Homeostatic changes of short-term plasticity of gabaergic synaptic transmission in rat hippocampal cell cultures.

It is well documented that prolonged alteration of activity in neuronal networks initiates a number of homeostatic mechanisms including compensatory changes of excitatory and inhibitory synaptic strength. We studied whether this also evokes compensatory changes of short-term synaptic transmission. Using patch-clamp technique in hippocampal cell cultures we examined the effects: of prolonged decrease of neuronal firing evoked by sodium channel blocker: tetrodotoxin (TTX) and ionotropic glutamate receptor antagonist - kynurenate; prolonged enhancement ofneuronal firing evoked by antagonist GABAA receptors - bicuculline on short-term depression of GABAergic synaptic transmission evoked by train of stimuli (5 Hz). We found that both TTX and kynurenate treatments enhance depression of GABAergic transmission, while bicuculline treatment does not. We conclude that alteration of depression of GABAergic transmission evoked by the prolonged decrease of neuronal activity may contribute to homeostatic plasticity in hippocampal neuronal networks.

Cannabinoid regulation in identified synapse of terrestrial snail.

In the terrestrial snail a direct monosynaptic glutamatergic connection between the primary sensory neuron and a premotor interneuron involved in withdrawal behaviour can be functionally identified using electrophysiological techniques. We investigated the involvement of cannabinoids in regulation of this synaptic contact. The results demonstrate that the specific binding sites for agonists to mammalian type 1 cannabinoid receptors (CB1Rs) exist in the snail's nervous system. Application of a synthetic cannabinoid agonist anandamide selectively changed the efficacy of synaptic contacts between the identified neurons. A decrease in the long-term synaptic facilitation of the synaptic contact elicited by high-frequency nerve tetanization in the presence of cannabinoid agonist anandamide was observed, suggesting a possible role of endocannabinoids in regulation of plasticity at this synaptic site. The selective antagonist of CB1Rs [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] AM251 bath application was changing the efficacy of the synaptic contact only when the postsynaptic neuron had been intracellularly activated before its application. This observation implies an involvement of endocannabinoids in plasticity phenomena induced by activity in the postsynaptic target. Additional support of endocannabinoid involvement in synaptic function at this site was given by experiments in which AM251 blocked the short-term suppression of synaptic excitation evoked by low-frequency nerve tetanization, a phenomenon qualitatively similar to cannabinoid-dependent synaptically evoked suppression of excitation demonstrated in the mammalian nervous system. The results of the present study suggest an involvement of cannabinoids in the regulation of synaptic efficacy. Further, anandamide could be a candidate for an endogenous neuromessenger involved in plasticity processes.

[Retrograde signaling in the GABA- and glutamatergic synapses of the brain].

According to classical concept, due to chemical synapses, information in the central nervous system is transferred in one direction: from presynaptic neurons to postsynaptic ones. Although several cases of information transfer in the opposite direction were known for a long time, those were considered as rare exceptions. However, recent results indicate that retrograde signaling between brain neurons is rather a common phenomenon. In this review we will focus on two related forms of short-term plasticity of GABAergic and glutamatergic synaptic transmission observed in several brain structures and mediated by retrograde messengers endocannabinoids. Namely, we will characterize phenomenon termed "depolarization-induced suppression of inhibition", observed at GABAergic synapses and related phenomenon observed at glutamatergic synapses named "depolarization-induced suppression of excitation".

Presence of depolarization-induced suppression of inhibition in a fraction of GABAergic synaptic connections in rat neocortical cultures.

Brief depolarization of postsynaptic neurons in hippocampus and cerebellum results in a transient depression of GABAergic inhibitory input, called "depolarization-induced suppression of inhibition" (DSI). We studied whether a similar phenomenon occurs in the rat neocortical neurons. Using patch-clamp technique in neocortical cell cultures, we examined the effects of a 5-second depolarization of postsynaptic neurons on evoked GABAergic inhibitory post-synaptic currents (IPSCs). We found that the depolarization evoked a suppression of IPSC amplitude in 6 out of 26 neuronal pairs tested. The suppression of IPSC amplitude lasted for approximately 70 seconds and was accompanied by changes of paired-pulse ratio and IPSC coefficient of variation (CV), which is suggestive of a presynaptic mechanism. These results are in agreement with previous observations in hippocampal cell cultures and suggest that neocortical neurons express DSI.

Presence of depolarization-induced suppression of inhibition in a fraction of gabaergic synaptic connections in rat neocortical cultures.

