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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 intravenous bicuculline and strychnine on inspiratory inhibitory responses in the cat

Single shock stimulation of the superior laryngeal nerve (SLN), intercostal nerve (ICN), phrenic nerve (PN) or within the medullary respiratory groups (DRG-VRG) produces a transient, short-latency attenuation of inspiratory motor activity. Trains of stimuli delivered to SLN and ICN cause premature termination of inspiration. This study examined involvement of glycine and GABAA receptors in these reflex inhibitions. Experiments were conducted in decerebrate, vagotomized, and paralyzed cats. Control responses of left PN activity to threshold single shock stimulation of SLN, PN, ICN and the DRG-VRG were recorded and the thresholds for SLN- and ICN-evoked inspiratory termination were determined. Five min after intravenous injection of bicuculline (1 mg/kg) or strychnine (50 micrograms/kg), the responses to stimulation were again recorded. This procedure was reiterated until the cumulative dose elicited marked convulsions. Neither drug affected the inspiratory terminating reflexes. Systemic bicuculline had no effect on transient inspiratory inhibition. However strychnine prolonged the onset latency and the duration of all four inhibitory responses. Since the degree of transient inhibition was not lessened (only delayed), it appears that these inspiratory inhibitory reflexes do not rely exclusively on actions of glycine or GABAA receptors.     

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.     

Muscarinic reduction of GABAergic synaptic potentials results in disinhibition of the AMPA/kainate-mediated EPSP in auditory cortex

The present study is concerned with the ability of muscarinic actions of acetylcholine (ACh) to modulate glutamate and γ-aminobutyric acid (GABA)-mediated synaptic transmission in the in vitrorat auditory cortex. Whole-cell patch clamp recordings were obtained from layer II–III pyramidal neurons, and the fast-EPSP (AMPA/kainate), fast-IPSP (GABA_A), and slow-IPSP (GABA_B), were elicited following a stimulus to deep gray/white matter. Acetyl-β-methylcholine (MCh), a muscarinic receptor agonist, applied by either superfusion or iontophoresis, produced an atropine-sensitive or in the amplitude of the fast-EPSP. The effect of MCh could be predicted by the response of the fast-EPSP to paired-pulse stimulation (i.e. a conditioning pulse followed 300 ms later by a test pulse). The fast-EPSP was decreased in amplitude by MCh in cases where the test-EPSP was suppressed in the pre-MCh condition, and increased in amplitude when the test-EPSP was facilitated. The fast- and slow-IPSPs were always reduced by MCh. In several experiments, the strength of synaptic inhibition was systematically modified by varying stimulus intensity. When the fast-EPSP was elicited in the absence of IPSPs, it was in amplitude by MCh. However, when the fast-EPSP was elicited in conjunction with large IPSPs it was in amplitude during MCh. Because the magnitude of the fast-EPSP is influenced by the degree of temporal overlap with IPSPs, it was hypothesized that enhancement of the fast-EPSP was the result of disinhibition produced asa consequence of muscarinic reduction of GABAergic IPSPs. This view was supported by the finding that MCh could reduce the amplitude of pharmacologically isolated GABAergic IPSPs (i.e. elicited in the absence of glutamatergic transmission). Our results suggest that ACh at muscarinic receptors can modify fast glutamatergic neurotransmission differently as a function of strength of inhibition, to suppress that produced by ‘weak’ inputs and enhance that produced by ‘strong’ inputs.     

Hemispheric asymmetries in spontaneous firing characteristics of substantia nigra pars reticulata neurons following a unilateral 6-hydroxydopamine lesion of the rat nigrostriatal pathway

Single unit activity of substantia nigra pars reticulata (SNR) neurons was recorded bilaterally in rats subjected to unilateral 6-hydroxydopamine lesions of the ascending mesostriatal dopaminergic pathway, resulting in an almost complete loss of dopaminergic neurons in the ipsilateral SN pars compacta. Firing rate and firing pattern of SNR neurons in lesioned rats were compared with respective data from sham-lesioned rats and naive controls. In lesioned rats, the mean firing rate of SNR neurons at the lesioned side was significantly reduced and there was an increase in the occurrence of bursting activity. In contrast, firing rate in the contralateral SNR was significantly increased without change in the frequency of bursting neurons. This asymmetrical change in spontaneous firing characteristics of SNR neurons following the lesion could be involved in the complex behavioral changes seen in this model of Parkinson's disease.     

Contrast adaptation in cat visual cortex is not mediated by GABA

The possible involvement of gamma-aminobutyric acid (GABA) in contrast adaptation in single cells in area 17 of the cat was investigated. Iontophoretic application of N-methyl bicuculline increased cell responses, but had no effect on the magnitude of adaptation. These results suggest that contrast adaptation is the result of inhibition through a parallel pathway, but that GABA does not mediate this process.     

Electrophysiological properties of rat septal region neurons during development in culture

We report on the development of membrane properties of septal region neurons from embryonic rats in serum-free culture during 1–25 days in vitro (DIV). Na⁺-dependent action potentials could be evoked within 1 day after plating and 3 different types of outward current were observed by means of the patch-clamp technique: I_K, I_A and I_C. In some neurons the neurotransmitter GABA evoked a chloride current after 2 DIV. In addition a cationic current elicited by glutamate appeared after 4 DIV. Within 8–12 DIV virtually all neurons were sensitive to both GABA and glutamate. Spontaneous action potentials and postsynaptic potentials occurred after 7–10 DIV but cultured septal neurons did not generate any pacemaker-like activity.     

Role of the cell recognition molecule, cognin, in GABAergic differentiation in chick retina

Previous work showed that GABAergic differentiation in developing chick retina depends on insulin and cell interactions. Here, we investigated whether it depended on cell signaling mediated by retina cognin, a 50 kDa cell recognition molecule. Cognin mediates cell adhesion in vitro and occurs on retinal neurons that become both GABAergic and cholinergic. We investigated two markers of GABAergic differentiation: glutamate decarboxylase (GAD) activity and high-affinity GABA uptake. Both increase during differentiation of retinal neurons in culture and can be easily measured. We blocked cognin-mediated cell signaling with cognin antibody and found a reduction of the developmental increase in GAD activity in cultures of retinal neurons from 7 and 11 day chick embryos. There was no reduction of high-affinity GABA uptake. This suggested that cognin-mediated signaling was necessary for the normal developmental increase in GAD but not for high-affinity GABA uptake. These results contrasted with our previous observations on cholinergic differentiation in cultured retinal neurons. We found that cognin antibody blocked the normal developmental increase in choline acetyltransferase (ChAT) only if the cells were exposed before embryonic day 7. Thus, while both GAD and ChAT activity appear to be controlled by cell signaling involving cognin, the periods of developmental sensitivity for the two differentiation markers are different. Antibodies to other adhesion molecules, Ng-CAM, and N-cadherin, did not similarly affect GAD activity. Antibodies to laminin at a 10-fold higher concentration inhibited GAD activity only in early embryonic retina. Tests for protein synthesis and “housekeeping” enzyme activity demonstrated that the cognin antibody effect was selective for neuronal differentiation pathways. Thus, GABAergic differentiation in developing retina is sensitive to cell signaling mediated in part by cognin.