(A) receptor activation induces excitation in TN-GnRH neurons, which may facilitate their neuromodulatory functions by increasing their spontaneous firing frequencies. The excitatory actions of GABA in the adult brain have recently been attracting much attention, and the easily accessible large TN-GnRH neurons should be a nice model system to analyze their physiological functions.

Enantiomeric N-substituted phthalimides with excitatory amino acids protect zebrafish larvae against PTZ-induced seizures.

Epilepsy is a chronic neurological disease with high prevalence and adverse impacts on the quality of life of patients and caregivers. Up to one-third of individuals with epilepsy do not respond to current pharmacotherapy, underscoring the importance of identifying new molecules for epilepsy control. Thalidomide, the first synthetized phthalimide, is a neuroactive molecule with anti-seizure drug properties. The phthalimide group has been studied in some N-phthaloyl amino acids due to its pharmacological properties. Here we examine enantiomers of phthaloyl aspartate (R and S) and phthaloyl glutamate (R and S) for anti-seizure effects using zebrafish as a model. The zebrafish model is rapidly growing in use as a preclinical screening tool for drug discovery in epilepsy. Pentylenetetrazol (PTZ) exposure was used to produce convulsive behavior in 7- and 10-days post-fertilization (dpf) zebrafish larvae; these ages correspond to before and after the blood-brain-barrier (BBB) is fully developed. Larvae were pre-treated for 60 min with: control, valproic acid sodium salt (SVP) 3 mM, or one of two concentrations of N-phthaloyl-R-glutamic acid (R-TGLU; 100, 316 µM) prior to PTZ addition. R-TGLU modified the locomotor phenotype and protected against PTZ in 7 and 10 dpf larvae at 316 µM, suggesting it crossed the BBB. We next tested the per se and anticonvulsant effect of the glutamate and aspartate phthalimides were tested at 237.1 and 316 µM concentration in 10dpf zebrafish. The four tested molecules produced an anticonvulsant effect at 237.1 µM concentration, however the behavioral changes that they induce suggest that they might act by different mechanisms.

Ion permeation through a voltage- sensitive gating pore in brain sodium channels having voltage sensor mutations.

Voltage-gated sodium channels activate in response to depolarization, but it is unknown whether the voltage-sensing arginines in their S4 segments pivot across the lipid bilayer as voltage sensor paddles or move through the protein in a gating pore. Here we report that mutation of pairs of arginine gating charges to glutamine induces cation permeation through a gating pore in domain II of the Na(V)1.2a channel. Mutation of R850 and R853 induces a K(+)-selective inward cationic current in the resting state that is blocked by activation. Remarkably, mutation of R853 and R856 causes an outward cationic current with the opposite gating polarity. These results support a model in which the IIS4 gating charges move through a narrow constriction in a gating pore in the sodium channel protein during gating. Paired substitutions of glutamine allow cation movement through the constriction when appropriately positioned by the gating movements of the S4 segment.

Patch sensor detection of glutamate release evoked by a single electrical shock.

We tried to detect minimal stimulation-induced glutamate overflow from the surface of a hippocampal slice using an outside-out patch electrode excised from pyramidal cell membranes. The amplitude of the stimulation-induced patch current was dependent on the distance between the slice surface and the tip of patch sensor. The current-voltage relations of the stimulation-induced patch current were similar to those of the current evoked puff by application of L-glutamate to the patch. This indicates that the stimulation-induced patch current was produced by glutamate released from presynaptic terminals, and thus this technique may be useful in the study of transmitter release evoked by minimal electrical stimulation in brain slices.

AMPA and kainate receptors mediate mutually exclusive effects on GABA(A) receptor expression in cultured mouse cerebellar granule neurones.

