Biomedical subjects

Sergei Kirischuk

Publications and source records attributed to Sergei Kirischuk.

10 recordsLinked to original sources

PubMed2006

Ambient GABA constrains the strength of GABAergic synapses at Cajal-Retzius cells in the developing visual cortex.

At early stages of brain development, GABA plays a dual role. It fulfills important trophic functions and provides a major excitatory drive for the immature neuronal network. Here, we investigated whether GABA itself can limit the strength of excitatory GABAergic synapses on Cajal-Retzius (CR) cells in sagittal slices from the mouse visual cortex. (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845), a specific GABAB receptor (GABABR) blocker, increased the frequency of spontaneous Ca2+ transients and spontaneous and miniature IPSCs (mIPSCs) but did not affect mIPSC amplitudes or kinetics. CGP55845 significantly increased evoked IPSC (eIPSC) amplitudes and decreased the paired-pulse ratio (PPR). Baclofen, a specific GABABR agonist, produced opposite effects. The size of the readily releasable pool was not affected by these GABABR modulators. The same CGP55845 actions were observed at physiological temperatures, but they were abolished after glutamate decarboxylase block with 3-mercaptopropionic acid (3-MP). These results indicate that presynaptic GABABRs dynamically regulate GABA release probability. SNAP-5114, a specific GABA transporter-2/3 (GAT-2/3) blocker, enhanced mIPSC frequencies, decreased PPR, and increased eIPSC amplitudes without changing eIPSC kinetics. These effects were blocked by CGP55845 and 3-MP. NO-711, a specific GAT-1 blocker, prolonged eIPSC decay and decreased eIPSC/mIPSC amplitudes. These NO-711-mediated effects were not sensitive to CGP55845 and 3-MP. We conclude that the strength of GABAergic inputs to CR cells is constrained by GABABRs that are persistently activated by ambient GABA. The latter is also provided by GAT-2/3 operating in the reversed mode. Presynaptic GAT-1 functions in the uptake mode and possibly provides GABA for presynaptic vesicle filling.

Animals↗

PubMed2006

N-ethylmaleimide increases release probability at GABAergic synapses in layer I of the mouse visual cortex.

The sulphydryl alkylating agent N-ethylmaleimide (NEM) has been often used as an uncoupler of pertussis toxin-sensitive G-proteins. However, the effects of NEM on gamma-aminobutyric acid (GABA)ergic synaptic transmission remain controversial. Using the whole-cell patch-clamp technique, GABA(A) receptor-mediated postsynaptic currents (IPSCs) have been recorded from Cajal-Retzius (CR) cells in layer I of the neonatal mouse visual cortex. NEM increased the frequencies of both spontaneous and miniature IPSCs (mIPSCs) without an effect on the median mIPSC amplitudes or mIPSC kinetics. The NEM actions on mIPSCs did not depend on the extracellular Ca(2+), Ca(2+) release from intracellular stores, adenylyl cyclase and protein kinase A activities. NEM increased the mean amplitudes of evoked IPSCs and strongly decreased the paired-pulse ratio. The size of the readily releasable pool of presynaptic vesicles (RRP) was estimated using a high-frequency stimulation protocol. The RRP size was not affected by NEM. In addition, NEM significantly decreased the latency between the stimulus and the onset of GABA release. These results suggest that NEM selectively increases GABA release probability. At postnatal day 2, mIPSCs were observed only in about 30% of CR cells. NEM application revealed, however, that more than 90% of CR cells receive GABAergic inputs. Therefore, NEM seems to be a useful tool to verify the existence of 'silent' GABAergic synapses.

Animals↗

PubMed2005

Time-matched pre- and postsynaptic changes of GABAergic synaptic transmission in the developing mouse superior colliculus.

