Postsynaptic Currents in Dorsal Root Ganglion Neurons Co-cultured with Spinal Cord Neurons

In co-cultured dorsal root ganglion (DRG) neurons and spinal cord neurons from newborn rats, using a voltage-clamp technique in the whole-cell configuration enabled us to observe in DRG neurons the effects evoked by extracellular local electrical stimulation of cells corresponding to spinal cord neurons in their morphological characteristics. Such stimulation caused the appearance of postsynaptic currents (PSC) in DRG neurons in 9% of the cases. The mean delay of these currents (measured from the stimulus leading edge) was 4.7 ± 0.29 msec, the mean time to peak was 2.6 ± 0.77 msec, and the decay time constant = 14.5 ± 1.04 msec. The reversal potential of evoked PSC (ePSC) was close to the equilibrium potential for chloride ions estimated by the Nernst equation. Application of 20 M bicuculline induced practically complete and reversible ePSC block. The conclusion was drawn that these currents arise due to activation of the chloride channels operated by GABA receptors and, hence, represent an inhibitory PSC. Thus, one may deem it proved that spinal cord neurons can establish functional inhibitory synapses with DRG neurons.     

Induction of Long-Term Depression of Synaptic Transmission in a Co-Culture of DRG and Spinal Dorsal Horn Neurons of Rats

In a co-culture of dissociated neurons of lumbar dorsal root ganglia (DRG) and spinal dorsal horn (DH) neurons of newborn rats, we examined peculiarities of induction of long-term depression (LTD) of synaptic transmission through synapses formed by primary afferents on DH neurons. Induction of LTD was provided by low-frequency (5 sec⁻¹) microstimulation of single DRG neurons. Ion currents were simultaneously recorded in pre- and post-synaptic cells using a dual whole-cell path-clamp technique. Parameters of evoked excitatory and inhibitory postsynaptic currents (eEPSCs and eIPSCs, respectively) initiated in DH neurons by intracellular stimulation of DRG neurons were analyzed. Monosynaptic eEPSC mediated by activation of AMPA receptors demonstrated no sensitivity to blockers of NMDA and kainate receptors (20 μM D_L-AP5 and 10 μM SIM 2081, respectively), but were entirely blocked upon applications of 10 μM DNQX. Monosynaptic glycinergic eIPSCs found in some of the DH neurons were blocked by 1 μM strychnine and were insensitive to 10 μM bicuculline and blockers of glutamatergic neurotransmission, D_L-AP5 and DNQX. Long-lasting (360 sec) low-frequency stimulation of DRG neurons did not affect the amplitude of glycineinduced eIPSCs in DH neurons. At the same time, such stimulation of DRG neurons evoked a drop in the amplitude of AMPA-activated eEPSCs in DH neurons to 41.6 ± 2.5%, on average, as compared with the analogous index in the control. This effect lasted at least 20 min after stimulation. Long-term depression of glutamatergic transmission in DH neurons was observed at the holding potential of −70 mV and did not change after applications of 10 μM bicuculline and 1 μM strychnine. The LTD intensity depended on the duration of low-frequency stimulation of primary afferent neurons. Sequential stimulation of DRG neurons lasting 120, 160, 200, and 240 sec resulted in decreases in the eEPSC amplitude in DH neurons to 85.6 ± 3.9, 62.7 ± 4.3, 51.8 ± 3.5, and 41.6 ±2.5% with respect to control values. Our findings show that use-dependent induction of homosynaptic LTD of glutamatergic transmission is possible at the level of a separate pair of synaptically connected DRG and DH neurons under co-culturing conditions. Such LTD of glutamatergic synaptic transmission mostly mediated by activation of AMPA receptors depends on the duration of activation of a presynaptic DRG neuron and does not need depolarization of a postsynaptic DH neuron.     