Studies on animal models of epilepsy and cerebellar ataxia, e.g., stargazer mice (stg) have identified changes in the GABAergic properties of neurones associated with the affected brain loci. Whether these changes contribute to or constitute homeostatic adaptations to a state of altered neuronal excitability is as yet unknown. Using cultured cerebellar granule neurones from control [+/+; alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor (AMPAR)-competent, Kainate receptor (KAR)-competent] and stg (AMPAR-incompetent, KAR-competent), we investigated whether non-NMDA receptor (NMDAR) activity regulates GABA(A) receptor (GABAR) expression. Neurones were maintained in 5 mmol/L KCl-containing basal media or depolarizing media containing either 25 mmol/L KCl or the non-NMDAR agonist kainic acid (KA) (100 micromol/L). KCl- and KA-mediated depolarization down-regulated GABAR alpha1, alpha6 and beta2, but up-regulated alpha4, beta3 and delta subunits in +/+ neurones. The KCl-evoked but not KA-evoked effects were reciprocated in stg neurones compatible with AMPAR-regulation of GABAR expression. Conversely, GABAR gamma2 expression was insensitive to KCl-mediated depolarization, but was down-regulated by KA-treatment in a 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-reversible manner in +/+ and stg neurones compatible with a KAR-mediated response. KA-mediated up-regulation of GABAR alpha4, beta3 and delta was inhibited by L-type voltage-gated calcium channel (L-VGCC) blockers and the Ca2+/calmodulin-dependent protein kinase inhibitor, 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinoline sulfonic acid ester (KN-62). Up-regulation of GABAR alpha4 and beta3 was also prevented by calcineurin (CaN) inhibitors, FK506 and cyclosporin A. Down-regulation of GABAR alpha1, alpha6 and beta2 was independent of L-VGCC activity, but was prevented by inhibitors of CaN. Thus, we provide evidence that a KAR-mediated and at least three mutually exclusive AMPAR-mediated signalling mechanisms regulate neuronal GABAR expression.

C6 glioma cells differentiated by retinoic acid overexpress the glutamate transporter excitatory amino acid carrier 1 (EAAC1).

The transport of excitatory amino acids (EAA) in CNS is performed by a family of high affinity, sodium dependent carriers. One of these transporters, excitatory amino acid carrier 1 (EAAC1), is known to be regulated by several mechanisms that modify carrier abundance on the plasma membrane. Much less is known on EAAC1 regulation at the level of gene expression. Here we report that, in C6 rat glioma cells, a line recently described to contain neural stem-like cells, EAAC1 is markedly induced by all trans-retinoic acid (ATRA), a well known differentiating agent. Consistently, ATRA stimulates EAA transport, with the maximal effect observed at concentrations>or=1 microM. After 4 days of treatment with 10 microM ATRA, the transport Vmax is fivefold enhanced, Slc1a1 mRNA is increased by 400% compared with control, EAAC1 carrier is sixfold overexpressed and the C6 culture is greatly enriched of cells with bipolar morphology strongly positive for EAAC1 immunoreactivity. Compared with untreated cells, ATRA-treated C6 cells express less Slc1a3 mRNA, for the transporter GLAST, but significantly higher levels of Slc1a2 mRNA, for the transporter GLT-1, although no expression of either protein is detected with Western blot in both untreated and ATRA-treated cells. Consistently, the inhibition pattern of aspartate transport and its stimulation by phorbol esters are indicative of a transport process due to EAAC1 operation. Under the conditions adopted, ATRA treatment causes the induction of proteolipid protein, an oligodendrocytic marker. These results indicate that, in C6 cells, ATRA stimulates the expression of EAAC1, possibly as a step toward oligodendrocytic differentiation, and constitute the first demonstration of the induction of this transporter by a differentiating agent.

Two modes of respiratory rhythm generation in the newborn rat brainstem-spinal cord preparation.

Two respiration-related rhythm generators, the pre-Bötzinger complex inspiratory and the parafacial pre-inspiratory rhythm generators, have been identified in the medulla of rodents that produce intrinsic periodic bursts. Although both generators can be independently active under specific conditions, they interact as a coupled oscillator system to regulate the respiratory rhythm. Here, we summarize different mechanisms of modulation of the respiratory rhythm in the brainstem-spinal cord preparation of newborn rats and discuss factors determining rhythm generator dominance. We show two different modes of respiratory rhythm generation in the brainstem-spinal cord preparation that depends on the background stimulation level.