Developmental changes in the kinetics of GABAergic postsynaptic currents have been reported for various brain structures. However, it has remained unclear whether these modifications are matched by presynaptic changes. We addressed this question by analysing evoked IPSCs (eIPSCs) in mouse superior colliculus slices between postnatal day (P) 1 and 22. eIPSCs were elicited by electrical stimulation and measured in the whole-cell patch-clamp configuration. IPSCs were analysed using the binomial model of synaptic transmission. The readily releasable pool (RRP, N) was estimated from the cumulative eIPSC amplitude histograms during 50-Hz stimulation. Median delayed IPSC (dIPSC) amplitude was used as a quantal amplitude (q) estimate. The mean release probability (p) was determined as the mean eIPSC amplitude divided by the product of RRP and q. The experiments revealed that GABAergic synapses pass through two distinct periods of functional adjustment: (i) P1-3 (coincidental with the onset of glutamatergic spontaneous activity and a switch from depolarizing to hyperpolarizing GABA action) displayed a significant decrease of p, associated with an increase in the paired-pulse ratio (eIPSC(2)/eIPSC(1)); and (ii) P6-15 (the period before and shortly after eye opening) is characterized by a drastic reduction of IPSC duration. On the presynaptic side, it was accompanied by a down-regulation of asynchronous release in favour of stimulus-locked synchronous release. We conclude that postsynaptic modifications of GABAergic synaptic transmission in the superior colliculus (SC) are indeed accompanied by presynaptic changes, and this may guarantee the necessary efficacy of inhibition during the developmental reconstruction of the synaptic network in the SC.

Action Potentials↗

PubMed2005

Developmental downregulation of low-voltage-activated Ca2+ channels in Cajal-Retzius cells of the mouse visual cortex.

Cajal-Retzius (CR) cells have been demonstrated to fulfil an important secretory function in the developing neocortex. On the other hand, the contribution of CR cell electrical activity during cortex development is still unclear. Using the whole-cell patch-clamp technique, we studied low-voltage-activated (LVA) Ca(2+) channels in CR cells in the layer I of the mouse visual cortex. CR cells were found to display a transient Ca(2+) current (I(T)) in response to a depolarization step from -100 mV to -40 mV. I(T) showed: (i) typical for LVA Ca(2+) channels voltage dependence of activation (half-activation at -55 mV) and inactivation (half-inactivation at -76 mV); (ii) fast activation and inactivation kinetics, with time constants of 1.4 and 28 ms, respectively, at -40 mV; (iii) fast recovery from steady-state inactivation (time constant: 290 ms); (iv) a complete block by 1 microm mibefradil; and (v) a partial block (to 55%) by 100 microm Ni(2+). The density of I(T) dramatically decreased between postnatal day (P) 1 and P9. Immunostaining demonstrated the presence and postnatal downregulation of the alpha(1G)-subunit of LVA Ca(2+) channels in CR cells. Experiments performed in the current-clamp mode revealed that mibefradil delayed an action potential generation in response to a suprathreshold depolarizing current at P1, but not at P8-9. We suggest that LVA Ca(2+) channels might influence CR cell excitability during the first postnatal week and thereby contribute to the shaping of synaptic connectivity in the neocortical layer I.

Age Factors↗

PubMed2003

Intraterminal Ca2+ concentration and asynchronous transmitter release at single GABAergic boutons in rat collicular cultures.

Neurotransmitter release in response to a single action potential has a precise time course. A significant fraction of the releasable vesicles is exocytosed synchronously, within a few milliseconds after the arrival of an action potential. If repeatedly activated, stimulus-locked phasic synchronous release declines, but synaptic transmission can be maintained through tonic asynchronous transmitter release. The desynchronisation of release during repetitive activation is generally attributed to a build-up of intraterminal Ca2+ concentration. However, the precise relationship between presynaptic Ca2+ level and asynchronous release rate at small central synapses has remained unclear. Here we characterise this relationship for single GABAergic terminals in rat collicular cultures. In the presence of tetrodotoxin, inhibitory postsynaptic currents (IPSCs) and presynaptic Ca2+ transients were recorded in response to direct presynaptic depolarisation of individual boutons. Repetitive stimulation indeed resulted in a shift from phasic to asynchronous neurotransmitter release. A clear dominance of the asynchronous release mode was observed after 10 pulses. The steady-state asynchronous release rate showed a third-power dependency on the presynaptic Ca2+ concentration, which is similar to that of evoked release. The Ca2+ sensor for asynchronous release exhibited a high affinity for Ca2+ and was far from saturation. These properties of the Ca2+ sensor should make the asynchronous release very sensitive to any modification of presynaptic Ca2+ concentration, including those resulting from changes in presynaptic activity patterns. Thus, asynchronous release represents a powerful but delicately regulated mechanism that ensures the maintenance of appropriate inhibition when the readily releasable pool of vesicles is depleted.