GABA-Mediated Inhibition in Cultured Neurons of the Rat Brain Cortex

In cultured pyramidal neurons of the rat brain cortex, we recorded (in the whole-cell configuration) postsynaptic currents (PSC) evoked by direct electrical microstimulation of an axon of the interneuron adjacent to the pyramidal cell. Application of 5 M bicuculline rapidly, entirely, and reversibly blocked these currents. Linear changes in the holding potential on the membrane of the postsynaptic cell resulted in linear changes in the amplitude of averaged currents. The currents underwent reversion when the holding potential was –16 mV, which was close to the reversal potential for Cl^- ions at their respective concentrations in the extra- and intracellular solutions. We conclude that the recorded currents are inhibitory PSC (IPSC) mediated by GABA release. The amplitudes of the recorded currents varied from a measurable minimum (8 pA) to more than 150 pA at a holding potential on the postsynaptic cell membrane of –80 mV. Times to peak of the high- and low-amplitude currents showed no significant differences, being about 6.4 msec on average. Decays of the current could be satisfactorily approximated by a monoexponential function with a mean time constant of 17 msec. The time constants of IPSC decay were distributed accordingly to the Gaussian law. In some cases, the amplitude distributions of IPSC were unimodal ((with a rightward asymmetry), but in most cases they were clearly polymodal. The amplitude distribution can be described by the sum of several Gaussian distributions; the distance between modes of the Gaussians was 25 ± 6 pA, on average. The obtained estimates of the amplitude of monoquantal GABA-induced IPSC in neurons of the brain cortex allow us to conclude that in various CNS regions the dimension of the vesicles in GABA-ergic synapses formed by inhibitory interneurons is identical.     

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABA_A receptors (GABA_ARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABA_ARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABA_AR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABA_AR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABA_ARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.     

Maintenance of high-frequency transmission at purkinje to cerebellar nuclear synapses by spillover from boutons with multiple release sites

Cerebellar Purkinje neurons maintain high firing rates but their synaptic terminals depress only moderately, raising the question of how vesicle depletion is minimized. To identify mechanisms that limit synaptic depression, we evoked 100 Hz trains of GABAergic inhibitory postsynaptic currents (IPSCs) in cerebellar nuclear neurons by stimulating Purkinje axons in mouse brain slices. The paired-pulse ratio (IPSC(2)/IPSC(1)) of the total IPSC was approximately 1 and the steady-state ratio (IPSC(20)/IPSC(1)) was approximately 0.5, suggesting a high response probability of postsynaptic receptors, without an unusually high release probability. Three-dimensional electron microscopic reconstructions of Purkinje boutons revealed multiple active zones without intervening transporters, suggestive of "spillover"-mediated transmission. Simulations of boutons with 10-16 release sites, in which transmitter from any site can reach all receptors opposite the bouton, replicated multiple-pulse depression during normal, high, and low presynaptic Ca influx. These results suggest that release from multiple-site boutons limits depletion-based depression, permitting prolonged, high-frequency inhibition at corticonuclear synapses.     

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.     

Assessment of the Reliability of Amplitude Histograms for Inhibitory Synaptic Currents in Rat Cortex Culture

We analyzed in detail the quantum parameters of evoked inhibitory postsynaptic currents (eIPSC) recorded from synaptically connected cultured cortex neurons using a whole-cell patch-clamp technique. The IPSC were evoked using minimum extracellular stimulation of a presynaptic unit with a frequency of 0.2 sec^-1 at the holding potential of -80 mV. Amplitude histograms for eIPSC demonstrated clearly detectable equally spaced peaks. For each histogram, we used a method based on autocorrelation analysis and Monte Carlo simulation to determine whether peaks in the amplitude histograms can result due to finite sampling from the sum of the Gaussian distributions. The autocorrelation function allowed us to measure the peak spacing (and, hence, the mean quantum size) for each histogram; this parameter was found to be 10 pA.     

Evoked inhibitory postsynaptic currents in the dynamics of development of cultured hippocampal neurons of rats

On a low-density culture of the hippocampal neurons of rats, we studied inhibitory transmission through the synaptic connection of a cell pair; a patch-clamp technique in the whole-cell configuration and direct extracellular stimulation of the neurites were used. We found that the mean amplitude of evoked inhibitory postsynaptic currents (eIPSC) and variability of their amplitudes significantly increased within the culturing period. The duration of the current rising phase also increased concurrently with the growth and differentiation of the neurons, but this change was non-monotonic. The coefficient of variation of the current amplitudes, as well as the time constant of current decay (the latter reflects the properties of a postsynaptic unit), showed no clear changes in the course of culturing of the neurons. Our data show that in the course of synaptogenesis the number of unitary inhibitory synaptic contacts between cultured hippocampal neurons increases, while modifications of the transmission mechanism at the level of unitary synaptic contact are less significant.     

