Synaptic transmission in hippocampal neurons: numerical reconstruction of quantal IPSCs.
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ATP facilitates spontaneous glycinergic IPSC frequency at dissociated rat dorsal horn interneuron synapses.
1. The ATP action on spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) was investigated in rat substantia gelatinosa (SG) neurons mechanically dissociated from the 2nd layer of the dorsal horn in which their presynaptic glycinergic nerve terminals remained intact. 2. ATP reversibly facilitated the frequency of the mIPSCs in a concentration-dependent manner without affecting their amplitude distribution. The ATP agonist, 2-methylthioATP (2MeSATP), mimicked the ATP action, while another ATP receptor agonist, alphabeta-methylene-ATP (alpha,beta-meATP), had no effect on mIPSCs. 3. The ATP receptor antagonists, suramin (1 x 10-6 M) and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1 x 10-5 M), completely blocked the facilitatory effect of ATP on glycine release (102.0 +/- 11.2 % and 99.3 +/- 16.2 %, n = 6, respectively) without altering the current amplitude distributions. 4. N-Ethylmaleimide (NEM), a sulphydryl alkylating agent, suppressed the inhibitory effect of adenosine on mIPSC frequency (111.2 +/- 13. 3 %, n = 4) without altering the current amplitude distribution. However, ATP still facilitated the mIPSC frequency (693.3 +/- 245.2 %, n = 4) even in the presence of NEM. 5. The facilitatory effect of ATP (1 x 10-5 M) on mIPSC frequency was not affected by adding 1 x 10-4 M Cd2+ to normal external solution but was eliminated in a Ca2+-free external solution. 6. These results suggest that ATP enhances glycine release from nerve terminals, presumably resulting in the inhibition of SG neurons which conduct nociceptive signals to the CNS. This presynaptic P2X-type ATP receptor may function to prevent excess excitability in SG neurons, thus preventing an excessive pain signal and/or SG cell death.
Adenosine Triphosphate↗
Mice lacking the major adult GABAA receptor subtype have normal number of synapses, but retain juvenile IPSC kinetics until adulthood.
There is a large variation in structurally and functionally different GABA(A) receptor subtypes. The expression pattern of GABA(A) receptor subunits is highly regulated, both temporarily and spatially. Especially during development, profound changes in subunit expression have been described. In most brain areas, the GABA(A) receptor alpha1 subunit replaces the alpha2 and/or alpha3 subunit as major alpha subunit. This is accompanied by a marked decrease in the open time of GABA(A) receptors and hence in the duration of postsynaptic responses. We describe here the development of GABAergic, synaptic transmission in mice lacking the alpha1 subunit. We show that alpha1 is to a large extent--but not entirely--responsible for the relatively short duration of postsynaptic responses in the developing and the mature brain. However, alpha1 already affects GABAergic transmission in the neonatal cerebral cortex when it is only sparsely expressed. It appears that the alpha1 -/- mice do not show a large reduction in GABAergic synapses but do retain long-lasting postsynaptic currents into adulthood. Hence, they form a good model to study the functional role of developmental GABA(A) receptor subunit switching.
Age Factors↗
Activation of nicotinic acetylcholine receptors increases the frequency of spontaneous GABAergic IPSCs in rat basolateral amygdala neurons.
The basolateral amygdala (BLA) is a critical component of the amygdaloid circuit, which is thought to be involved in fear conditioned responses. Using whole cell patch-clamp recording, we found that activation of nicotinic acetylcholine receptors (nAChRs) leads to an action potential-dependent increase in the frequency of spontaneous GABAergic currents in principal neurons in the BLA. These spontaneous GABAergic currents were abolished by a low-Ca2+/high-Mg2+ bathing solution, suggesting that they are spontaneous inhibitory postsynaptic currents (sIPSCs). Blockade of ionotropic glutamate receptors did not prevent this increased frequency of sIPSCs nor did blockade of alpha7 nAChRs. Among the nAChR agonists tested, cystisine was more effective at increasing the frequency of the sIPSCs than nicotine or 1,1-dimethyl-4-phenyl piperazinium iodide, consistent with a major contribution of beta4 nAChR subunits. The nicotinic antagonist, dihydro-beta-erythroidine, was less effective than d-tubocurarine in blocking the increased sIPSC frequency induced by ACh, suggesting that alpha4-containing nAChR subunits do not play a major role in the ACh-induced increased sIPSC frequency. Although alpha2/3/4/7 and beta2/4 nAChR subunits were found in the BLA by RT-PCR, the agonist and antagonist profiles suggest that the ACh-induced increase in sIPSC frequency involves predominantly alpha3beta4-containing nAChR subunits. Consistent with this, alpha-conotoxin-AuIB, a nAChR antagonist selective for the alpha3beta4 subunit combination, inhibited the ACh-induced increase in the frequency of sIPSCs. The observations suggest that nicotinic activation increases the frequency of sIPSCs in the BLA by acting mainly on alpha3beta4-containing nicotinic receptors on GABAergic neurons and may play an important role in the modulation of synaptic transmission in the amygdala.
