Ernest Puil

S-Glutamate: its interactions with spinal neurons.

2. Observations with extracellular recording techniques ................ 2.1. Etfects of S-glutamate applied by super-perfusion or perfusion .......... 2.1.1. Techniques ............................. 2.1.2. Primary afferent terminal excitability ................. 2.1.3. Primary afferent depolarization and dorsal root responses ........ 2.1.4. Ventral root responses ........................ 2.2. Effects of S-glutamate and closely related analogues applied by microiontophoresis . 2.2.1. Techniques problems in interpretation of results ............ 2.2.2. Responses of neurons in dorsal and ventral horns to S-glutamate ...... 22.3. Effects of other aminoacids structurally related to glutamate ........ 2.2.3.1. Time course and termination of excitatory effects ........ 2.2.3.2. Some problems in comparison of apparent potencies of agonists . . 2.2.3.3, Responsiveness of spinal neurons to S-glutamate and S-aspartate . . 2.2.3.4. Other structurally related excitatory amino acids .........

Anaesthetic suppression of transmitter actions in neocortex.

1 The effects of general anaesthetics were investigated on neuronal sensitivities to transmitter substances, which were determined by iontophoretic applications of acetylcholine, glutamate, N‐methyl‐d‐aspartate (NMDA) and γ‐aminobutyrate (GABA) during intracellular recording in in vitro slice preparations of neocortex (guinea‐pig). 2 In most of the 65 neurones studied, perfusion of isoflurane (0.5–2.5 minimum alveolar concentration (MAC)) or Althesin (25–200 μm) and, in some cases, halothane (0.5–2 MAC), markedly reduced the depolarizing responses and associated membrane conductance changes evoked by dendritic applications of acetylcholine, glutamate, NMDA and GABA. 3 The order of depression was acetylcholine > glutamate or NMDA ≫ GABA. This selectivity could also be assessed from the EC50 for the isoflurane‐induced depression of the just‐maximal responses to acetylcholine, which was 0.9 MAC compared with an EC50 =1.9 MAC for the suppression of glutamate responses. The selectivity was less pronounced in the case of the actions of Althesin, where the EC50s were 75 μm for the depression of acetylcholine responses and 90 μm for the depression of glutamate responses. 4 The hyperpolarizing responses observed when GABA was applied near the perikaryon in 7 neurones, were slightly reduced (∼ 15%) in 4, and unchanged in 3 neurones during anaesthetic application. 5 The pronounced depression of the responsiveness to the putative arousal transmitters and an observed blockade of acetylcholine‐induced potentiation of glutamate actions suggest that anaesthetics produce unconsciousness, at least in part, by interfering with subsynaptic mechanisms of neocortical activation.

