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PubMed2003

Functional localization of cannabinoid receptors and endogenous cannabinoid production in distinct neuron populations of the hippocampus.

The possible localization of cannabinoid (CB) receptors to glutamatergic and GABAergic synaptic terminals impinging upon GABAergic interneurons in the CA1 region of the rat hippocampus was examined using the electrophysiological measurement of neurotransmitter release in brain slices. Whereas activation of cannabinoid receptors via the application of the cannabinoid agonist WIN55,212-2 significantly and dose-dependently reduced evoked IPSCs recorded from interneurons possessing somata located in the stratum radiatum (S.R.) and stratum oriens (S.O.) lamellae, evoked glutamatergic EPSCs were unaffected in both neuronal populations. However, in agreement with previous reports, WIN55,212-2 significantly reduced EPSCs recorded from CA1 pyramidal neurons. Additional experiments confirmed that the effects of WIN55,212-2 on IPSCs were presynaptic and that they could be blocked by the CB1 receptor antagonist SR141716A. The involvement of endogenous cannabinoids in the presynaptic inhibition of GABA release was also examined in the interneurons and pyramidal cells using a depolarization-induced suppression of inhibition (DSI) paradigm. DSI was observed in CA1 pyramidal neurons under control conditions, and its incidence was greatly increased by the cholinergic agonist carbachol. However, DSI was not observed in the S.R. or S.O. interneuron populations, in either the presence or absence of carbachol. Whereas DSI was not present in these interneurons, the inhibitory inputs to these cells were modulated by the synthetic cannabinoid WIN55,212-2. These data support the hypothesis that cannabinoid receptors are located on inhibitory, but not excitatory, axon terminals impinging upon hippocampal interneurons, and that CA1 pyramidal neurons, and not interneurons, are capable of generating endogenous cannabinoids during prolonged states of depolarization.

Animals↗
PubMed2004

Diversity beyond variance: modulation of firing rates and network coherence by GABAergic subpopulations.

Computational modelling studies have revealed that heterogeneity in interneuronal networks can powerfully modulate firing rates, responses to excitatory inputs, theta-gamma oscillations and network synchrony. In these previous studies, heterogeneity was represented by the degree of variance in various interneuronal synaptic and cellular parameters. However, a major characteristic of gamma-amino-butyric-acid-ergic (GABAergic) synaptic and interneuronal populations is the presence of distinct subgroups, which variance-based approaches cannot fully address. Here we apply an information theory-based measure of diversity, the Shannon-Wiener diversity index (equivalent to entropy), which takes into account both the number and relative abundance of categories within a population. Computational modelling data and experimental dynamic clamp results show that increasing the diversity index in the somatically injected inhibitory post synaptic currents (IPSC) peak conductances modulates the firing rates of CA1 pyramidal cells in a predictable manner that depends on both the mean and variance of the IPSC conductance. Furthermore, increases in the diversity index in interneuronal populations strongly decreased network coherence, even when population variance, the previously applied measure of heterogeneity, remained unchanged. These modelling and experimental results reveal a new approach to the study of interneuronal heterogeneity, and demonstrate the modulation of principal cell firing rates and interneuronal network coherence by parameter clustering at both the synaptic and cellular levels.

Action Potentials↗
PubMed2001

GHB depresses fast excitatory and inhibitory synaptic transmission via GABA(B) receptors in mouse neocortical neurons.

Gamma-hydroxybutyrate (GHB) is a drug of abuse which induces sedation and euphoria. However, overdoses can severely depress the level of consciousness or can be fatal especially when combined with other substances. Studies have suggested that the GHB-effects are mediated via actions on thalamocortical pathways and local neocortical circuits, although the effect of GHB at the level of single neocortical neurons is not clear. Using whole-cell patch-clamp recordings, we studied the effects of GHB on neocortical neurons in brain slices from 12- to 33-day-old mice. We found that GHB depressed the frequency and amplitude of GABAergic and glutamatergic spontaneous inhibitory and excitatory post-synaptic currents (IPSCs and EPSCs) driven by presynaptic action potential firing, while the amplitude and frequency of Ca(2+) entry-independent miniature IPSCs were not affected. Using minimal stimulation, GHB reduced the probability of release at inhibitory synapses onto neocortical layer 2/3 pyramidal cells. Also, GHB directly hyperpolarized layer 2/3 non-pyramidal cells by up to 11 mV and inhibited action potential firing. All these effects of GHB were mediated via GABA(B)-receptors. In conclusion, GHB activates both pre- and postsynaptic GABA(B)-receptors in neocortical neurons participating in fast synaptic transmission, leading to a powerful depression of neocortical network activity. We propose that GABA(B)-receptor antagonists may be useful in the treatment of acute GHB intoxication.

