Effects of pharmacological manipulation on neurotransmitter and other amino acid levels in the CSF of the Senegalese baboonPapio papio

The concentrations of GABA, glutamate, aspartate, glycine, taurine, glutamine, asparagine and alanine were determined in the CSF of 10 Senegalese baboons (Papio papio) following initial ketamine anaesthesia and subsequent administration (4 h later) of different compounds known to alter either inhibitory or excitatory neurotransmission. Ketamine itself was apparently without effect as the administration of a second dose of ketamine did not significantly alter the levels of any of the amino acids studied, although GABA levels tended to decrease. The presence of haemolysed material in occasional samples was associated with high GABA, glutamate, aspartate, taurine and asparagine levels. Therefore only haemolysate-free samples were included for analysis. Of the compounds administered, gamma-vinyl GABA had the most evident effect on CSF amino acid levels, increasing GABA (greater than 5-fold) and decreasing glutamate (greater than 50%), aspartate (40-50%), asparagine (20%) and alanine (30-35%) levels. The changes in GABA, glutamate and aspartate were still apparent 24 h post-gamma-vinyl GABA administration. In contrast, sodium valproate did not significantly alter the CSF levels of any of the amino acids studied. Upon acute administration allylglycine decreased the CSF concentrations of GABA and alanine, but not glutamate. These alterations are unlikely related to the occurrence of allylglycine-induced convulsions (in 2 of 4 experiments) as electroconvulsive shock did not alter CSF amino acid levels. During the experimental period encompassing the allylglycine injection (8 weeks), basal (initial post-ketamine, pre-drug sample) amino acid levels were abnormal with large increases in glutamate, GABA, aspartate and taurine whereas asparagine levels were below the limit of detection. Diazepam administration was followed by a significant increase in taurine and a decrease in aspartate levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

The laminar distribution of amino acids in the caudal cerebellum and electrosensory lateral line lobe of weakly electric fish (Gymnotidae)

We have studied the distribution of the putative amino acid neurotransmitters glutamate, aspartate, gamma-aminobutyric acid (GABA), glycine, taurine and beta-alanine in the caudal cerebellar lobe and electrosensory lateral line lobe (ELL) of weakly electric gymnotid fish. In the caudal lobe of the cerebellum, the levels of the various amino acids in the granular and molecular layers are comparable to the levels in the rat cerebellum, with the exception of taurine which is present in greater amounts in the gymnotid. In the ELL, these amino acids are differentially distributed in the various layers of this structure. Glutamate and taurine are enriched in the molecular layer, whereas GABA, aspartate, and beta-alanine are enriched in the deep neuropil + granular layers. Glycine is slightly enriched in the pyramidal cell layer.     

Effects of baclofen and γ-aminobutyric acid on different types of medullary respiratory neurons

Effects of baclofen and γ-aminobutyric acid on medullary respiratory neurons were investigated. Medullary inspiratory neurons of the dorsal and ventral respiratory groups were stimulated by baclofen, 0.5–2 mg/kg, and depressed by doses greater than 4–6 mg/kg. Expiratory neurons were depressed by doses of baclofen which increased phrenic nerve activity. Microelectrophoresis of baclofen (5 mM, pH 3) depressed medullary inspiratory neurons. It is suggested that low i.v. doses of baclofen increase inspiratory activity by disinhibition of medullary neurons whereas higher doses directly depress medullary inspiratory neurons.     

Inhibitions mediated by glycine and GABAA receptors shape the discharge pattern of bulbar respiratory neurons

Experiments were performed to identify the glycinergic or GABAergic nature, and the timing of discharge, of the neurons which produce chloride-dependent inhibitions on other bulbar respiratory neurons (RNs) during their silent and active phases. RNs recorded extracellularly in pentobarbital-anesthetized or decerebrate cats, were subjected to iontophoretic applications of glutamate, of the glycine antagonist strychnine, and of the GABA_A receptor antagonist bicuculline. Both antagonists induced discharge or increased discharge frequency in restricted parts of the respiratory cycle without affecting the discharge frequency in other parts of the cycle. Strychnine most often elicited activity in late-inspiration and early-expiration, but also in early inspiration and in late expiration. Bicuculline was most often effective throughout the entire discharge period of each neuron with no effect during the silent period, although it also acted selectively during late-inspiration in inspiratory neurons, an effect attributed to GABA_A receptor blockade. The convergence of glycinergic afferent inputs during late inspiration and early expiration suggests that glycinergic neurons may play an important role in the inspiratory to expiratory phase transition.     

