Pre- and post-synaptic activity of glutamate on preparations of the frog semicircular canal

Glutamate (Glu) has at least two sites of action in the frog semicircular canal: the hair cell (presynaptic) and the primary afferent nerve fibres (postsynaptic). Glu's action on the hair cell results in an increased release of the natural transmitter which is responsible for a substantial increase in the frequency of firing in primary afferents. Glu produces a long-lasting depolarization in the afferent nerve fibres which does not by itself elicit any afferent discharge of impulses when the release of the natural transmitter is prevented. The difficulty of reconciling some of the observations made of the effects of Glu in semicircular canals with its presumed role as an afferent transmitter in this organ is discussed.     

Glutamate excitatory effects on ampullar receptors of the frog

Summary The action of glutamate on frog ampullar receptors was investigated to assess the potential role of this excitatory amino acid as an afferent transmitter in the hair cell system. Intracellular recordings from single afferent units in the isolated labyrinth revealed that glutamate and the glutamate receptor agonists, N-methyl-D-aspartic acid, quisqualic acid and kainic acid increase dose-dependently the frequency of the resting afferent discharge of EPSPs and spikes and produce long lasting depolarizations. After blocking synaptic transmission by using 5 mM Co²⁺, the same compounds elicited only depolarizations of amplitude comparable to those observed in normal saline. Quisqualic acid and kainic acid were much more potent than N-methyl-D-aspartic acid in increasing the frequency of afferent discharge and in causing axonal depolarizations. The depolarization caused by glutamate was reduced dose-dependently by the competitive non-NMDA receptor antagonist 6-cyano-7-nitroquinaxoline-2,3 dione and disappeared almost completely in Na⁺-free Ringer solution. These results are consistent with the hypothesis that glutamate is the afferent transmitter in vestibular organs and indicate that receptors mainly of the non-NMDA type are present not only at postsynaptic level but also in hair cells. Presynaptic glutamate receptors may function as autoreceptors controlling by a positive feed-back mechanism the release of the afferent transmitter.     

Properties of the postsynaptic depolarization produced by glutamate on the vestibular receptors in frogs

In order to investigate the possible role of glutamate (Glu) as afferent transmitter in the vestibular system, this agent was tested on sensory organs of frog semicircular canals. Intracellular recordings from single afferent axons in isolated labyrinths showed that, after blocking chemical transmission with high Mg++ (12 mM), micro-injections of Glu (5 mM-15 ul) elicited a long lasting postsynaptic depolarization. The amplitude of this depolarization was reduced dose dependently after addition of the amino acid antagonists Kynurenic acid or gamma-D-glutamylglycine to the bath. When the Na+ concentration in the bath was progressively reduced, the depolarization decreased gradually and disappeared almost completely in Na(+)-free Ringer. The removal of K+ affected the depolarization to a lesser extent: in K(+)-free Ringer depolarization decreased only by 30-40%. On the contrary, the complete substitution of Ca++ ions in the bath was without effect. Our results suggest that in the frog semicircular canals the postsynaptic depolarization induced by Glu involves the activation of non NMDA type of amino acid receptors, probably coupled to channels selective for Na+ and K+ ions. The present findings are consistent with the hypothesis that Glu or a related substance may be the transmitter released at the afferent synapses of the vestibular receptors.     

Ultrastructure of the lateral-line sense organs of the ratfish,Chimaera monstrosa

Summary The ultrastructure of the lateral-line neuromasts in the ratfish, Chimaera monstrosa is described. The neuromasts rest at the bottom of open grooves and consist of sensory, supporting, basal and mantle cells. Each sensory cell is equipped with sensory hairs consisting of a single kinocilium and several stereocilia. There are several types of sensory hair arrangement, and cells with a particular arrangement form patches within the neuromast. There are two types of afferent synapse. The most common afferent synapse has a presynaptic body and is typically associated with an extensive system of anastomosing tubules on the presynaptic side. When the tubules are absent, vesicles surround the presynaptic body. These synapses are often associated into synaptic fields, containing up to 35 synaptic sites. The second type of afferent synapse does not have a presynaptic body and is not associated with the tubular system. The afferent synapses of the second type do not form synaptic fields and are uncommon. The efferent synapses are either associated with a postsynaptic sac or more commonly with a strongly osmiophilic postsynaptic membrane. The accessory cells are similar to those in the acoustico-lateralis organs of other aquatic vertebrates. A possibility of movement of the presynaptic bodies and of involvement of the tubular system in the turnover of the transmitter is discussed. A comparison of the hair tuft types in the neuromasts of Ch. monstrosa with those in the labyrinth of the goldfish and of the frog is attempted.     