Amygdala↗
Noise analysis of miniature IPSCs in adult rat brain slices: properties and modulation of synaptic GABAA receptor channels.
1. The properties of synaptic gamma-aminobutyric acid (GABA)A receptor channels were resolved by using tight-seal, whole-cell recordings from granule cells of the dentate gyrus in adult rat hippocampal slices and by applying the technique of nonstationary noise analysis to study miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of tetrodotoxin (TTX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and D-2-amino-5-phosphonovaleric acid (D-AP5). This technique allowed us to extract information about the conductance, the number, and the kinetics of ligand gated channels underlying elementary synaptic currents. 2. To ascertain the validity of the nonstationary noise analysis method we have first tested it on computer simulated mIPSCs with different channel activation, lifetime kinetics, and opening probabilities. Using intraburst mean open times, shorter than the time to the first opening following activation, caused a large variance at the peak due to the stochastic channel properties. This resulted in a skewed mean current-variance relationship, which precluded proper estimation of unit conductance and especially the number of channels open at the peak of mIPSCs. Regardless of the probability of channel opening, accurate estimates of the unit conductance and the number of channels underlying each simulated mIPSC were obtained when channels had mean open times longer than the time to first opening. 3. Once the validity of the nonstationary analysis had been ascertained, it was used on mIPSCs recorded at 35 degrees C. The unit conductance of the synaptic GABAA channels was 28 +/- 1 (SE) pS and the average number of channels underlying mIPSCs was 46 +/- 4. The mean current-variance relationship was not skewed at higher amplitudes, suggesting that the intrinsic variance at the peak of the GABAA mIPSCs is low and that the open time of the channels is longer than the time to first opening. The estimated unit conductance of the channels was constant over a wide range of holding potentials. 4. The amplitude distribution of mIPSCs with rapid 10-90% rise times (290 +/- 20 microseconds) was clearly skewed towards low values. This skew was not due to filtering of electrotonically distant currents. Current-variance analysis revealed that the skewness resulted from differences in the number of GABAA receptor channels and not from the heterogeneity of unitary conductances at various synapses. Selection of mIPSCs with slower rise times yielded smaller unit conductance estimates.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Properties of unitary IPSCs in hippocampal pyramidal cells originating from different types of interneurons in young rats.