Electrophysiological responses of trigeminal root ganglion neurons in vitro

The membrane electrical properties of neurons and their responses to endogenous compounds or other neuroactive substances were investigated in vitro with intracellular recording techniques in slices of trigeminal root ganglia of guinea-pigs. The mean resting membrane potential of these neurons was -60 mV. Intracellular injections of hyperpolarizing current pulses evoked time-dependent rectification with varying degrees of dependence on membrane voltage in 107 of 110 neurons. Membrane potential oscillations were observed following the termination of the hyperpolarizing pulses and after similar injections of depolarizing current. This phenomenon appeared to be voltage-dependent at levels that were subthreshold for spike genesis; the more pronounced oscillations were evident at the more depolarized levels and were insensitive to tetrodotoxin applications. Two groups of neurons could be distinguished on the basis of certain characteristics in their action potentials. The majority exhibited short duration (0.6 ms) spikes with mean amplitude of 72 mV in response to intracellular depolarizing current. The brief (3 ms) afterhyperpolarizations that followed such spikes were blocked by intracellular injections of Cs+ or by bath applications of tetraethylammonium. Action potentials in the minority group exhibited a hump in their repolarization phase. The humped spikes had a mean peak amplitude of 78 mV and a longer duration (2 ms). Both the duration (6 ms) and the amplitude (16 mV) of the afterhyperpolarization were significantly greater in this latter group of neurons. Some fast spikes were easily blocked whereas others, including humped spikes, were resistant to tetrodotoxin (10(-6) M). Spikes which were resistant, were also not affected by perfusion with Co2+ (10(-3) M) and were reduced in amplitude during perfusion with Na+-deficient solution. Bath applications of S-glutamate (10(-4)-10(-2) M) depolarized only two of ten neurons by less than 3 mV. Similarly, 5-hydroxytryptamine produced a small depolarization in only two of thirteen neurons. Perfusion of gamma-aminobutyrate (10(-5)-10(-2) M) resulted in an increase in input conductance that waned despite continued application and was associated with a depolarization (2-14 mV) in 44/50 neurons. In some neurons, gamma-aminobutyrate application enhanced their repetitive firing ability, possibly as a result of the increased oscillatory behavior of the membrane at certain depolarized potentials. The effects of gamma-aminobutyrate were blocked by the GABAA-receptor antagonist, bicuculline (10(-4) M) but were unaffected by the GABAB-receptor agonist, baclofen (10(-4) M).(ABSTRACT TRUNCATED AT 400 WORDS)

Pentobarbital modulates intrinsic and GABA-receptor conductances in thalamocortical inhibition.

We investigated interactions of an anesthetic barbiturate, pentobarbital, with non-ligand gated channels and identified inhibitory synaptic transmission in thalamic neurons. Using whole cell voltage-clamp, current-clamp and single channel recording techniques in rat ventrobasal neurons of slices and dispersed preparations, we determined the mechanisms of pentobarbital actions on ionic currents and inhibitory postsynaptic currents (IPSCs), mediated by aminobutyric acid (GABA). We investigated pentobarbital effects on intrinsic currents using hyperpolarizing voltage commands from rest and tetrodotoxin blockade of action potentials. At concentrations near 8 microM, pentobarbital increased input conductance and induced net outward current, I(PB), at potentials near action potential threshold. The reversal potential of I(PB) was -75 mV, implicating K+ and other ions. Cs+ (3 mM) which inhibits both K+ currents and inward rectifier (Ih), completely blocked IPB, whereas the selective Ih blocker, ZD-7288 (25 microM), or Ba2+ (2 mM) which suppresses only K+ currents, reduced IPB. Pentobarbital inhibited the Ih, consistent with a ZD-7288-induced shift in reversal potential for IPB toward K+ equilibrium potential. Pentobarbital increased the inward K+ rectifier, IKir, and leak current, Ileak. We determined the susceptibility of IPSCs, evoked by reticular stimulation, to antagonism by bicuculline, picrotoxinin and 2-hydroxysaclofen and identified their receptor subclass components. At EC50 = 53 microM, pentobarbital increased the duration of IPSCs. The prolonged IPSC duration during pentobarbital was attributable to enhanced open probability of GABAA channels, because combined with GABA, pentobarbital application increased mean channel open time without affecting channel conductance. At concentrations up to 100 microM, pentobarbital did not directly activate GABAA receptors. The concentration-response relationships for pentobarbital effects on the intrinsic currents and IPSCs overlapped, implying multiple sites of action and possible redundancy in anesthetic mechanisms. This is the first study to show that an i.v. anesthetic modulates the intrinsic currents, Ih, IKir, and Ileak, as well as IPSC time course in the same neurons. These effects likely underlie inhibition in thalamocortical neurons during pentobarbital anesthesia.