NMDA receptors in afferent synapses of frog semicircular canals

The effects of competitive (2-amino-phosphonovaleric acid) and noncompetitive (Mg²⁺, ketamine, kynurenic acid) antagonists of N-methyl-D-aspartate (NMDA) receptors on synaptic transmission were studied in afferent synapses of the frog semicircular canals. All of these antagonists reduced the rate of background activity in the nerve of posterior semicircular canal by 30–50%, which confirms the presence of glutamate NMDA receptors in the hair cell synapses in the frog semicircular canals.Neurofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 168–169, May–June, 1993.     

Absence of the effect of opioid peptides on muscarine receptors in the frog vestibular apparatus

We studied the effects of opioid peptide leu-enkephaline, a specific antagonist of acetylcholine receptors atropine, and non-selective opiate antagonist naloxone on synaptic transmission and responses evoked by acetylcholine in semicircular organs of the frog. A decrease in frequency of acetylcholine (0.1-5.0 microM) responses under leu-enkephaline (10 nM) id not differ from the frequency decline induced by leu-enkephaline alone. Atropine (1 microM) left the response to leu-enkephaline intact while blocking the excitatory effect of acetylcholine. No modification of the acetylcholine response under leu-enkephaline was observed in the presence of naloxone (1 microM). The findings suggest that no interaction exists between the acetylcholine-mediated excitatory action on resting activity in the isolated semicircular canal preparation and the suppressive action of leu-enkephaline.     

Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses

Kainate receptors (KARs) are a poorly understood family of ionotropic glutamate receptors. A role for these receptors in the presynaptic control of transmitter release has been proposed but remains controversial. Here, KAR agonists are shown to enhance fiber excitability, and a number of experiments show that this is a direct effect of KARs on the presynaptic fibers. In addition, KAR activation inhibits evoked transmitter release from mossy fiber synapses. Synaptic release of glutamate from either neighboring mossy fiber synapses or associational/commisural (A/C) synapses results in the activation of these presynaptic ionotropic KARs. These results, along with previous studies, indicate that KARs, through the endogenous release of glutamate, mediate excitatory postsynaptic potentials (EPSPs), alter presynaptic excitability, and modulate transmitter release.     

Lack of evidence of an interaction between leu-enkephalin and muscarinic-like responses in the frog semicircular canal

Using multiunit recording of action potentials from the whole vestibular nerve, we studied whether opioid peptide leu-enkephalin (Enk) may modulate muscarinic-like responses in semicircular organs of the frog. When acetylcholine (ACh) (0.1-1 mM) was applied with 1 nM Enk, the maximal frequency increase induced by ACh was reduced.However,the frequency decrease of ACh responses under Enk did not differ from the frequency decline in basal spike discharge induced by Enk alone. Administration of atropine (1 microM) left the response to Enk intact and blocked the excitatory effect of ACh. No modification of the ACh response under Enk was observed in the presence of the non-selective opioid receptor antagonist naloxone (10 microM). This study suggests that no interaction exists between the ACh-mediated excitatory action on resting activity in the isolated semicircular canal preparation and the suppressive action of Enk.     

Neurochemical transmission in the dorsal column nuclei

The transmitter chemistry of the dorsal column nuclei is reviewed, with special emphasis on the monosynaptic component of the dorsal column-medial lemniscal pathway. It is maintained that in this anatomically addressed system concerned mainly with fast, secure sensory transmission, amino acids represent the predominant mechanism used for chemical relay of primary afferent impulses. The major excitatory primary afferent transmitter is most likely glutamic acid, whereas gamma-aminobutyric acid (GABA) fulfills adequately the role of transmitter of recurrent, postsynaptic and presynaptic inhibition. Recent immunohistochemical and physiological evidence indicates that 5-hydroxytryptamine, originating mainly from neurons of the raphé nuclei, plays a modulatory role in dorsal column transmission of innocuous sensory information. The basic synaptic elements involved in transmission across this relay, along with their corresponding chemical identities, are presented in the form of a speculative model.     

Physical dimensions influence the functional property of the semicircular canal

The functional dependence of the semicircular canal upon its physical dimensions was evaluated by measuring the internal radius (r), the radius of curvature (R) and the cupula radius (rC) of the posterior canal in 10 freshly dissected frog labyrinths. These values have been compared to the same parameters of the cat labyrinth. The coefficients I, B, K in the Steinhausen equation were determined for both animals. The A ratio between cupula deflection and endolymph displacement was also calculated by utilizing the Bernard equation. The A ratio is three times larger in the frog than in the cat. It follows that if the same acceleration produces similar endolymph displacements in the posterior canal of both animals, the cupula deflection will be larger in the frog. The solution of the Steinhausen equation in the presence of a constant acceleration, however, reveals that the same stimulus intensity will result in a larger endolymph displacement in the cat posterior canal; similarly, the endolymph displacement directly depends on the duration of the stimulating period in both animals. Contrary to the Bernard assumption, these effects generate a G ratio (psi frog/psi cat) which is less than the Q ratio (A frog/A cat). Moreover, G decreases on increasing the duration of the stimulating period. For stimuli of short duration the semicircular canal of a small animal is expected to exhibit a higher sensitivity than that of a larger one. However, the definitive primary afferent discharge will be largely controlled by the receptor/generator potential properties.     