Brief depolarization of postsynaptic neurons in hippocampus and cerebellum results in a transient depression of GABAergic inhibitory input, called "depolarization-induced suppression of inhibition" (DSI). We studied whether a similar phenomenon occurs in the rat neocortical neurons. Using patch-clamp technique in neocortical cell cultures we examined the effects of a 5-second depolarization of postsynaptic neurons on evoked GABAergic inhibitory post-synaptic currents (IPSCs). We found that the depolarization evoked a suppression of IPSC amplitude in 6 out of 26 neuronal pairs tested. The suppression of IPSC amplitude lasted for approximately 70 seconds and was accompanied by changes of paired-pulse ratio and IPSC coefficient of variation (CV), which is suggestive of a presynaptic mechanism. These results are in agreement with previous observations in hippocampal cell cultures and suggest that neocortical neurons express DSI.

[Effect of synaptic transmission blockade and hypoglycemia on neuron impulse activity in cultured rat hippocampal neurons]. / Vplyv blokady synaptychnoï peredachi ta hipohlikemiï na impul'snu neironnu aktyvnist' u kul'turi neironiv hipokampa shchura.

Spontaneous neuronal activity was studied in rat hippocampal cell cultures using patch clamp technique in cell-attached or loose-patch configuration. It was found that in spite of relatively low average frequency (1-2 Hz) of neuronal activity in the cell cultures, neurons often fire doublets or triplets of action potentials with interspike interval of 60 ms and less. Interspike interval histograms were substantially better fitted by double exponential decay functions than by single exponential ones. On average, estimated decay time constant for the fits were tau1 approximately 36, ms and tau2 approximately 1000 ms respectively. Spontaneous neuronal firing to a large extent depended on glutamatergic excitation and GABA(A)ergic inhibition: a blocker of AMPA/kainate receptor CNQX (10 microM) either substantially decreased or completely blocked spontaneous action potentials; a blocker of GABA(A) receptors bicuculine (10 microM) increased neuronal firing. Effect of glucose deprivation on action potential frequency was also studied. It was found that glucose deprivation reduces AP frequency to 25% of control. Taken together, these results support an idea that hypoglycaemia alters synaptic transmission in hippocampus.

Postsynaptic mechanism may contribute to inhibitory acetylcholine effect on GABAergic synaptic transmission in hippocampal cell cultures.

The effect of acetylcholine (ACh) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections. Spontaneous IPSCs and IPSCs evoked by extracellular stimulation of a single presynaptic neuron were recorded. ACh inhibited the evoked IPSCs in most of the connections, although facilitation was also observed. Regardless of inhibitory or facilitatory effects on the evoked IPSCs, an enhanced spontaneous synaptic input to the postsynaptic neurons was usually observed. ACh-induced changes in the evoked IPSCs were usually accompanied by changes in paired pulse depression (PPD), which are thought to reflect presynaptic mechanisms of modulation. However, the time course of PPD changes did not always match that of the IPSC changes, suggesting a contribution of other, possibly postsynaptic, mechanism(s). To analyze this possibility, effects of ACh on responses to direct application of exogenous GABA were studied. In a proportion of the neurons (40%) ACh reversibly decreased GABA responses, indicating that postsynaptic mechanisms may also contribute to the inhibitory ACh effect on GABAergic transmission. We conclude that several different modulatory mechanisms of ACh action participate in the regulation of GABAergic transmission at the level of synaptic connection of a single GABAergic neuron.

Some receptor properties in motor neurons of an Aplysia withdrawal reflex.

Glutamate or a glutamate-related substance is the neurotransmitter used at the majority of the excitatory junctions of the neuronal network mediating the gill and siphon withdrawal reflex in Aplysia. In this report, we have studied some receptor properties of the major postsynaptic elements of the network, the motor neurons. We have examined the effect of a compound interfering with glutamate receptor, concanavalin A (con A). We found that con A treatment transforms the mainly hyperpolarizing responses to L-glutamate in motor neurons to prolonged depolarizing ones; these latter responses are sensitive to CNQX. We have also examined whether con A could enhance the CNQX sensitive excitatory postsynaptic potentials in these motor neurons. We found, by contrast, that con A did not alter the synaptic responses. The possible implications of the differential effect of con A on the glutamate responses and the synaptic responses are discussed.

Analysis of potentiation in the cerebral ganglion of Aplysia.