Whole cell recordings were used in hippocampal slices of young rats to examine unitary inhibitory postsynaptic currents (uIPSCs) evoked in CA1 pyramidal cells at room temperature. Loose cell-attached stimulation was applied to activate single interneurons of different subtypes located in stratum oriens (OR), near stratum pyramidale (PYR), and at the border of stratum radiatum and lacunosum-moleculare (LM). uIPSCs evoked by stimulation of PYR and OR interneurons had similar onset latency, rise time, peak amplitude, and decay. In contrast, uIPSCs elicited by activation of LM interneurons were significantly smaller in amplitude and had a slower time course. The mean reversal potential of uIPSCs was -53.1 +/- 2.1 (SE) mV during recordings with intracellular solution containing potassium gluconate. With the use of recording solution containing the potassium channel blocker cesium, the reversal potential of uIPSCs was not significantly different (-58.5 +/- 2.6 mV), suggesting that these synaptic currents were not mediated by potassium conductances. Bath application of the gamma-aminobutyric acid-A (GABA(A)) receptor antagonist bicuculline (25 microM) reversibly blocked uIPSCs evoked by stimulation of all interneuron subtypes. In bicuculline, the mean peak amplitude of uIPSCs recorded with potassium gluconate was reduced to 3.5 +/- 4.4% of control (n = 7). Similarly, with cesium methanesulfonate, the mean amplitude in bicuculline was 2.9 +/- 3.1% of control (n = 13). Application of the GABA(B) receptor antagonist CGP 55845A (5 microM) resulted in a significant and reversible increase in the mean amplitude of uIPSCs recorded with cesium-containing intracellular solution. Thus uIPSCs from all cell types appeared under tonic presynaptic inhibition by GABA(B) receptors. Paired stimulation of individual interneurons at 100- to 200-ms intervals did not result in paired pulse depression of uIPSCs. For individual responses, a significant negative correlation was observed between the amplitude of the first and second uIPSCs. A significant paired pulse facilitation (154.0 +/- 8.0%) was observed when the first uIPSC was smaller than the mean of all first uIPSCs. A small, but not significant, paired pulse depression (90.8 +/- 4.0%) was found when the first uIPSC was larger than the mean of all first uIPSCs. Our results indicate that these different subtypes of hippocampal interneurons generate Cl(-)-mediated GABA(A) uIPSCs. uIPSCs originating from different types of interneurons may have heterogeneous properties and may be subject to tonic presynaptic inhibition via heterosynaptic GABA(B) receptors. These results suggest a specialization of function for inhibitory interneurons and point to complex presynaptic modulation of interneuron function.
Animals↗
Analysis of EPSCs and IPSCs carrying rhythmic, locomotor-related information in the isolated spinal cord of the neonatal rat.
To understand better the synaptic language used by neurons in active networks, we have analyzed postsynaptic currents (PSCs) received by interneurons in the isolated spinal cord from neonatal rats during 5-hydroxytryptamine- and N-methyl--aspartate-induced fictive locomotion. Using a computer algorithm, we identified PSCs in rhythmically active interneurons in laminae VII and X. To test whether the PSCs actually participated in the transmission of the cyclic, locomotor-related signal, we constructed an analytic current trace based on only the identified events. Each identified PSC was fitted by a mathematical function, and the shape of this function was added to a baseline with time delays given by the time positions of the identified PSCs. By averaging the resulting analytic current trace over several cycles, we showed that the identified PSCs built a cyclic signal locked to the rhythmic activity recorded from the ventral roots. Furthermore, subtraction of the analytic from the original current trace reduced the amplitude of the cyclic signal received by these cells. Thus the identified PSCs contributed to the cyclic information, allowing us to analyze how they built the compound cyclic signal. Most often there was an inverse relationship between the contribution from excitatory and inhibitory PSCs during the cyclic modulation, indicating that there was a reciprocal regulation of the presynaptic inhibitory and excitatory cells. Comparing the most inhibitory and most excitatory halves of the locomotor related cycle, there was a considerably larger modulation of the frequency of PSCs than of their amplitude. The small and sometimes insignificant modulation of PSC amplitude suggests that facilitation and depression had little importance for the information transfer. The modest amplitude modification also suggests that the large range of available PSC amplitudes seen in these neurons was not used very efficiently to code the cyclic information.
Animals↗
PGM1 deficiency is linked to sarcomeric and mitochondrial dysfunction in patient-derived iPSC-cardiomyocytes.
BACKGROUND: PGM1-congenital disorder of glycosylation (PGM1-CDG) is frequently associated with cardiomyopathy. Although galactose therapy corrects glycosylation defects, cardiac dysfunction typically persists, suggesting a glycosylation-independent mechanism. Recent evidence of mitochondrial abnormalities in PGM1-deficient human and murine heart, together with the association of PGM1 with the Z-disk protein LDB3 (ZASP/Cypher), suggests a critical role for PGM1 in cardiomyocyte structural and energetic homeostasis. We hypothesized that PGM1-related cardiomyopathy arises from a glycosylation-independent disruption of Z-disk-mitochondrial coupling driven by loss of PGM1-LDB3 interactions, resulting in mitochondrial energy failure and impaired contractile function. METHODS: Induced pluripotent stem cell-derived cardiomyocytes (iCMs) were generated from PGM1-deficient patient fibroblasts. Multielectrode array (MEA) recordings, untargeted (glyco)proteomics, and pathway analysis were performed to assess functional and molecular changes. Key findings were validated using tracer metabolomics and mitochondrial respiration assays. RESULTS: PGM1-deficient iCMs exhibited reduced beating frequency, impaired contractility, and prolonged contraction kinetics. Proteomic analyses revealed depletion of Z-disk components, including LDB3. AlphaFold3 structural modeling predicted a direct interaction between PGM1 and LDB3, implicating PGM1 in Z-disk integrity, which was confirmed in vitro. In addition, mitochondrial proteins were severely depleted, prompting us to investigate mitochondrial function. Functional validation confirmed extensive metabolic rewiring, energy depletion, and severely impaired mitochondrial respiration. Finally, the in silico drug repurposing identified possible therapeutic options that could target PGM1-deficient cardiomyopathy. CONCLUSION: Our data suggests PGM1 is key regulator of cardiomyocyte function, linking sarcomeric Z-disk integrity with mitochondrial metabolism. These mechanistic insights offer a foundation for developing targeted therapies for PGM1-CDG and potentially other cardiomyopathies involving Z-disk dysfunction.