Glutamate-induced suppression of inhibitory synaptic transmission in cultivated hippocampal neurons

In a dissociated culture of rat hippocampal neurons (14 to 24 daysin vitro), modulation effects of glutamate on GABA_A-ergic inhibitory transmission were studied with the use of simultaneous patch-clamp whole-cell recording from monosynaptically connected neuron pairs. In all experiments (n=49), 1.5-min-long or longer extracellular application of 0.5 to 100 μM glutamate suppressed evoked inhibitory postsynaptic currents (IPSC). This suppression usually included fast (seconds) and slow (τ=1.3 min) phases. In 83.7% of the cases studied, IPSC did not return to the control values during the entire subsequent recording period (from 10 to 64 min). When glutamate was applied in the presence of blockers of glutamate ionotropic receptors, DL-APV or CNQX, the fast phase of the effect was removed, while some suppression of inhibitory neuronal responses, although weaker, was preserved (n=19); in most cases (73.3%) this residual suppression was slow and long-lasting. It is concluded that both types of glutamate receptors, ionotropic and metabotropic, are involved in modulation of GABA_A-ergic synaptic transmission. The first above receptor type provides fast and reversible suppression, while the effect provided by the second type is slow and long-lasting.     

Quantum Properties of GABA Release in Cultured Neurons from the Rat Cortex

We analyzed the properties of inhibitory synaptic transmission between neurons in low-density cultures of cortical cells. Miniature, spontaneous, and evoked inhibitory postsynaptic currents were studied using a whole-cell path-clamp technique at a holding potential of -80 mV. These postsynaptic currents were identified as GABA release-activated Cl^- currents mediated by GABA_A receptors. Fitting amplitude histograms of these currents with Gaussian curves and an autocorrelation technique revealed the presence of equidistant peaks corresponding to a mean quantum size of 10 pA.     

Potassium channel blocker-induced presynaptic modulation of inhibitory synaptic transmission in hippocampal neurons of rats

The effects of blockers of voltage-gated potassium channels, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), on inhibitory postsynaptic currents (IPSC) evoked by local electrical stimulation of zones of unitary synaptic terminals on hippocampal neurons were studied using a voltage-clamp technique under conditions of low density cell culture. At activation of the transmitter release in the absence of action potentials (when the terminals are in a tetrodotoxin-containing medium), external application of 5 mM 4-AP reversibly increased the averaged IPSC amplitude by 90±30%, while a similar effect of 10 mM TEA reached only 20±7%. The amplitudes of individual evoked IPSC varied between 10 and more than 150 pA. Amplitude histograms of IPSC in all studied neurons (n=14) were of a polymodal nature and could not described by a Gaussian law. An increase in the averaged IPSC amplitude under the influence of potassium channel blockers cannot be described as resulting only from modification of the number of trials without transmitter release (blank events). The mechanism of potassium channel blocker-induced facilitation of IPSC evoked by single synaptic terminals is discussed.     

Quantum Characteristics of Neurotransmitter Release in GABA-ergic Synapses of Cultured Neurons of the Rat Spinal Cord

Using the whole-cell patch-clamp technique and stimulation of a single presynaptic terminal, we studied peculiarities of GABA release in inhibitory synapses of cultured neurons of the rat spinal cord. Analyzing the amplitude distributions of evoked inhibitory postsynaptic currents, we estimated the main quantum parameters of transmitter release. It was demonstrated that the minimum transmitter release in GABA-ergic synapses of spinal neurons cultured 9 to 11 days is multiquantum (packets containing at least 2 or 3 quanta). The distribution of the number of released quanta sufficiently agreed with that theoretically calculated according to the Poisson law. It is hypothesized that the minimum simultaneous two (three-)-quantum release of GABA in synapses of spinal neurons can be related to synchronous involvement of two closely adjacent excited terminals, each of which possesses one active zone, or of one terminal with two active zones.     