Identification of different types of respiratory neurones in the dorsal brainstem nucleus tractus solitarius of the rat.

In Nembutal anaesthetised, spontaneously breathing rats, stereotaxic mapping of the nucleus tractus solitarius (NTS) for respiratory neuronal activity was undertaken. Eight different types of respiratory cells were found between 0.25 and 1.5 mm lateral to midline, extending 0.5 mm caudal to 1.5 mm rostral to obex, and 0.4-1.5 mm below the dorsal surface. A study of the respiratory motor (diaphragm EMG) and neuronal responses to excitatory amino acid (EAA) stimulation of the NTS areas was undertaken. Electrical stimulation of the vagus nerve was employed to study the NTS cellular responses to activation of pulmonary afferents. The effects of chemical activation of the midbrain periaqueductal grey (PAG) on NTS respiratory neuronal activity were investigated. EAA microinjections into the ventrolateral NTS rostral to the obex resulted in an increase in respiratory motor frequency along with increases to inspiratory cell discharge, whilst microinjections into the medial NTS caudal to the obex caused respiratory depression. EAA stimulation of calamus scriptorius produced apnea. NTS inspiratory neurones were inhibited following stimulation of ipsilateral vagus nerve, suggesting their involvement in the Hering-Breuer reflex pathway. PAG stimulation caused excitation of the NTS inspiratory cells indicating the presence of an excitatory respiratory pathway between the two nuclei. Following beta-adrenergic antagonist pre-treatment of ventrolateral NTS, EAA microinjections into PAG did not evoke a cardiorespiratory effect. Based on the various findings the role of NTS in organising respiration in the rat is discussed.

Inhibition by the chromaffin cell-derived peptide serine-histogranin in the rat's dorsal horn.

The heptadecapeptide histogranin, synthesized by adrenal chromaffin cells, is implicated in the analgesia produced by transplanting chromaffin cells into the spinal cord, including block of hyperalgesia mediated by NMDA-subtype glutamate receptors. To examine the neurophysiological basis for this analgesia, we applied the stable analog [Ser(1)]-histogranin (SHG) by iontophoresis near extracellularly recorded wide-dynamic range (WDR) neurons in anesthetized rats. When SHG was applied during peripheral electrical stimulation of A and C fibers at 0.1Hz, the C-fiber response was significantly inhibited but the A-fiber response was unaffected. SHG also opposed the NMDA-receptor-dependent post-tetanic facilitation (wind-up) of C-fiber responses produced by increasing the rate of peripheral afferent stimulation to 1Hz for 20s. To test whether block of NMDA-subtype receptors could be wholly or partially responsible for this suppression, SHG was applied during sequential pulsed iontophoresis of three agonists targeting distinct excitatory synaptic receptors: NMDA, kainate and substance P. All three excitatory effects were reversed by SHG; this reversal outlasted the 10-30min observation period when higher SHG doses were applied (>60nA). Histogranin therefore probably produces prolonged spinal analgesia by opposing the basal and potentiating synaptic effects of C-fibers on dorsal horn neurons. Actions besides or in addition to NMDA-receptor antagonism (e.g., agonism at inhibitory postsynaptic receptors or block of voltage-gated cation channels on C-fibers) are implied by the diversity of excitatory transmitters opposed by SHG.

The origin of spontaneous activity in developing networks of the vertebrate nervous system.

Spontaneous neuronal activity has been detected in many parts of the developing vertebrate nervous system. Recent studies suggest that this activity depends on properties that are probably shared by all developing networks. Of particular importance is the high excitability of recurrently connected, developing networks and the presence of activity-induced transient depression of network excitability. In the spinal cord, it has been proposed that the interaction of these properties gives rise to spontaneous, periodic activity.