Humans↗
Simultaneous NMDA-dependent long-term potentiation of EPSCs and long-term depression of IPSCs in cultured rat hippocampal neurons.
A fundamental issue in understanding activity-dependent long-term plasticity of neuronal networks is the interplay between excitatory and inhibitory synaptic drives in the network. Using dual whole-cell recordings in cultured hippocampal neurons, we examined synaptic changes occurring as a result of a transient activation of NMDA receptors in the network. This enhanced transient activation led to a long-lasting increase in synchrony of spontaneous activity of neurons in the network. Simultaneous long-term potentiation of excitatory synaptic strength and a pronounced long-term depression of inhibitory synaptic currents (LTDi) were produced, which were independent of changes in postsynaptic potential and Ca2+ concentrations. Surprisingly, miniature inhibitory synaptic currents were not changed by the conditioning, whereas both frequency and amplitudes of miniature EPSCs were enhanced. LTDi was mediated by activation of a presynaptic GABAB receptor, because it was blocked by saclofen and CGP55845 [(2S)-3-{[(15)-1-(3, 4-dichlorophenyl)ethyl]amino-2-hydroxypropyl)(phenylmethyl)phosphinic acid]. The cAMP antagonist Rp-adenosine 3 ', 5 ' -cyclic monophosphothioate abolished all measured effects of NMDA-dependent conditioning, whereas a nitric oxide synthase inhibitor was ineffective. Finally, network-induced plasticity was not occluded by a previous spike-timing-induced plasticity, indicating that the two types of plasticity may not share the same mechanism. These results demonstrate that network plasticity involves opposite affects on inhibitory and excitatory neurotransmission.
2-Amino-5-phosphonovalerate↗
Opioids suppress IPSCs in neurons of the rat medial septum/diagonal band of Broca: involvement of mu-opioid receptors and septohippocampal GABAergic neurons.
The medial septum/diagonal band region (MSDB), which provides a major cholinergic and GABAergic input to the hippocampus, expresses a high density of opioid receptors. Behaviorally, intraseptal injections of opioids produce deficits in spatial memory, however, little is known about the electrophysiological effects of opioids on MSDB neurons. Therefore, we investigated the electrophysiological effects of opioids on neurons of the MSDB using rat brain slices. In voltage-clamp recordings with patch electrodes, bath-applied met-enkephalin, a nonselective opioid receptor agonist, decreased the number of tetrodotoxin and bicuculline-sensitive inhibitory synaptic currents in cholinergic- and GABA-type MSDB neurons. A similar effect occurred in brain slices containing only the MSDB, suggesting that opioids decrease GABA release primarily by inhibiting spontaneously firing GABAergic neurons located within the MSDB. Accordingly, in extracellular recordings, opioid-sensitive, spontaneously firing neurons could be found within the MSDB. Additionally, in intracellular recordings a subpopulation of GABA-type neurons were directly inhibited by opioids. All effects of met-enkephalin were mimicked by a mu receptor agonist, but not by delta or kappa agonists. In antidromic activation studies, mu-opioids inhibited a subpopulation of septohippocampal neurons with high conduction velocity fibers, suggestive of thickly myelinated GABAergic fibers. Consistent with the electrophysiological findings, in double-immunolabeling studies, 20% of parvalbumin-containing septohippocampal GABA neurons colocalized the mu receptor, which at the ultrastructural level, was found to be associated with the neuronal cell membrane. Thus, opioids, via mu receptors, inhibit a subpopulation of MSDB GABAergic neurons that not only make local connections with both cholinergic and noncholinergic-type MSDB neurons, but also project to the hippocampus.