Immunohistochemical localization of γ-aminobutyric acid in the rat pituitary gland and related hypothalamic regions

The distribution on γ-aminobutyric acid (GABA) containing neurons in the rat pituitary gland and related hypothalamic areas was immunohistochemically investigaed using antibodies raised against GABA conjugated to bovine serum albumin by glutaraldehyde. A dense network of GABA-like immunoreactive fine varicose nerve fibers was observed within the posterior and intermediate lobes of the pituitary gland, surrounding endocrine cells and capillaries, but not in the anterior lobe. In the pituitary stalk, the dense varicose fibers ran along the anterior wall of the posterior lobe into the posterior and intermediate lobes. A small number of GABA-like immunoreactive cell bodies were evident in the intermediate lobe. GABA-like immunoreactive fibers occurred at low to high density in most parts of the hypothalamus. GABA-like immunoreactive neurons were observed in some regions related to the pituitary gland (such as periventricular nucleus, paraventricular nucleus, arcuate nucleus and accessory magnocellular nucleus). These results provide morphological evidence for the presence of GABAergic neurons in the rat hypothalamo-pituitary system.     

Effects of GABAB receptor agonists and antagonists on the bulbar respiratory network in cat

We examined the involvement of the GABA_B receptor in central respiratory mechanisms. Respiratory neurons (RNs) from the ventral respiratory group in the medulla of the cat were subjected to iontophoretic applications of the GABA_B receptor agonist baclofen and the antagonists saclofen and CGP 35348.In all types of RNs baclofen decreased the firing rate. This reduction was antagonized by CGP 35348. Application of either antagonist increased the spontaneous discharge in both inspiratory and expiratory RNs. CGP 35348 excited 57% of the neurons tested, on the average by 34% with ejection currents of 100 nA. Saclofen excited 6 of 9 neurons tested. Baclofen administered systemically (8–12 mg/kg i.v.) to either anesthetized, decerebrate or intact freely moving cats, induced a selective lengthening of the inspiratory phase, an effect comparable to the apneusis induced by the NMDA antagonist MK-801. Baclofen also produced either a pronounced decrease in the amplitude of phrenic nerve discharge or an apnea, both of which were reversed by increasing paCO₂. The results suggest that endogenously released GABA acting on GABA_B receptors may be involved in the control of respiratory neuronal discharge.     

Strychnine alters the fusiform cell output from the dorsal cochlear nucleus

Anatomical and physiological evidence suggests that fusiform cells, the major output neurons of the dorsal cochlear nucleus (DCN), receive significant inhibitory input. Fusiform cells often display strongly non-monotonic rate-intensity functions and pauser-buildup or buildup tone-evoked temporal responses, patterns which may be mediated by inhibitory neurotransmitters. Other neurons located within the fusiform cell layer or in the more superficial molecular layer display varied rate-intensity functions and temporal responses. Neurons displaying response properties characteristic of fusiform cells are sensitive to iontophoretic application of the inhibitory amino acid neurotransmitter, glycine. Application of the glycine receptor antagonist, strychnine, alters the non-monotonic portion of the rate-intensity function at doses which do not alter spontaneous activity or near-threshold tone-evoked responses. These neurons are also sensitive to GABA and the GABAB agonist, (-)-baclofen, but are insensitive to the GABAA antagonist, bicuculline. DCN neurons which display monotonic rate-intensity functions and temporal response properties different than those associated with fusiform cells are sensitive to bicuculline, (-)-baclofen, and GABA. These data suggest that a glycinergic input onto fusiform cells may control the non-monotonic nature of the response of these neurons near characteristic frequency and therefore may contribute significantly to the nature of the output of the DCN.     