Action Potentials↗
PubMed2025

Dynamic changes in chromosome and nuclear architecture during maturation of normal and ALS C9orf72 motor neurons.

We have investigated changes in chromosome conformation, nuclear organization, and transcription during differentiation and maturation of control and mutant motor neurons harboring hexanucleotide expansions in the C9orf72 gene that cause amyotrophic lateral sclerosis (ALS). Using an in vitro reprogramming, differentiation and neural maturation protocol, we obtained highly purified populations of post-mitotic motor neurons for both normal and diseased cells. As expected, as fibroblasts are reprogrammed into iPSCs, and as iPSCs differentiate into motor neurons, chromatin accessibility, chromosome conformation, and nuclear organization change along with large-scale alterations in transcriptional profiles. We find that the transcriptome changes extensively during the first three weeks of post-mitotic neuronal maturation, with thousands of genes changing expression, but then is relatively stable for the next three weeks. In contrast, chromosome conformation and nuclear organization continue to change over the entire 6-week maturation period: chromosome territoriality increases, long-range interactions along chromosomes decrease, compartmentalization strength increases, and centromeres and telomeres increasingly cluster. In motor neurons derived from ALS patients such changes in chromosome conformation were much reduced. Chromatin accessibility changes also showed delayed maturation. The transcriptome in these cells matured relatively normally but with notable changes in expression of genes involved in lipid, sterol and mitochondrial function. We conclude that neural maturation is associated with large scale post-mitotic changes in gene expression, chromosome conformation and nuclear organization, and that these processes are defective in motor neurons derived from ALS patients carrying C9orf72 hexanucleotide repeat expansions.

Journal Article↗
PubMed2004

Endocannabinoid transport tightly controls 2-arachidonoyl glycerol actions in the hippocampus: effects of low temperature and the transport inhibitor AM404.

The control of endocannabinoid actions on cortical neurons by a putative carrier-mediated uptake is still poorly understood at the level of synaptic transmission. We investigated the effect of an endocannabinoid, 2-arachidonoyl glycerol (2-AG), on inhibitory postsynaptic currents (IPSCs) in hippocampal slices under physiological conditions, and when uptake was altered by a selective blocker or lower temperature. Bath application of 2-AG (20 micro m) caused a 40% reduction in the amplitude of IPSCs evoked in the perisomatic region of CA1 pyramidal neurons at room temperature; this effect could be blocked by a specific CB(1) receptor antagonist, AM251. By contrast, a smaller (20%) but significant suppression of inhibitory transmission was found by 2-AG at 33-35 degrees C. This reduced blocking effect at physiological temperature could be brought back to 40% by coapplying the endocannabinoid uptake blocker, AM404 (10 or 20 micro m) with 2-AG. In parallel experiments, we measured [(3)H]2-AG uptake at different temperatures in primary cultures prepared from cortical neurons. These data confirmed a striking inhibition of [(3)H]2-AG uptake at room temperature compared with values observed at 37 degrees C. Uptake could be significantly modified by anandamide, 2-AG and AM404, suggesting a common transporter for the two endocannabinoids. These findings together demonstrate the presence of an effective endocannabinoid uptake in cortical neurons, which could considerably alter the spatial and temporal constraints of endocannabinoid signalling at physiological temperature, and which may critically change the interpretation of findings at room temperature.

Animals↗
PubMed2004

Exposure to interferon-gamma during synaptogenesis increases inhibitory activity after a latent period in cultured rat hippocampal neurons.