Opisthotonos following propofol: A nonepileptic perspective and treatment strategy

In this report of opisthotonos during recovery from propofol anaesthesia, we relate clinical observations with scientific considerations, and propose a strategy for treatment of this rare side effect. Following a brief operative procedure, a healthy 29-yr-old woman developed recurrent opisthotonos while recovering from anaesthesia with alfentanil, propofol, and nitrous oxide. In contrast to accumulating reports, the patient remained conscious during each episode of back extension and retrocollis. The preservation of consciousness and similarities to strychnine-induced opisthotonos suggest to us that the mechanism may have a brainstem and spinal origin. Recent investigations show that propofol potentiates the inhibitory transmitters glycine and γ-aminobutyric acid (GABA) which would enhance spinal inhibition during anaesthesia. Postanaesthetic opisthotonos, however, may be due to a propofol-induced tolerance to inhibitory transmitters. This rebound phenomenon would lead to an acute, enduring refractoriness in inhibitory pathways of the brainstem and spinal cord, resulting in increased activity of extensor motoneurons. We recommend a therapeutic strategy that restores inhibition by glycine and GABA at multiple sites; the preferred therapeutic agents would be diazepam and physostigmine. The episodes are usually short-lived, but two of the reviewed 17 patients developed recurrent retrocollis for four and 23 days following antiepileptic drug therapy. Since high doses of phenytoin and carbamazepine can result in opisthotonos, we recommend that anticonvulsants be reserved for post-anaesthetic patients with electroencephalographic evidence of seizure activity.RésuméDans ce rapport d’opisthotonos survenant au cours du réveil d’une anesthésie réalisée au propofol, nous décrivons les observations cliniques avec leurs considérations scientifiques, et proposons une stratégie de traitement de cet effet inusité. Après une chirurgie brève, une patiente de 29 ans, en bonne santé, développe un opisthonos récurrent alors qu’elle se réveille d’une anesthésie réalisée avec alfentanil, propofol et protoxyde d’azote. Contrairement aux observations accumulées, elle reste consciente à chaque épisode d’extension dorsale et cervicale. La persistence de la conscience ainsi que la similitude de cet opisthonos avec celui induit par la strychnine nous suggère que le mécanisme peut avoir son origine du tronc cérébral et de la moelle. Des investigations récentes montrent que le propofol potentialise la glycine et l’acide γ-aminobutyrique (GABA), neurotransmetteurs inhibiteurs, ce qui pourrait accentuer l’inhibition médullaire pendant l’anesthésie. L’opisthonos postanesthésique peut être secondaire à une tolérance aux transmetteurs inhibiteurs induite par le propofol. Ces phénomènes de rebond pourraient conduire à une résistance aiguë et durable aux influx inhibiteurs du tronc cérébral et de la moelle, résultant en une activité accrue des motoneurones extenseurs. Nous recommandons une stratégie thérapeutique qui restaure l’inhibition de la glycine et du GABA à des sites multiples: les agents thérapeutiques de choix seraient le diazepam et la physostigmine. Les épisodes sont habituellement brefs, mais deux des 17 patients revus ont développé une extension du cou récurrente à quatre et 23 jours après un traitement antiépileptique. Puisque des doses élevées et phénytoïne et de carbamazépine peuvent entraîner un opisthonos, nous recommandons que les anticonvulsivants soient réservés aux patients qui présentent une activité comitiale à l’élearoencéphalogramme après leur anesthésie.

Firing properties of spherical bushy cells in the anteroventral cochlear nucleus of the gerbil

In gerbils, spherical bushy cells (SBCs) encode low frequency sound signals into a temporal firing pattern. To investigate the support for the timing in this temporal code, we characterized the membrane electrical properties of visually identified SBCs in brainstem slices. A brief depolarizing subthreshold transient potential (TP) triggered, with relatively invariant latency, a single spike at the onset of a response to depolarizing current pulses. The activation of a subthreshold Na+-conductance, sensitive to blockade with tetrodotoxin, and a high threshold Ca2+-conductance, sensitive to blockade with Co2+ or Cd2+, accelerated the rising phase and amplified the TP. A K+-conductance, sensitive to blockade by 4-aminopyridine (4-AP, 50 microM), shaped the decay of the TP. Following a single spike, voltage-gated activation of transient and sustained K+-conductances suppressed any tendency to repetitively discharge. A reduction in either K+-conductance due to application of 4-AP or tetraethylammonium (TEA, 10 mM), converted the single spike mode to repetitive firing during the depolarizing pulses. A persistent, tetrodotoxin-sensitive Na+-conductance amplified steady-state depolarizing responses. A hyperpolarization-activated conductance, greatly decreased by extracellular Cs+ (3 mM) but resistant to Ba2+ (up to 1 mM), filtered the responses to hyperpolarizing current inputs. A depolarized membrane potential promoted repetitive firing in SBCs. This state, expected in pathophysiological conditions, would corrupt the temporal code.