Animals↗

PubMed2003

GABA(A) receptor activity and PKC control inhibitory synaptogenesis in CNS tissue slices.

The molecular signals inducing the formation of inhibitory synapses early in development have remained enigmatic. Here we focus on the role of PKC and GABA(A)R receptor activation in the formation of GABAergic synapses in a natural cellular environment. Rapid synaptogenesis was observed in horizontal slices from the superior colliculus of embryonic (E19) rat, when GABA still acts as a depolarizing transmitter, excitatory synaptic activity is absent, and the number of already existing inhibitory synapses is very small. The vast majority of newly formed synapses expressed a GABAergic phenotype. Pharmacological block of GABA(A)R activation and Ca2+ influx through nifedipine-sensitive Ca2+ channels significantly enhanced the number of synaptic contacts, increased the immunoreactivity for GAD65, promoted synaptic accumulation of GABA(A)R clusters, and stimulated the generation of miniature IPSCs. The inhibitory synapse formation in situ was unconditionally prevented by PKC blockade and stimulated by PKC activation. Thus, a negative feedback relationship must exist between PKC and GABA(A)R activation. This new model of experimental synaptogenesis in brain slices promises to be a fruitful approach toward a better understanding of intracellular signaling cascades involved in the activity-regulated synapse formation, a problem of great clinical and theoretical relevance.

Animals↗

PubMed2003

Pre- and postsynaptic contribution of GABAC receptors to GABAergic synaptic transmission in rat collicular slices and cultures.

The mammalian superior colliculus (SC) is reported to contain gamma-aminobutyric acid (GABA)C receptors (GABACRs) at high concentration. However, their role in GABAergic synaptic transmission is not yet known. The aim of the present study was: (i) to clarify whether GABACRs are activated by endogenous GABA; and (ii), to determine whether GABACRs play a role in inhibitory synaptic transmission. Experiments were performed on acute horizontal slices from the postnatal rat SC or on collicular neurons in dissociated cell culture. In both preparations, bicuculline-resistant current responses to exogenous GABA and currents elicited by cis-4-aminocrotonic acid (CACA) were blocked by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA), a GABACR antagonist. The CACA-induced currents exhibited a linear current-voltage relationship and reversed at the Cl- equilibrium potential. These results indicate that functional GABACRs are present in the somato-dendritic membrane of collicular neurons. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded using the whole-cell patch clamp technique. TPMPA significantly decreased mIPSC amplitudes in slices, but not in cultured neurons. As TPMPA decreased also the coefficient of variation of mIPSCs, we suggest that somatodendritic GABACRs are located extrasynaptically but can be involved in the generation of IPSCs if GABA diffusion is constrained. In cultures, individual connections were activated by focal electrical stimulation of single neurons, and evoked inhibitory postsynaptic currents (eIPSCs) were recorded. Paired-pulse stimulation revealed that TPMPA significantly decreased the paired-pulse ratio at short (50 ms) interstimulus intervals, and this effect was inversely dependent on the amplitude of the first eIPSC. We conclude that presynaptic GABACRs are activated by endogenous GABA and can alleviate the short-term depression resulting from a preceding episode of GABA release. Thus, in GABAergic synapses of the SC GABACRs are involved in pre- and postsynaptic functions and may therefore contribute to the activity-dependent adjustment of GABAergic inhibition.

Action Potentials↗

PubMed2002

Presynaptic and postsynaptic mechanisms underlie paired pulse depression at single GABAergic boutons in rat collicular cultures.