Certain disorders of the nervous system may have their origin in disturbances in the development of synaptic connections and network structure that may not become overt until later in life. As inflammatory cytokines can influence synaptic activity in neuronal cultures, we analysed whether cytokine exposure during synaptogenesis can lead to imbalances in a neuronal network. Short-term application of interferon-gamma (IFN-gamma), but not tumour necrosis factor-alpha, during peak synaptogenesis (but not before or after) in Sprague-Dawley rat hippocampal cultures, caused both a decrease in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) and an increase in the frequency of spontaneous inhibitory postsynaptic currents (IPSCs). These effects were only detected in recordings made weeks later. This was not due to a depression of glutamatergic synapses or to a change in the relative number of neurons containing glutamic acid decarboxylase (GAD). There was an increase in the average amplitude of miniature IPSCs, and in GAD-expressing neurons the amplitude of miniature EPSCs were larger as well as the responses to glutamate. This indicates that IFN-gamma-treatment induced increased inhibition via postsynaptic changes. These effects of IFN-gamma treatment were not observed when neuronal nitric oxide synthase was inhibited. Our study therefore shows that exposure to IFN-gamma during a restricted period of development, which coincides with the peak of excitatory synaptogenesis, can cause progressive changes in synaptic activity in the network. Thus, cytokine exposure at a critical period of development may constitute a 'hit-and-run' mechanism for certain nervous system disorders that become manifest after a latency period.

Animals↗
PubMed2004

Potentiation by angiotensin II of spontaneous excitatory postsynaptic currents in rat supraoptic magnocellular neurones.

The physiological actions of angiotensin II in the supraoptic (SON) and paraventricular nuclei have been widely demonstrated, including the modulation of firing rate and release of arginine vasopressin and oxytocin. Here, we investigated whether angiotensin II modulates synaptic inputs into the SON. To do this, we measured spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) from rat SON neurones in thin slice preparations using the whole-cell patch-clamp technique. Angiotensin II reversibly increased the frequency of spontaneous EPSCs in a dose-related manner without affecting the amplitude, indicating that angiotensin II potentiated EPSCs via a presynaptic mechanism. Angiotensin II-induced potentiation of EPSCs was unaffected in the presence of tetrodotoxin. On the other hand, angiotensin II did not cause significant effects on IPSCs. The potentiation of EPSCs by angiotensin II was potently suppressed by previous exposure to the angiotensin type 1 (AT1) receptor antagonist, losartan. Our results suggest that angiotensin II potentiates the excitatory synaptic inputs into SON neurones, via the AT1 receptors.

Angiotensin II↗
PubMed1994

Glycinergic inhibitory synaptic currents and related receptor channels in the zebrafish brain.

To extend our study of the inhibitory synaptic network we have developed an isolated whole-brain preparation of the 52-h-old zebrafish (Brachydanio rerio) in which the structural and functional integrity of the brain is preserved. We report the characterization of quantal inhibitory events and the correlation of their properties with those of the underlying activated channels. During whole-cell recordings of the Mauthner cells, applications of 10(-6) M tetrodotoxin greatly reduced the frequency and amplitude of the spontaneously occurring synaptic events, which were dominated by Cl--dependent inhibitory postsynaptic currents (IPSCs). Lowering Ca2+ and adding Mg2+ to tetrodotoxin-containing solutions resulted in a further decrease in amplitude of the recorded synaptic currents, the remaining ones being considered as miniature IPSCs (mIPSCs). Applications of 0.5-1 microM strychnine in the presence of tetrodotoxin eliminated > 90% of the inhibitory currents in the preparation. The amplitude histograms of these mIPSCs exhibited two initial equally spaced peaks, followed by a skewed distribution for higher values. The first two components were well fitted by the sum of two Gaussian curves, giving a mean quantal amplitude of 35.7 pA (at a holding potential of -50 mV) and a coefficient of variation of 0.25 for the first peak. Outside-out recordings showed at least two classes of glycine receptor channels, one having multiple conductance levels with a main state of 81-86 pS and another displaying only one opening level of 41-43 pS. These two mean conductance states had similar mean open times, of 0.6-1 and 4.5-6 ms respectively. In addition, three mean closed times were observed for the 41-43 pS level. The shortest group (0.6-1 ms) was considered as representing gaps within bursts. Burst analysis revealed three mean burst durations, of 0.6, 4 and 35 ms. Comparisons of the amplitude of the first class of mIPSCs and of the open channel conductances indicated that one quantum opens 14-22 channels. Moreover, the correspondence between the mean decay time of mIPSCs and the mean open time or medium burst duration (4-5 ms) suggests that glycine-activated channels open only once in response to a single exocytosis. The pre- and postsynaptic origins of mIPSCs amplitude fluctuations are discussed in the context of multivesicular release versus the hypothesis of postsynaptic receptor saturation.