Quantal analysis was used to characterize synaptic transmission between A and B neurons in the cerebral ganglion of Aplysia in control and during slow developing potentiation, a form of synaptic plasticity exhibited by these synapses. Control values of mean quantal content (m) and quantal size (q) estimated by the method of coefficient of variation (CV) were m approximately 6, q approximately 56 microV in the solution with Ca2+/Mg2+ = 5/200 and m approximately 18, q approximately 41 microV in the solution with Ca2+/Mg2+ = 55/150. There was a good correlation between an increase in the amplitude of excitatory synaptic potential and an increase in calculated quantal content (m(cv)) during potentiation. A decrease of Ca2+/Mg2+ ratio in the bath solution allowed observation of transmission failures and in some cases regular peaks on excitatory postsynaptic potential amplitude histograms. The latter provided more direct estimate of the quantal size. Induction of the potentiation in this solution, however, became difficult. In cases of successful potentiation induction, probability of failures was less than in control; distances between histogram peaks, reflecting quantal size remained the same. The results obtained in this study support a hypothesis that potentiation of the synaptic transmission between A and B neurons of Aplysia is primarily due to an increase of transmitter release.

Cloning and characterization of Aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in Aplysia californica.

Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.

The synaptic junctions of LE and RF cluster sensory neurones of Aplysia californica are differentially modulated by serotonin.

The monosynaptic component of withdrawal reflexes in Aplysia californica, from sensory neurones to motor neurones, is a critical site of the synaptic modulation occurring during short-term and long-term behavioural changes. There are four clusters of sensory neurones (LE, rLE, RE, RF) innervating the receptive field for the gill and siphon withdrawal reflex. The receptive fields of these cells are located on the siphon, the mantle, the branchial cavity and the gill itself. In most studies, the synapses made by the sensory neurones of the LE cluster of the abdominal ganglion or the VC cluster of the pleural ganglion have been investigated and shown to be facilitated by the neuromodulator serotonin (5-hydroxytryptamine, 5-HT). In this report, we have examined the effect of 5-HT on the synaptic junctions of the RF cluster neurones. The duration of action potentials in these cells, unlike those of the other clusters, is barely affected by serotonin. We found that while the LE synapses are facilitated by 5-HT (10 micromol l-1), the RF synapses are not. In fact, the RF-L14 connections are actually depressed by 5-HT; this effect is not due to shunting in the postsynaptic neurone. The RF-L7 connections are also depressed by 5-HT, although the effect is smaller. The RF-L14 connections are blocked by the non-NMDA receptor agonist CNQX (100 micromol l-1), suggesting that the transmitter and the postsynaptic receptors involved are similar to those present on the LE or VC cluster cells. The absence of serotonin-induced facilitation of the RF cluster cells may provide the animal with a means of reducing the nonspecific effects of aversive sensitization and therefore of allowing a greater specificity and more flexibility in plastic behavioural changes.

Cloning and functional expression of an Aplysia 5-HT receptor negatively coupled to adenylate cyclase.

Serotonin (5-HT) is involved in the control of various behaviors in Aplysia californica, including reproduction, feeding, locomotion, circadian rhythm, synaptic plasticity, and synaptic growth. The large variety of functions of 5-HT is mediated by different receptor subtypes that are coupled to different second-messenger systems. Here, we report the cloning of a cDNA coding for an Aplysia G-protein-coupled 5-HT receptor (5-HTap1). Its deduced amino acid sequence resembles those of the 5-HT1 receptor subfamily. When expressed in stable cell lines, 5-HTap1 exhibits high-affinity binding for the serotonergic radioligand [N-methyl-3H]lysergic acid diethylamide. This binding is competed by several 5-HT agonists and antagonists, and the pharmacological profile of inhibition has some similarities with those of 5-HT1 and 5-HT7 receptors. Application of 5-HT or its agonists 5-carboxamidotryptamine maleate and (+/-)-8-hydroxy-2-(di-n-propyl-amino) tetralin hydrobromide on cells transformed with 5-HTap1 produced a dose-dependent inhibition of forskolin-stimulated cAMP accumulation. 5-HTap1 is thus negatively coupled to adenylate cyclase. The production of antiserum against the 5-HTap1 receptor allowed us to examine its expression in animal tissues. The receptor protein is detected in every tissue examined, although it seems only weakly expressed in some samples. The receptor is also found in every ganglia of the nervous system, both in the sheath and in the neurons. 5-HTap1 mRNA is absent from the sheath, indicating that the protein observed there is probably located on the nerve terminals.