Animals↗
Activity deprivation reduces miniature IPSC amplitude by decreasing the number of postsynaptic GABA(A) receptors clustered at neocortical synapses.
Maintaining the proper balance between excitation and inhibition is necessary to prevent cortical circuits from either falling silent or generating epileptiform activity. One mechanism through which cortical networks maintain this balance is through the activity-dependent regulation of inhibition, but whether this is achieved primarily through changes in synapse number or synaptic strength is not clear. Previously, we found that 2 d of activity deprivation increased the amplitude of miniature EPSCs (mEPSCs) onto cultured visual cortical pyramidal neurons. Here we find that this same manipulation decreases the amplitude of mIPSCs. This occurs with no change in single-channel conductance but is accompanied by a reduction in the average number of channels open during the mIPSC peak and a reduction in the intensity of staining for GABA(A) receptors (GABA(A)Rs) at postsynaptic sites. In addition, the number of synaptic sites that express detectable levels of GABA(A)Rs was decreased by approximately 50% after activity blockade, although there was no reduction in the total number of presynaptic contacts. These data suggest that activity deprivation reduces cortical inhibition by reducing both the number of GABA(A)Rs clustered at synaptic sites and the number of functional inhibitory synapses. Because excitatory and inhibitory synaptic currents are regulated in opposite directions by activity blockade, these data suggest that the balance between excitation and inhibition is dynamically regulated by ongoing activity.
Animals↗
[Onset of anemia in IPSC and IUCD users].
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Biology↗
The minimal inhibitory synaptic currents evoked in neonatal rat motoneurones.
1. Tight-seal whole-cell recordings were made from lumbar motoneurones visually identified in thin slices of neonatal rat spinal cord. The inhibitory postsynaptic currents (IPSCs) were evoked by extracellular stimulation of a neighbouring internuncial neurone in the presence of glutamate receptor antagonists. 2. Glycinergic IPSCs were recorded in the presence of bicuculline. The IPSCs appeared in an all-or-none manner as the graded stimulus intensity exceeded a certain threshold. Their latencies showed a unimodal distribution with a mean of 0.81 ms at 37 degrees C. Thus, the observed IPSCs are suggested to be monosynaptically evoked unitary IPSCs. The mean conductance of unitary IPSCs was 2.9 +/- 1.2 nS (+/- S.D.). 3. When the external Ca2+ concentration ([Ca2+]o) was reduced, the number of failures in response to stimulation increased, thereby reducing the mean amplitude of IPSCs. The mean amplitude of IPSCs was linearly related to the [Ca2+]o (0.35-1.4 mM) with a mean slope of 3.1 +/- 0.67 on double logarithmic co-ordinates. 4. The amplitude of individual IPSCs decreased with decrease in [Ca2+]o. However, below 0.7 mM [Ca2+]o, the mean amplitude of IPSCs (excluding failures) reached a steady minimum level. The mean conductance of these IPSCs measured in 0.5 mM [Ca2+]o was 657 +/- 281 pS. 5. The minimal IPSCs had a coefficient of variation of 0.50 +/- 0.13. No clear correlation was observed between the rise time and the amplitude of minimal IPSCs evoked in individual motoneurones, indicating that the amplitude variability is not due to the different synaptic locations. 6. Spontaneous miniature IPSCs were recorded from motoneurones in the presence of tetrodotoxin. The miniature IPSCs had a mean conductance of 739 +/- 278 pS, being comparable to the minimal evoked IPSCs. 7. Under various internal and external Cl- concentration, the reversal potential of the IPSCs (EIPSC) approximately coincided with the Cl- equilibrium potential. A 730-fold change in the potassium concentration gradient across the membrane did not affect the EIPSC. The permeability ratio of K+ to Cl- (Pk/PCl) was less than 0.05. 8. It is concluded that the IPSCs are carried almost exclusively by Cl- and that the minimal evoked IPSCs represent the quantal response of the transmitter.