Selective depression of low-release probability excitatory synapses by sodium channel blockers

Sodium channels (NaChs) play a central role in action potential generation and are uniquely poised to influence the efficacy of transmitter release. We evaluated the effect of partial NaCh blockade on two aspects of synaptic efficacy First, we evaluated whether NaCh blockade accounts for the ability of certain drugs to selectively depress glutamate release. Second, we evaluated the contribution of NaChs to intraneuronal variability in glutamate release probability (p(r)). The antiglutamate drug riluzole nearly completely depresses glutamate excitatory postsynaptic currents (EPSCs) at concentrations that barely affect GABAergic inhibitory postsynaptic currents (IPSCs). NaCh inhibition explains the selective depression. Unlike other presynaptic depressants, partial NaCh blockade increases paired-pulse EPSC depression. This result is explained by selective depression of low-p(r) synapses. We conclude that local variations in the action potential contribute to p(r) variability among excitatory synapses.     

Fast and slow excitation of inhibitory cells in the CA3 region of the hippocampus

Pyramidal cells form excitatory synaptic connections with local inhibitory neurons in the hippocampus. This recurrent synapse plays a crucial stabilizing role in the control of hippocampal activity, since it transforms pyramidal cell population. Using a combination of dual recording from presynaptic and postsynaptic cells and anatomical techniques, we show that these synaptic connections often comprise a single site for liberation of excitatory transmitter. The resulting excitatory postsynaptic potentials (EPSCs) have a fast time course and a similar amplitude to miniature EPSCs recorded in tetrodotoxin and cobalt. In contrast, activation of metabotropic glutamate receptors (mGluRs) by transmitter liberated during repetitive activation of these synapses produces an excitation with a much slower time course. In addition to somatodendritic mGluRs, which excite inhibitory cells, a different species of mGluR is present on inhibitory cell terminals. This mGluR is activated by higher concentrations of the agonist t-1-amino-cyclopentyl–1,3-decarboxylate and acts to reduce γ-aminobutyric acid release. mGluRs, thus, have a dual action to enhance and to depress synaptic inhibition in the hippocampus. © 1995 John Wiley & Sons, Inc.     

Phosphatases modulate transmission and serotonin facilitation at synapses: Studies with the inhibitor okadaic acid

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 μM OA. At lower doses (0.5–5 μM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, Which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

Phosphatases modulate transmission and serotonin facilitation at synapses: studies with the inhibitor okadaic acid.

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 microM OA. At lower doses (0.5-5 microM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

High extracellular concentrations of potassium and rubidium modulate the synaptic transmission in the frog labyrinth

High extracellular K or Rb levels (20 mM) produce an increase in the resting EPSP and spike frequencies recorded intra cellularly from single fibres of the posterior nerve in the isolated frog labyrinth. The afferent discharge facilitation proved to be inversely related to the fibre's initial resting activity. The K effect is systematically larger than the Rb effect. High sensitive and scarcely sensitive units may be identified with respect to K and Rb action. The present findings suggest that, according to previous models of hair cell functioning, the K and Rb effects are mediated by a raise in intracellular Ca concentration which sustains an increased transmitter release at the cyto-neural junction.     

Flexible processing of sensory information induced by axo-axonic synapses on afferent fibers.

Recent experiments indicate that afferent information is processed in the intraspinal arborisation of mammalian group I fibres. During muscle contraction, Ib inputs arising from tendon organs are filtered out by presynaptic inhibition after their entry in the spinal cord. This paper reviews the mechanisms by which GABAergic axo-axonic synapses, i.e., the morphological substrate of presynaptic inhibition, exert this filtering effect. Using confocal microscopy, axo-axonic synapses were demonstrated on segmental Ib collaterals. Most synapses were located on short preterminal and terminal branches. Using a simple compartmental model of myelinated axon, the primary afferent depolarisation (PAD), generated by such synapses, was predicted to reduce the amplitude of incoming action potentials by inactivating the sodium current, and this prediction was experimentally verified. A further theoretical work, relying on cable theory, suggests that the electrotonic structure of collaterals and the distribution of axo-axonic synapses allow large PADs (about 10 mV) to develop on some distal branches, which is likely to result in a substantial presynaptic inhibition. In addition, the electrotonic structure of group I collaterals is likely to prevent PAD from spreading to the whole arborisation. Such a non-uniform diffusion of the PAD accounts for differential presynaptic inhibition in intraspinal branches of the same fibre. Altogether, our experimental and theoretical works suggest that axo-axonic synapses can control the selective funnelling of sensory information toward relevant targets specified according to the motor task.     