Kinetic characteristics of inhibitory postsynaptic currents in cultured chick embryo spinal cord neurons

Decay of inhibitory postsynaptic currents (IPSC) was analyzed in dissociated culture of chick embryo spinal cord. Differences in the kinetic characteristics of low-amplitude and giant IPSC were revealed. Decay of currents in the first group was single-exponential, while decay in the second group was double-exponential. The time constant of single-exponential current decay increased during membrane depolarization and decreased during rise in temperature of the solution. Decay of the double-exponential currents depended little on potential, while temperature changes acted only on its slow component. Strychnine in submaximum concentrations produced not only a decrease in amplitude of giant IPSC, but also a deceleration of decay due to the slow component. The regularity of these phenomena suggests that decay of giant IPSC, as distinguished from that of low-amplitude currents, is determined by removal of transmitter from the synaptic cleft.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 427–435, July–August, 1991.     

Effect of nifedipine in high concentrations on inhibitory synaptic transmission

Effect of nifedipine on inhibitory postsynaptic currents (IPSC) was studied in cultured hippocampal neurons. Nifedipine, if used in low concentrations, caused no essential changes in the IPSC amplitude. If used in high concentrations (50 or 100 μM), this calcium channel blocker reduced the IPSC amplitude, on the average, by 35 and 42%, respectively. The calcium current component sensitive to nifedipine at high concentrations was found to be insensitive to the agents, which block calcium channels of N- and P/Q types. It is concluded that the L-type calcium channels sensitive to nifedipine in low concentrations are absent in the presynaptic membrane of inhibitory synapses, whereas the only component of calcium current sensitive to this blocking agent in a high concentration, as well as the ω-CTx-GVIA- and ω-Aga-IVA-sensitive components of this current, participate in the transmission of inhibitory synaptic influences on the neurons studied.     

Whisker Deprivation Drives Two Phases of Inhibitory Synapse Weakening in Layer 4 of Rat Somatosensory Cortex

Inhibitory synapse development in sensory neocortex is experience-dependent, with sustained sensory deprivation yielding fewer and weaker inhibitory synapses. Whether this represents arrest of synapse maturation, or a more complex set of processes, is unclear. To test this, we measured the dynamics of inhibitory synapse development in layer 4 of rat somatosensory cortex (S1) during continuous whisker deprivation from postnatal day 7, and in age-matched controls. In deprived columns, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and evoked IPSCs developed normally until P15, when IPSC amplitude transiently decreased, recovering by P16 despite ongoing deprivation. IPSCs remained normal until P22, when a second, sustained phase of weakening began. Delaying deprivation onset by 5 days prevented the P15 weakening. Both early and late phase weakening involved measurable reduction in IPSC amplitude relative to prior time points. Thus, deprivation appears to drive two distinct phases of active IPSC weakening, rather than simple arrest of synapse maturation.     

Quantal Characteristics of Synaptic GABA Release under Conditions of Stimulation of Single Synaptic Terminals of Cultured Cortical Cells of the Rat

We studied evoked inhibitory postsynaptic currents (eIPSC) using local electrical stimulation of single presynaptic terminals of cultured rat neocortical neurons. According to pharmacological and kinetic properties, these currents were qualified as GABA_A-activated. Using autocorrelation analysis of distributions of the eIPSC amplitudes, which were in all cases polymodal, we examined quantal characteristics of the above eIPSC. These results were compared with the values of quantal parameters (N, p, Q, and m) of the current families obtained using approximation by binomial distribution. Amplitude histograms of spontaneous miniature IPSC recorded under conditions of the minimum quantal release of the neurotransmitter were normal (close to Gaussian) with the mode within a 10 pA range, which is very close to analogous parameters calculated using autocorrelation and binomial techniques.     

Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)

Voltage-gated sodium channels play important roles in modulating dorsal root ganglion (DRG) neuron hyperexcitability and hyperalgesia after peripheral nerve injury or inflammation. We report that chronic compression of DRG (CCD) produces profound effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents, which are different from that by chronic constriction injury (CCI) of the sciatic nerve in small DRG neurons. Whole cell patch-clamp recordings were obtained in vitro from L₄ and/or L₅ dissociated, small DRG neurons following in vivo DRG compression or nerve injury. The small DRG neurons were classified into slow and fast subtype neurons based on expression of the slow-inactivating TTX-R and fast-inactivating TTX-S Na⁺ currents. CCD treatment significantly reduced TTX-R and TTX-S current densities in the slow and fast neurons, but CCI selectively reduced the TTX-R and TTX-S current densities in the slow neurons. Changes in half-maximal potential (V_1/2) and curve slope (k) of steady-state inactivation of Na⁺ currents were different in the slow and fast neurons after CCD and CCI treatment. The window current of TTX-R and TTX-S currents in fast neurons were enlarged by CCD and CCI, while only that of TTX-S currents in slow neurons was increased by CCI. The decay rate of TTX-S and both TTX-R and TTX-S currents in fast neurons were reduced by CCD and CCI, respectively. These findings provide a possible sodium channel mechanism underlying CCD-induced DRG neuron hyperexcitability and hyperalgesia and demonstrate a differential effect in the Na⁺ currents of small DRG neurons after somata compression and peripheral nerve injury. This study also points to a complexity of hyperexcitability mechanisms contributing to CCD and CCI hyperexcitability in small DRG neurons.     

Spontaneous synaptic activity in cell culture of chick embryo spinal cord

The spontaneous development of synaptic activity (SSA) was studied in cell cultures of chick embryo spinal cord. The complicated time structure of the SSA, an important early-stage characteristic of which was giant inhibitory postsynaptic currents (IPSC), was demonstrated. The ionic nature and pharmacological sensitivity of these IPSC suggest that glycine is their transmitter. Emergence of excitatory postsynaptic currents (EPSC) and complex antagonistic relationships between excitatory and inhibitory SSA was detected later. Possible mechanisms for maintenance of synaptic activity during the inhibitory function are discussed. Correlations between the regularities of synaptic transmission development that we have disclosed and neuronal circuit electrical activity are examined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 280–290, May–June, 1991.     

Study of role of inhibitory interneurons in mechanisms of regulation of sensory synapses formed by thalamic and cortical inputs on pyramidal cells of the dorsolateral amygdala nucleus

The work deals with study of role of inhibitory interneurons in the process of regulation of sensory currents converging on soma of pyramidal cells of the dorsolateral amygdala nucleus as well as of role of these interneurons in mechanism of regulation of plasticity of amygdala synapses. It has been shown that the part of the spontaneous inhibitory postsynaptic currents recorded on the dorsolateral amygdala pyramidal cells is relatively high and amounts to about a half of the total amount of the recorded events. Analysis of the evoked postsynaptic responses has shown the interneurons to regulate activity and duration of these responses due to the postsynaptic membrane hyperpolarization as a result of activation of GABA_A-receptors. Also studied was role of interneurons in providing mechanisms of the long-term potentiation of the synaptic responses evoked by stimulation of cortical and thalamic inputs. Block of effect of interneurons with help of picrotoxin has been shown to lead to an increase of evoked potentiation of synaptic responses.     

Analysis of excitatory and inhibitory components of postsynaptic currents recorded in the pyramidal neurons and interneurons of the rat hippocampus

Postsynaptic currents recorded in the whole-cell configuration with patch-clamp method are actually the sum ofexcitatory (EPSC) and inhibitory (IPSC) components. An approach has been developed allowing the quantitative evaluation of the amplitude and the time course of EPSC and IPSC without treatment of the brain slice with pharmacological inhibitors. The approach is based on the substantial difference in the equilibrium potential values of incoming cationic and anionic currents as the existence of linear regions of corrent-voltage dependence of these currents. The comparison of the results obtained with the classical pharmacological method and with the suggested one demonstrated their coincidence. It allows analysing the postsynaptic currents in sigle neurons without altering the synaptic transmission in the whole brain slice. The contribution of inhibitory currents in the composite synaptic response of intemeurons turned out to be smaller in comparison with pyramidal neurons of CA1 field of the rat hippocampus.