Modulation of bursts and high-threshold calcium spikes in neurons of rat auditory thalamus

Neurons in the ventral partition of the medial geniculate body are able to fire high-threshold Ca2+-spikes. The neurons normally discharge such spikes on low-threshold Ca2+-spikes after the action potentials of a burst. We studied membrane mechanisms that regulate the discharge of high-threshold Ca2+-spikes, using whole-cell recording techniques in a slice preparation of rat thalamus. A subthreshold (persistent) Na+-conductance amplified depolarizing inputs, enhancing membrane excitability in the tonic firing mode and amplifying the low-threshold Ca2+-spike in the burst firing mode. Application of tetrodotoxin blocked the amplification and high-threshold Ca2+-spike firing. A slowly inactivating K+ conductance, sensitive to blockade with 4-aminopyridine (50-100 microM), but not tetraethylammonium (2-10 mM), appeared to suppress excitability and high-threshold Ca2+-spike firing. Application of 4-aminopyridine increased the low-threshold Ca2+-spike and the number of action potentials in the burst, and led to a conversion of the superimposed high-threshold Ca2+-spike into a plateau potential. Application of the Ca2+-channel blocker Cd2+ (50 microM), reduced or eliminated this plateau potential. The tetrodotoxin sensitive, persistent Na+-conductance also sustained plateau potentials, triggered after 4-aminopyridine application on depolarization by current pulses. Our results suggest that high-threshold Ca2+-spike firing, and a short-term influx of Ca2+, are regulated by a balance of voltage-dependent conductances. Normally, a slowly inactivating A-type K+-conductance may reduce high-threshold Ca2+-spike firing and shorten high-threshold Ca2+-spike duration. A persistent Na+-conductance promotes coupling of the low-threshold Ca2+-spike to a high-threshold Ca2+-spike. Thus, the activation of both voltage-dependent conductances would affect Ca2+ influx into ventral medial geniculate neurons. This would alter the quality of the different signals transmitted in the thalamocortical system during wakefulness, sleep and pathological states.

Barbiturate activation and modulation of GABAA receptors in neocortex

We determined if anesthetic and anti-epileptic barbiturates inhibit neurons by different mechanisms. Current- and voltage-clamp recordings were made from somatosensory neurons of neocortex and some thalamocortical neurons in coronal brain slices of rats. We compared effects of pentobarbital, amobarbital, and phenobarbital on inhibitory postsynaptic currents (IPSCs) mediated by gamma-aminobutyric acid (GABA), input conductance, and evoked action potential firing. In neocortex, pentobarbital (EC(50)=41 microM) and amobarbital (EC(50)=103 microM) increased the decay time constant of GABA(A)ergic IPSCs. At higher concentrations, pentobarbital and amobarbital shunted firing by increasing input conductance through agonism at GABA(A) receptors. At anti-epileptic concentrations, phenobarbital increased the IPSC decay time constant (EC(50)=144 microM), and shunted firing by agonism at GABA(A) receptors (EC(50)=133 microM). In thalamocortical neurons, similar concentrations of phenobarbital had negligible effects on GABA(A)ergic IPSCs, conductance, and firing. In contrast to their thalamic actions, barbiturates inhibit neocortical neurons mostly through GABA receptors. Neocortical enhancement of inhibition by pentobarbital and amobarbital, combined with actions on thalamocortical neurons, may contribute to redundant mechanisms of anesthesia. The ability of phenobarbital at anti-epileptic concentrations to inhibit neocortical firing by direct activation and modulation of GABA(A) receptors relates to its specialized therapeutic effects.