Differential distribution of GABAA receptor subunits on bulbospinal serotonergic and nonserotonergic neurons of the ventromedial medulla of the rat

Spinally projecting neurons of the ventromedial medulla (VMM) compose an important efferent pathway for the modulation of nociception. These neurons receive a substantial γ-aminobutyric acid (GABA)-ergic input, but the GABA receptor that mediates this input is unknown. This study examined the distribution of GABA_A receptor α1 and α3 subunits in serotonergic and nonserotonergic neurons of the VMM that project to the dorsal horn in the rat. A pledget of Gelfoam^TM soaked in Fluoro-Gold was placed at the thoracolumbar junction of the spinal cord to label spinally projecting neurons. Alternate sections of the medulla were then incubated with a mixture of antisera to either serotonin and the α1 subunit, or to serotonin and the α3 subunit of the GABA_A receptor. Nearly 30% of spinally projecting neurons in the VMM were immunoreactive for the α1 subunit. A similar percentage of spinally projecting neurons in the VMM were immunoreactive for the α3 subunit, although diffuse cellular labeling combined with intense staining of processes in the neuropil precluded a rigorous semi-quantitative estimation of this population. No α1-subunit-immunoreactive neurons colocalized serotonin. In contrast, serotonergic neurons were immunoreactive for the α3 subunit. However, these double-labeled neurons were a modest percentage of the serotonergic population. A small percentage of spinally projecting serotonergic neurons was immunoreactive for the α3 subunit. These results suggest that significant numbers of spinally projecting serotonergic and nonserotonergic neurons of the VMM possess GABA_A receptors that differ in their respective subunit compositions and that both classes of neurons may mediate the antinociception produced by the microinjection of GABA_A receptor antagonists in the VMM. J. Comp. Neurol. 384:337–348, 1997. © 1997 Wiley-Liss, Inc.     

Modulation of the excitability of septohippocampal terminals in the rat: relation to neuronal discharge rate

The excitability of the axonal terminals of medial septal neurons projecting to the dentate gyrus has been studied in the anesthetized rat under various experimental conditions: spontaneous or drug-induced variations in neuronal soma discharge rate, conditioning stimulation of afferent pathways (perforant path, commissural pathway, fimbria-fornix). It has been observed that terminals excitability is inversely correlated to the level of neuronal ongoing activity. These effects were observed on virtually all septal neurons projecting to the dentate gyrus. Since about one half of the septohippocampal neurons are likely to be cholinergic, it follows that such a phenomenon is not transmitter specific.     

Immunohistochemical demonstration of GABA containing neurons in the guinea pig ileum using purified GABA antiserum

Antisera against GABA were prepared by immunizing rabbits with GABA conjugated to bovine serum albumin by glutaraldehyde and the antisera were then purified using a GABA immobilized epoxy-activated affinity column. Affinity chromatography and GABA-immobilized epoxy-activated agarose gels were made use of for the reduction of the cross-reactivities of GABA antiserum against endogenous amino acids. The purified GABA antiserum showed remarkably less cross-reactivity. Using this purified GABA antiserum, we noted numerous GABA-like immunoreactive (GABA-positive) nerve fibers in the myenteric meshworks and a few GABA-positive fibers exiting from the myenteric plexus of guinea pig ileum. In the myenteric ganglia, there were GABA-positive nerve cells and GABA-positive varicose fibers surrounding or running along the non-immunoreactive nerve cells. The direct visualization of enteric GABA neurons provides further support for the proposal that GABA is a neurotransmitter in the guinea pig small intestine.     

Neurotransmitter actions on oral pontine tegmental neurons of the rat: an in vitro study

The actions of neurotransmitters involved in the sleep-wakefulness cycle on neurons located in the ventral part of the oral pontine tegmentum were studied in a rat brain-slice preparation. Results show that glutamate and histamine evoke depolarizations and spike firing while serotonin and γ-aminobutyric acid evoke hyperpolarizations. The excitatory and inhibitory actions of these neurotransmitters increase pontine neuron activity during the conditions occurring during paradoxical sleep.     

GABA-induced responses in electrophysiologically characterized neurons within the rat rostro-ventrolateral medulla in vitro