The effect of acetylcholine and serotonin on calcium transients and calcium currents in identified Helix pomatia L. neurons.

The results presented demonstrate that in D neurons of the snail Helix pomatia L., acetylcholine (ACh) (10 divided by 100 microM) and serotonin (5-HT) (0.1 divided by 1000 microM) applications reduce both the basal intracellular concentration level ([Ca2+]in) and the amplitudes of calcium transients induced by membrane depolarization. It is likely that the mechanism of [Ca2+]in changes in the suppression of calcium inward currents (ICa). Influences of Ach and 5-HT on ICa were studied. Both effects were dose-dependent (ACh--0.01 divided by 100 microM and 5-HT--0.1 divided by 1000 microM). The half-maximal effects (IC50) were evoked by ACh concentration of 0.15 microM and 5-HT--15 microM. Furthermore we have also shown that in some cells 5-HT could evoke a transient increase in ICa (IC50 = 2 microM). The effects of Ach and 5-HT were nonadditive--the subsequent application of ACh after 5-HT, and vice versa, produced no inhibitory effects. This may indicate that both substances act through a common intermediate (possibly, G-protein).

Patch-clamp recording of cAMP-induced membrane current noise in Helix pomatia neurons.

Membrane current noise evoked by intracellular cAMP injection was studied in isolated Helix pomatia neurons with the patch-clamp technique. Fluctuation analysis was used to estimate the elementary current amplitude (i) and the single channel conductance. It was found that i decreased linearly with cell depolarization and the extrapolated reversal potential was approximately -12 mV. The calculated single-channel conductance was 0.9 +/- 0.14 pS, a value quite different from those obtained for cAMP-activated channels in Pleurobranchaea neurons.

Dopamine inhibits transmission between the interneuron initiating pacemaker activity in a bursting neuron and the bursting neuron on the snail Helix pomatia.

1. The effect of external application of dopamine on connection between a peptidergic interneuron initiating bursting pacemaker activity in non-active burster RPa1 and the bursting neuron itself of the snail Helix pomatia has been investigated under clamp conditions. 2. External application of dopamine in a dose-dependent manner (Kd 15 microM) produces a reversible inhibition of slow inward current (SIC) in the RPa1 neuron evoked by stimulation of peptidergic interneuron. At the same time, an increase of duration and amplitude of interneuronal action potential was observed. 3. Stimulation of the anal nerve evokes in RPa1 neuron postsynaptic current consisting of at least three components: two fast ones with reversal potentials -50 and -70 mV and a long-lasting one with no reversal potential between -50 and -95 mV. 4. It is concluded that inhibition of SIC by dopamine is due to the processes occurring in the postsynaptic RPa1 neuron. 5. Based on the hypothesis that bursting activity of RPa1 neuron results from the activation of presynaptic unidentified peptidergic interneuron(s) with persistent activity and on data presented it is suggested that inhibition of bursting activity evoked by application of dopamine or anal nerve stimulation is a consequence of decreased efficiency of synaptic transmission between the interneuron initiating bursting activity and the burster.

Role of cyclic adenosine monophosphate in simple forms of plasticity in the edible snail.

The participation of the adenylate cyclase system in the short-lived changes in the efficiency of synaptic transmission of a functionally identified synapse of the edible snail has been investigated. It was established that imidazole (a phosphodiesterase activator) in a concentration of 5 mM and tolbutamide (an inhibitor of cAMP-dependent phosphorylation) in a concentration of 2 mM do not alter the rate of the depression of EPSPs which is elicited by rhythmic stimulation at a frequency of 0.1 Hz, and do not block heterosynaptic facilitation. At the same time, both of these substances decrease the amplitude of EPSPs. The possibility of the modulation of the efficiency of synaptic transmission through the adenylate cyclase system is discussed.

[The role of cyclic adenosine monophosphate in simple forms of plasticity in the edible snail]. / Rol' tsiklicheskogo adenozinmonofosfata v prostykh formakh plastichnosti u vinogradnoi ulitki.

cAMP-dependence of synaptic depression and facilitation was investigated in functionally identified synaptic connection in the snail. It was found that 5 mM imidazole (phosphodiesterase activator) as well as 2 mM tolbutamide (inhibitor of cAMP-dependent protein kinase) do not change the rate of EPSPs depression during rhythmic (0.1 Hz) nerve stimulation, and do not affect facilitation. But treatment with both these drugs decreases EPSPs amplitude. Possibility of cAMP-dependent modulation of synaptic effectiveness is discussed.