Animals↗
Characterisation of inhibitory and excitatory postsynaptic currents of the rat medial superior olive.
The medial superior olive (MSO) is part of the binaural auditory pathway, receiving excitatory projections from both cochlear nuclei and an inhibitory input from the ipsilateral medial nucleus of the trapezoid body (MNTB). We characterised the excitatory and inhibitory synaptic currents of MSO neurones in 3- to 14-day-old rats using whole-cell patch-clamp methods in a brain slice preparation.A dual component EPSC was mediated by AMPA and NMDA receptors. The AMPA receptor-mediated EPSC decayed with a time constant of 1.99+/-0.16 ms (n = 8). Following blockade of glutamate receptors, a monosynaptic strychnine-sensitive response was evoked on stimulation of the MNTB, indicative of a glycine receptor-mediated IPSC. GABAA receptors contributed to IPSCs in rats under 6 days old (bicuculline blocked 30% of the IPSC). In older rats little or no bicuculline-sensitive component was detectable, except in the presence of flunitrazepam. These glycinergic IPSCs showed a reversal potential that varied with changes in [Cl-]i, as predicted by the Nernst equation. The IPSC exhibited two developmentally relevant changes. (i) At around postnatal day 6, the GABAA receptor-mediated component declined, leaving a predominant glycine-mediated IPSC. The isolated glycinergic IPSC decayed with time constants of 7.8+/-0.3 and 38.3+/-1.7 ms, with the slower component contributing 7.8+/-0.6% of the peak amplitude (n = 121, 3-11 days old, -70 mV, 25 deg C). (ii) After day 11 the IPSC fast decay accelerated to 3.9+/-0.3 ms (n = 12) and the magnitude of the slow component declined to less than 1%. Spontaneous miniature glycinergic IPSCs (mIPSCs) were variable in amplitude and were of large conductance (1.83+/-0.19 nS, n = 8). The amplitude was unchanged on lowering [Ca2+]o. The time course of evoked and spontaneous miniature glycinergic IPSCs were compared. The 10-90% rise times were 0.7 and 0.6 ms, respectively. The evoked IPSC decayed with a fast time constant of 7.2+/-0.7 ms, while the mIPSC decayed with a fast time constant of 5.3+/-0.4 ms in the same seven cells.The glycinergic IPSC decay was voltage dependent with an e-fold change over 118 mV. The temperature dependence of the IPSC decay indicated a Q10 value of 2. Picrotoxin and cyanotriphenylborate had little or no effect on IPSCs from 6- to 14-day-old animals, implying homomeric channels are rare. We conclude that the MSO receives excitatory inputs mediated by AMPA and NMDA receptors and a strong glycinergic IPSC which has a significant contribution from GABAA receptors in neonatal rats. Functionally, the IPSC could increase membrane conductance during the decay of binaural glutamatergic EPSCs, thus refining coincidence detection and interaural timing differences.
Animals↗
Fluctuations of inhibitory postsynaptic currents in Purkinje cells from rat cerebellar slices.
1. Paired patch-clamp recordings were performed in cerebellar slices to study IPSCs evoked by interneurones (stellate and basket cells) in Purkinje cells. 2. IPSCs were first examined while keeping the presynaptic cell in the cell-attached mode. In some of these experiments IPSCs had a remarkably broad amplitude distribution. Large variabilities were associated with large mean IPSC amplitudes and were preferentially obtained with presynaptic basket cells. Action currents recorded from the presynaptic interneurone in the cell-attached mode were reproducible indicating that the variability occurs downstream of action potential generation. 3. The variability of IPSC amplitudes was observed independently of the instantaneous firing rate of the presynaptic cell, and could therefore not be ascribed to synaptic fatigue. 4. Highly variable IPSC amplitude were also obtained if the presynaptic interneurone was placed in whole-cell recording with a potassium-based intracellular solution. 5. If the recording pipette were filled with Cs+ ions a large increase in the mean of the IPSCs was observed within seconds after establishing the whole-cell recording. In Cs(+)-dialysed cells it was possible to modulate the mean IPSC amplitudes by altering the characteristics of the presynaptic stimulation. IPSC amplitudes obtained in response to presynaptic voltage pulses to 0 mV were large and had little scatter. IPSCs obtained in response to pulses near +60 mV, close to the reversal potential of presynaptic Ca2+ currents, had a much lower mean amplitude and also had little scatter. Thus presynaptic application of Cs+ ions both increases the mean amplitude and decreases the variability of the postsynaptic response. 