Rostro-ventrolateral medulla (RVL) neurons were recorded using conventional intracellular recording techniques in brain slices maintained in vitro at 32°C and classified into 3 major groups. The first group included neurons having endogenous pacemaker-like (PL) activity with regular firing frequency (mean = 8 Hz) and a linear current-voltage relationship (I–V). The second group of neurons were slowly and irregularly firing (IF) or quiescent, presenting membrane potential oscillations and their I–V usually displayed an inward rectification. These neurons had a relatively longer action potential duration. The third group included silent neurons (S) with no apparent membrane oscillations and they differed from the first two groups by having relatively shorter action potential duration and amplitude and lower cell input resistance. When recorded with KCl-filled electrodes, the majority of silent neurons displayed a time-dependent inward rectification. With KAc-filled electrodes, irregular slow hyperpolarizing and depolarizing spontaneous potentials could be recorded primarily on PL and IF neurons, respectively. Moreover, fast spontaneous inhibitory postsynaptic potentials (PSPs) were detected in about 15% of PL and S neurons. They generally exhibited a regular pattern and were depolarizing when KCl-filled electrodes were used for recording. The amplitude of these inhibitory PSPs was reversibly reduced by the GABAA antagonists bicuculline, SR 95531 and picrotoxin. With KAc-filled electrodes, pressure-applied GABA (20 mM) evoked complex responses. In PL neurons, it consisted of a fast hyperpolarization followed by a slower depolarization that were both sensitive to SR 95531 and picrotoxin. The response was terminated by a long-lasting hyperpolarization that was reduced, but not abolished, by the GABA_B antagonist CGP 35348. In IF and S neurons, GABA application usually produced a fast followed by a slow monophasic hyperpolarization and depolarization, respectively. The fast component of these responses was sensitive to the GABA_A antagonists. Pressure application of isoguvacine (10 mM) always induced monophasic responses in all types of neurons recorded. Baclofen (1–30 μM) reduced the firing frequency and hyperpolarized PL and IF neurons, an effect that was antagonized by CGP 35348 (50–100 μM); however, it had little effect on silent neurons. It is concluded that RVL neurons have heterogeneous electrophysiological characteristics. Their predominant synaptic input and GABA responsiveness might be additional criteria to identify the excitatory and inhibitory elements in the RVL circuitry. All neuronal types seem to have functional GABA_A and GABA_B receptors; however, only a subpopulation is under tonic inhibitory control in vitro, probably from local GABAergic pacemaker intemeurons. Our results further emphasize the role of GABA as an important neurotransmitter in the RVL network.     

Corticosterone reduces the excitability of hippocampal pyramidal cells in vitro

In the rat brain, the hippocampus (HC) is a major target area for corticosterone (CT). In this study, we investigated the effects of CT in the in vitro slice preparation of the rat HC. Population spikes (PS) evoked by stratum radiatum stimulation were recorded in CA1. Bath-applied CT (10(-7)-10(-5) M) induced a decrease in the PS amplitude. This effect occurred within 10-15 min after the onset of CT perfusion, reached a plateau after 20-40 min and was reversible after 20 min washout. Neither dexamethasone (10(-5) M) nor vehicle produced any significant change in PS amplitude. Paired-pulse stimulation showed that the degree of inhibition of the second PS produced by the conditioning stimulus was either unchanged or decreased by CT. In the latter case, CT also reduced the inhibitory effect of gamma-aminobutyric acid on PS and enhanced the excitatory action of the opioid peptide D-Ala2-D-Leu5-enkephalin. These results show that CT reduces the excitability of HC pyramidal cells with a time course which may be compatible with a genomic action of CT. The fact that paired-pulse inhibition was either not changed or reduced, suggests that the decrease in PS size by CT is not due to an indirect excitatory effect of CT on inhibitory interneurons. Instead, CT may hyperpolarize pyramidal cells thus lowering their excitability and depressing the interneurons involved in recurrent inhibition.     

Immunohistochemical localization of γ-aminobutyric acid- and aspartate-containing neurons in the guinea pig vestibular nuclei

The immunohistochemical distributions of γ-aminobutyric acid (GABA)- and aspartate-containing neurons were studied in the guinea pig vestibular nuclei using purified antisera to GABA and aspartate, respectively. Most GABA-containing neurons had small cell bodies and were scattered throughout all regions of the vesticular nuclei. The largest number of these cells was found in the medial nucleus. Intraventricular injection of colchicine markedly increased GABA-like immunoreactivity in these cell bodies. GABA-containing terminals were distributed throughout all 4 subdivisions of the nuclei, with the richest localization found around the floor of the fourth ventricle. Various sized aspartate-containing neurons were noted in the vestibular nuclei and small cells were present in the superior, medial and lateral nucleus. Medium-sized cells were observed throughout the vestubular nuclei. Giant cells in the lateral nucleus also contained aspartate and were surrounded by GABA-like immunoreactive terminals, thereby suggesting the modulation of aspartate-containing neurons by GABAergic fibers from Purkinje cells.     