Paired pulse depression (PPD) is a common form of short-term synaptic plasticity. The aim of this study was to characterise PPD at the level of a single inhibitory bouton. Low-density collicular cultures were loaded with the Ca2+ indicator Oregon Green-1, active boutons were stained with RH414, and action potentials were blocked with TTX. Evoked IPSCs (eIPSCs) and presynaptic Ca2+ transients were recorded in response to direct presynaptic depolarisation of an individual bouton. The single bouton eIPSCs had a low failure rate (< 0.1), large average quantal content (3-6) and slow decay (tau(1) = 15 ms, tau(2) = 81 ms). The PPD of eIPSCs had two distinct components: PPD(fast) and PPD(slow) (tau = 86 ms and 2 s). PPD(slow) showed no dependence on extracellular Ca2+ concentration, or on the first eIPSC's failure rate or amplitude. Most probably, it reflects a release-independent inhibition of exocytosis. PPD(fast) was only observed in normal or elevated Ca2+. It decreased with the failure rate and increased with the amplitude of the first eIPSC. It coincided with paired pulse depression of the presynaptic Ca2+ transients (tau = 120 ms). The decay of the latter was accelerated by EGTA, which also reduced PPD(fast). Therefore, a suppressive effect of residual presynaptic Ca2+ on subsequent Ca2+ influx is considered the most likely cause of PPD(fast). PPD(fast) may also have a postsynaptic component, because exposure to a low-affinity GABA(A) receptor antagonist (TPMPA; 300 microM) counteracted PPD(fast), and asynchronous IPSC amplitudes were depressed for a short interval following an eIPSC. Thus, at these synapses, PPD is produced by at least two release-independent presynaptic mechanisms and one release-dependent postsynaptic mechanism.

Animals↗

PubMed2002

Synaptic anchoring of glycine receptors in developing collicular neurons under control of metabotropic glutamate receptor activity.

The role of GlyRs in supramedullar neurons is still enigmatic: although present in the plasma membrane, they seem to be excluded from synaptic transmission. Two distinct stages of inhibitory synaptogenesis in cultured collicular neurons were defined on the basis of strychnine sensitivity of IPSCs, presence of EPSCs, KCC2 expression, and transmitter-induced Ca(2+) responses. In the absence of KCC2 and glutamatergic transmission, newborn VIAAT-labeled boutons contained GlyT2, released glycine, and accumulated postsynaptic gephyrin and GlyRs. With ongoing maturation, when neurons expressed KCC2 and displayed glutamatergic activity, inhibitory synapses contained GAD65, released GABA, and accumulated postsynaptic gephyrin and GABA(A)Rs, but not GlyRs. This activity- and transmitter-specific regulation of GlyR distribution could be challenged by chronic blockade of group I mGluRs. It is thus concluded that glutamatergic transmission can contribute to the regulation of synaptic GlyR stabilization during inhibitory synapse development.

Animals↗

PubMed2002

Inter-bouton variability of synaptic strength correlates with heterogeneity of presynaptic Ca(2+) signals.

The elevation of presynaptic calcium concentration is a crucial step in excitation-secretion coupling. However, the amplitudes of action-potential-induced presynaptic calcium transients can display high variability among different terminals. The aim of this study was to clarify whether, at individual boutons, synaptic strength correlates with the average amplitude of presynaptic calcium transients. Low-density collicular cultures were loaded with the calcium indicator Oregon Green bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) 1. Action potentials were blocked with tetrodotoxin. Presynaptic terminals were identified with FM4-64, a use-dependent vesicle marker. Presynaptic calcium influx was elicited by a focal electrical stimulation of single boutons. Whole cell patch-clamp and calcium imaging techniques were used to record GABAergic evoked inhibitory postsynaptic currents (eIPSCs) and presynaptic fluorescence changes in the stimulated terminal. To make the eIPSCs from different boutons comparable, they were normalized to the mean value of miniature IPSCs (mIPSCs) of the postsynaptic cell. Records from 47 boutons showed that eIPSCs varied between 0.5 and 3.0 and presynaptic calcium transients varied between 0.1 and 1.3. However, there was a strong correlation between the mean amplitudes of eIPSCs and presynaptic calcium responses. The eIPSC-[Ca(2+)](pre) relationship allows to use the amplitudes of presynaptic calcium transients as an indicator of release efficacy and, in a set of contacts made by one axon, to predict the relative impact of individual terminals.

Animals↗