6. To test whether interneurones could be coupled by electrical synapses, paired recordings were performed from neighbouring interneurones. No correlation was found between the firing patterns of such paired recordings, indicating that electrical coupling among presynaptic neurones is not responsible for large IPSC fluctuations as recorded in Purkinje cells. 7. Finally, IPSC fluctuations were investigated in paired recordings from two Purkinje cells. IPSCs corresponding to the activity of common interneurones were identified on the basis of temporal correlation. By plotting the amplitudes of such common IPSCs in one cell against those obtained simultaneously in the other cell, the pattern of IPSCs due to a single presynaptic neurone could be identified. These results show that fluctuations of IPSCs due to the same interneurone in one postsynaptic Purkinje cell are independent of those occurring in another Purkinje cell. 8. The results indicate that the major source of fluctuations is localized within the axonal arborization of presynaptic interneurones. The results with presynaptic Cs+ require that the fluctuations involve the concerted release of several presynaptic vesicles. Two possible mechanisms for such multiquantal events are discussed: fluctuations in presynaptic depolarization, and fluctuations in a regenerative Ca2+ amplification mechanism.
Animals↗
Cell surface domain specific postsynaptic currents evoked by identified GABAergic neurones in rat hippocampus in vitro.
1. Inhibitory postsynaptic currents (IPSCs) evoked in CA1 pyramidal cells (n = 46) by identified interneurones (n = 43) located in str. oriens were recorded in order to compare their functional properties and to determine the effect of synapse location on the apparent IPSC kinetics as recorded using somatic voltage clamp at -70 mV and nearly symmetrical [Cl-]. 2. Five types of visualised presynaptic interneurone, oriens-lacunosum moleculare (O-LMC), basket (BC), axo-axonic (AAC), bistratified (BiC) and oriens-bistratified (O-BiC) cells, were distinguished by immunocytochemistry and/or synapse location using light and electron microscopy. 3. Somatostatin immunoreactive O-LMCs, innervating the most distal dendritic shafts and spines, evoked the smallest amplitude (26 +/- 10 pA, s.e.m., n = 8) and slowest IPSCs (10-90 % rise time, 6.2 +/- 0.6 ms; decay, 20.8 +/- 1.7 ms, n = 8), with no paired-pulse modulation of the second IPSC (93 +/- 4 %) at 100 ms interspike interval. In contrast, parvalbumin-positive AACs evoked larger amplitude (308 +/- 103 pA, n = 7) and kinetically faster (rise time, 0.8 +/- 0.1 ms; decay 11.2 +/- 0.9 ms, n = 7) IPSCs showing paired-pulse depression (to 68 +/- 5 %, n = 6). Parvalbumin- or CCK-positive BCs (n = 9) terminating on soma/dendrites, BiCs (n = 4) and O-BiCs (n = 7) innervating dendrites evoked IPSCs with intermediate kinetic parameters. The properties of IPSCs and sensitivity to bicuculline indicated that they were mediated by GABAA receptors. 4. In three cases, kinetically complex, multiphasic IPSCs, evoked by an action potential in the recorded basket cells, suggested that coupled interneurones, possibly through electrotonic junctions, converged on the same postsynaptic neurone. 5. The population of O-BiCs (4 of 4 somatostatin positive) characterised in this study had horizontal dendrites restricted to str. oriens/alveus and innervated stratum radiatum and oriens. Other BiCs had radial dendrites as described earlier. The parameters of IPSCs evoked by BiCs and O-BiCs showed the largest cell to cell variation, and a single interneurone could evoke both small and slow as well as large and relatively fast IPSCs. 6. The kinetic properties of the somatically recorded postsynaptic current are correlated with the innervated cell surface domain. A significant correlation of rise and decay times for the overall population of unitary IPSCs suggests that electrotonic filtering of distal responses is a major factor for the location and cell type specific differences of unitary IPSCs, but molecular heterogeneity of postsynaptic GABAA receptors may also contribute to the observed kinetic differences. Furthermore, domain specific differences in the short-term plasticity of the postsynaptic response indicate a differentiation of interneurones in activity-dependent responses.