Heterogeneity of AMPA receptors in the dorsal column nuclei of the rat

We have combined immunocytochemistry with retrograde tracing to demonstrate that projecting neurons in the gracile and cuneate nuclei express predominantly the GluR3 subunit of the AMPA receptor while interneurons in these nuclei express predominantly the GluR1 subunit. Interneurons expressing the GluR2 subunit are also present. It is speculated that the two classes of interneurons may release different inhibitory transmitters.     

Electrophysiological study of dorsal respiratory neurons in the medulla oblongata of the rat

There has been controversy whether the dorsal respiratory group (DRG), identified in the cat and several other species as a concentration of mainly inspiratory neurons located in the ventrolateral subnucleus of the solitary tract, also exists in the rat. The aim of this study was to re-examine this question by systematically exploring this region with extracellular microelectrodes, in anesthetized and artificially ventilated rats. One-hundred and forty-two units were recorded which fired in phase with central respiratory cycles (determined by recording from the phrenic nerve) and/or lung inflations. One-hundred and nineteen recordings were thought to be from neuronal cell bodies (confirmed in some cases by excitatory responses to microelectrophoretic administration ofdl-homocysteic acid), while the remaining 23 were from lung vagal afferents. Most neurons in the former group (87/119) were inspiratory. Out of 96 neurons tested for spinal projections only 14 (12 inspiratory, 2 expiratory) responded antidromically following stimulation at C₃ segment. These results confirm the existence of the DRG in the rat and demonstrate that neurons located in this region have firing patterns generally similar to those previously described in the cat. The main difference is the relative paucity in the rat of neurons projecting spinally below the C₂ level, which indicates that most DRG neurons in this species do not project directly to phrenic and intercostal motoneurons, but to other, as yet unidentified, neuronal groups within the brainstem or upper cervical segments.     

Effects of serotonin and substance P on bulbar respiratory neurons in vivo

We studied the effects of separate or co-applications by microiontophoresis of serotonin (5-HT) and substance P(4–11) onto brainstem respiratory neurons in anesthetized or decerebrate cats. 5-HT either produced an excitation (36%,n = 10) or an inhibition (43%,n = 12) or had no effect (21%,n = 6). SP(4–11) had predominantly an excitatory effect (84%,n = 26) or no effect. Fifteen respiratory neurons responded to both 5-HT and SP(4–11). Test applications of 5-HT were made during a long application of SP(4–11). We obtained ‘additive effects’ when the inhibitory effect of 5-HT was superimposed to the excitation of SP(4–11) with slight modification (n = 1) or without any modification (n = 2). In other cases, called ‘non-additive effects’, we observed a great modification of the responsiveness of the neuron to the inhibitory effect of 5-HT (n = 2) or a complete blockade of the excitatory effect of 5-HT (n = 2) during co-application. The remaining results presented a potentiation of 5-HT effect by SP(4–11) or a biphasic response to 5-HT during SP(4–11) application. The results indicate that both 5-HT and SP receptors coexist on the membrane of the same respiratory-related neurons in the brainstem of cat and suggest an interaction between both substances in vivo in the central respiratory system.     

GABA-like immunoreactivity in the chick vestibular end organs

gamma-Aminobutyric acid (GABA)-like immunoreactivity in the chick vestibular endorgans was examined using an antiserum against GABA coupled with glutaraldehyde to bovine serum albumin. GABA-like immunoreactivity was confined to the cytoplasm of the hair cells in both cristae and maculae. GABA-like immunoreactive cells were evenly distributed throughout the sensory epithelia, and no difference existed between type I and type II hair cells. The results provide evidence that GABA-like immunoreactivity is localized to sensory cells and raises the possibility that GABA may serve as an afferent neurotransmitter in the chick vestibular end organs.     

An aspect of the organization of the GABAergic system in the rat main olfactory bulb: laminar distribution of immunohistochemically defined subpopulations of GABAergic neurons

The organization of the GABAergic system in the rat main olfactory bulb was investigated immunohistochemically using antisera against glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), parvalbumin (PV) and methionin-enkephalin-Arg⁶-Gly⁷-Leu⁸ (ENK). Some GABAergic neurons were shown to contain TH immunoreactivity in the glomerular layer, PV immunoreactivity in the external plexiform layer and ENK-like immunoreactivity in the granule cell layer, indicating the stratified organization of the GABAergic subsystems.