Ultrastructure of synapses with different transmitter-releasing characteristics on motor axon terminals of a crab,Hyas areneas

Synaptic terminals on branches of an excitatory motor axon in a spider crab (Hyas areneas) were examined by electron microscopy to determine whether differences in size, structure, and number of synapses could be correlated with differences in transmitter release. Terminals releasing relatively large amounts of transmitter during low frequencies of nerve impulses ("high-output" terminals) had larger synapses, more prominent presynaptic dense bodies (active zones), and fewer synapses per unit length than terminals releasing relatively small amounts of transmitter ("low-output" terminals). Neither the difference in synaptic area, nor the quantitative differences in the active zones, were sufficient in themselves to explain the difference in synaptic efficacy, and it is postulated that a non-linear relationship may exist between structural features of the synapse and release of transmitter by a nerve impulse, and that differences other than those apparent from the ultrastructure could be involved. Greater facilitation at low-output terminals with high frequencies of nerve impulses may be due to greater reserves of "immediately available" transmitter, and to recruitment or activation of more individual synaptic contacts.     

Interrelations between auditory and vestibular receptors in the frog's labyrinth

Summary The influence of the efferent vestibular system being eliminated, the spontaneous activity of afferent fibres of the ampullary nerves of the horizontal and vertical anterior semicircular canals was recorded in the frog. By functionally eliminating either both papillae or all the vestibular receptors except for the papillae, and then using statistical methods, as well as by stimulating the papillae by sounds or the papillary nerve fibres by electrical stimulus, it has been shown that the auditory papillae have a facilitatory influence on the spontaneous afferent activity from the horizontal and vertical anterior canals. This influence is most likely mediated by receptor-receptor fibres arising from the auditory organs and innervating the semicircular canals.Abbreviations HC horizontal canal - VAC vertical anterior canal This research was supported by a grant from D.G.R.S.T. (Aide à la Recherche n 77.7.1127)     

Synaptosomal release of newly-synthetized or recently accumulated amino acids. Differential effects of kainic acid on naturally occurring excitatory amino acids and on [d-3H]aspartate

Kainic acid, a powerful neuroexcitant and neurotoxin, stimulates the release of naturally occurring excitatory amino acids, l-glutamate and l-aspartate, from hippocampal synaptosomes. The release stimulation affects in a similar way both the general pool of the two amino acids and the fraction of l-glutamate and l-aspartate, newly-synthetized from precursors or recently accumulated through the high-affinity uptake mechanism. Kainic acid exerts its stimulatory action on the basal release of the two amino acids as well as on the high K⁺-stimulated release of l-glutamate. Kainic acid has, however, different effects on the release of exogenously accumulated [d-³H]aspartate. In particular, the high K⁺-stimulated release of this false transmitter is strongly inhibited by 1 mM kainic acid. The present data confirm the presynaptic action of kainic acid on the general as well as on the recently-formed pools of naturally occurring excitatory amino acids. At the same time, our results suggest that [d-³H]aspartate is not a reliable substitute for l-glutamate and l-aspartate, in release studies and that the radioactivity released after preloading with [d-³H]aspartate does not necessarily reflect the release of naturally occurring excitatory amino acids.     

GABA- and glycine-immunoreactive synapses in spinal cord of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Using the method of the double immune label combined with two antibodies, i.e., monoclonal antibodies to gamma-aminobutyric acid (GABA) and polyclonal antibodies to glycine, the distribution of gamma-aminobutyric acid- and glycine-immunoreactive synapses on motoneurons and primary afferent axons was studied in the frog Rana temporaria spinal cord. An analysis of all labeled boutons on the dendrites and soma of motoneurons showed the existence of three categories of immunoreactive synapses as follows: 7% were labeled for GABA, 23% were labeled for glycine, and approximately 70% were immunoreactive to both GABA and glycine. These results confirm the predominant role of glycine in the postsynaptic inhibition of motoneuronal activity. Three similar populations of synaptic boutons were also founded on primary afferent axons, including one GABA-immunoreactive (25%) and one glycine-immunoreactive (5%); the majority of the immunoreactive synapses had the colocalization of two inhibitory transmitters. As a rule, the higher proportion of axo-axonal synapses was organized in synaptic triads. The possible simultaneous roles of glycine as a transmitter of postsynaptic inhibition and as a transmitter that mediates the process of the autoreception of glutamate in the axo-axonal synapses on the primary afferent fibers are discussed.