Glycinergic inhibition in thalamus revealed by synaptic receptor blockade.

Using juvenile rat brain slices, we examined the possibility that strychnine-sensitive receptors for glycine-like amino acids contributed to synaptic inhibition in ventrobasal thalamus, where gamma-aminobutyrate (GABA) is the prevalent inhibitory transmitter. Ventrobasal nuclei showed staining for antibodies against alpha1 and alpha2 subunits of the glycine receptor. Exogenously applied glycine, taurine and beta-alanine increased membrane conductance, effects antagonized by strychnine, indicative of functional glycine receptors. Using glutamate receptor antagonists, we isolated inhibitory postsynaptic potentials and currents (IPSPs and IPSCs) evoked by high-threshold stimulation of medial lemniscus. Like the responses to glycine agonists, these synaptic responses reversed near E(Cl). In comparative tests with GABA receptor antagonists, strychnine attenuated inhibition in a majority of neurons, but did not alter slow, GABA(B) inhibition. For complete blockade, the majority of fast IPSPs required co-application of strychnine with bicuculline or gabazine, GABA(A) receptor antagonists. Strychnine acting with an IC50 approximately = 33 nM, eliminated residual fast inhibition during selective GABA(A) receptor blockade with gabazine. The latency of onset for IPSPs was compatible with polysynaptic pathways or prolonged axonal propagation time. Strychnine lacked effects on monosynaptic, GABAergic IPSPs from zona incerta. The specific actions of strychnine implicated a glycine receptor contribution to fast inhibition in somatosensory thalamus.

Attenuation of glutamate-action, excitatory postsynaptic potentials, and spikes by intracellular QX 222 in hippocampal neurons

The effects of intracellular applications of QX 222, a quaternary analogue of lidocaine, were investigated in CA1 neurons of in vitro hippocampal slices of guinea-pig brain. QX 222 produced a strong depression of spontaneous, electrically-(by current injection) or orthodromically-evoked action potentials. These dose-dependent effects were characterized by a reduction in the rate of rise and amplitude of spikes, presumed to be mediated by a Na+-conductance. Although resting membrane conductance tended to diminish with prolonged applications of QX 222, marked changes in resting potential generally were not observed. The threshold for eliciting spikes by intracellular injection of depolarizing current was increased greatly by QX 222, reflecting the impairment of Na+-electrogenesis of spikes. The reduction of action potential amplitude by QX 222 may be partly attributable to enhanced inactivation of Na+-channels because brief depolarizing pulses preceded by strong tonic hyperpolarization, elicited spikes at a lower threshold and of considerably larger amplitude than in the absence of such tonic hyperpolarization. These observations on recovery are compatible with a removal of sodium inactivation. However, this experimental paradigm of current injection also might be expected to remove QX 222 molecules from their blocking sites at the inner end of Na+-channels. When spikes were abolished by QX 222, the depolarization evoked with application of S-glutamate by pressure ejection from an extracellular micropipette positioned close to the neuron was attenuated. This reversible blockade was reproducible in the 14 neurons where the interactions of QX 222 and glutamate were examined systematically. Excitatory postsynaptic potentials, evoked by stimulation of strata oriens or radiatum, were reduced in a similar manner by internal QX 222. These data confirm previous observations that voltage-dependent Na+-channels mediating spike genesis in CA1 neurons can be blocked by internal QX 222. However, QX 222 also apparently interferes with the functions of Na+-channels activated by glutamate-receptor interaction or by receptor interactions with neurotransmitter(s) associated with certain excitatory postsynaptic potentials in CA1 neurons.