Animals↗
Quantal analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study.
1. Synaptically connected neurones were identified in the granule cell layer of slices of 17- to 21-day-old rat hippocampus. Whole-cell current recording using the patch-clamp technique revealed synaptic currents ranging from less than 10 to 200 pA in symmetrical Cl- conditions, at a holding potential of -50 mV. These currents were blocked by 2 microM-bicuculline, indicating that they result from the activation of postsynaptic gamma-aminobutyric acid receptor (GABAA-receptor) channels. 2. Addition of tetrodotoxin (TTX, 1 microM) resulted in the loss of most currents of more than 40 pA in amplitude. Currents which disappeared after TTX treatment were assumed to be the result of spontaneous presynaptic action potentials. The currents seen in the absence of TTX are referred to as spontaneously occurring inhibitory postsynaptic currents (IPSCs); those remaining in the presence of TTX were defined as miniature IPSCs. 3. Similar currents were observed when recording in the whole-cell configuration while extracellular stimulation was applied to a nearby neurone. These currents were also completely blocked by 2 microM-bicuculline and by 0.5 microM-TTX. They were thus defined as stimulus-evoked IPSCs. 4. The half rise time of both miniature and stimulus-evoked IPSCs was fast (less than 1 ms). The time course of decay of both miniature IPSCs and stimulus-evoked IPSCs could be well fitted with the sum of two exponentials. At a membrane potential of -50 mV, the mean decay time constants of the two components were 2.0 +/- 0.38 and 54.4 +/- 18 ms (mean +/- S.D.) for miniature IPSCs (six cells) and 2.2 +/- 1.3 and 66 +/- 20 ms (three cells) for stimulus-evoked IPSCs. 5. Stimulus-evoked IPSCs varied in amplitude from less than ten to hundreds of picoamperes. In eight of eleven cells histograms of IPSC amplitudes showed several clear peaks which, when fitted with the sum of Gaussian curves, were found to be equidistant. This is consistent with the view that stimulus-evoked IPSC amplitudes vary in a quantal fashion. The quantal size varied between 7 and 20 pA, at a membrane potential of -50 mV. 6. Decreasing the Ca2+ and increasing the Mg2+ concentration in the extracellular solution decreased the number of peaks in the IPSC amplitude histogram but did not affect the size of the quantal event.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Presynaptic inhibition by dopamine of a discrete component of GABA release in rat substantia nigra pars reticulata.
1. Whole-cell patch clamp recordings were made from substantia nigra pars reticulata (SNr) neurones in rat midbrain slices. Monosynaptic IPSCs were evoked by electrical stimulation of the cerebral peduncle in the presence of the glutamate receptor antagonists CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and AP5 (2-amino-5-phosphonopentanoic acid). 2. IPSCs were predominantly outward at -70 mV (in 124/135 cells), with a reversal potential of -83 mV, a time to peak of 2.6 ms and a decay time constant of 6.5 ms. Faster inward IPSCs were also observed in thirty-five cells, with a time to peak of 1.0 ms, a decay time constant of 2.3 ms, and a reversal potential of -61 mV. Both IPSCs were sensitive to the GABAA receptor antagonists picrotoxin or bicuculline. 3. In cells recorded with Cs+-filled pipettes, the outward IPSC reversal potential was shifted to -76 mV, closer to the estimated Cl- equilibrium potential of -56 mV, while that of the inward IPSC was unchanged at -64 mV. 4. The outward IPSC was reversibly depressed by up to 100 % by dopamine in a concentration-dependent manner with an IC50 of 10.5 microM, while the inward IPSC was relatively insensitive. 5. Dopamine was without effect on cell holding current, or on outward IPSC reversal potential, but it increased paired-pulse IPSC facilitation, consistent with a presynaptic site of action. 6. The D1-like dopamine receptor agonist SKF 38393 (10 microM) depressed the outward IPSC by 43 %, while the D2-like dopamine receptor agonist quinpirole (10 microM) was without effect. 7. It is concluded that GABA-ergic synaptic input onto distal rather than proximal regions of SNr neurones is susceptible to presynaptic inhibition via a D1-like receptor. These inputs are probably from striato-nigral fibres, and their inhibition by dopamine is likely to influence the patterning of basal ganglia output.
Animals↗