Animals↗
PubMed1997

Electrophysiological evidence for multiple glycinergic inputs to neonatal rat sympathetic preganglionic neurons in vitro.

The time pattern of glycinergic inhibitory postsynaptic currents (IPSCs) in sympathetic preganglionic neurons was studied in thin transverse spinal cord slices of neonatal (1-10 days postnatal) rats by means of the patchclamp technique. Three time patterns could be distinguished: (i) large events [mostly > 400 pA (30-36 degrees C)] occurring at regular intervals, (ii) small events occurring at irregular intervals, and (iii) small events occurring in transient (1.5-10 s), high-frequency (> 15 Hz) bursts of synaptic activity. The large regular events had uniform kinetics which was consistent with the idea of a proximal site of origin for all of these events. They were reversibly inhibited in amplitude and frequency by extracellular application of a high concentration of acetylcholine (200 microM) or the specific nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium iodide (DMPP; 1 mM), but unaffected by glutamate (100 microM). IPSCs occurring in bursts had slower and less uniform kinetics, suggesting a more diverse site of origin. The frequency of events decreased during a burst. Similar bursts could be induced by extracellular application of glutamate receptor agonists. These results indicate that sympathetic pregnanglionic neurons in a thin, transverse spinal cord slice receive at least two different glycinergic inputs.

Animals↗
PubMed2004

Functional mapping of GABA A receptor subtypes in the amygdala.

The physiological significance of the large diversity of GABA A receptors is poorly understood. Using mice, which carry a point mutation that renders specific subtypes of GABA A receptors diazepam insensitive, it was recently discovered that particular types of GABA A receptors are involved in specific, behaviorally relevant signaling pathways. We have used these mice to study inhibitory synaptic transmission in the amygdala. GABA A receptor-mediated inhibitory postsynaptic currents (IPSCs) per se were not affected by the point mutations. Their modulation by diazepam, however, was altered depending on the genotype of the mice studied. Based on the different responses to diazepam, we found that IPSCs in the lateral/basolateral amygdala were mediated by both alpha2- and alpha1-subunit-containing GABA A receptors whereas those in the central amygdala were mediated only by alpha2-subunit-containing GABA A receptors. Immunohistochemical staining corroborated these findings at a morphological level. To investigate a possible link between interneuron and receptor diversity, we selectively depressed release from the subset of GABAergic terminals carrying type 1 cannabinoid receptors. These receptors are known to modulate amygdala-mediated behavior. Application of a type 1 cannabinoid receptor agonist resulted in a selective reduction of inhibitory current mediated by alpha1-subunit-containing GABA A receptors. Mice with specific diazepam-insensitive GABA A receptor subtypes therefore provide a novel tool to investigate GABA A receptor distribution and the organization of inhibitory circuits at a functional level. The crucial role of the amygdala for the mediation of anxiety is in agreement with the part that alpha2-subunit-containing GABA A receptors play in anxiolysis and their important function in this area of the brain.

Amygdala↗
PubMed2004

Unilateral cochlear ablation produces greater loss of inhibition in the contralateral inferior colliculus.

Bilateral cochlear ablation leads to a profound weakening of synaptic inhibition within the inferior colliculus (IC) of gerbils [Vale & Sanes (2000) J. Neurosci., 20, 1912-1921]. To examine whether unilateral deafening leads to similar functional alterations, we studied the effect of unilateral cochlear ablation on inhibitory synaptic properties both ipsilateral and contralateral to the deafened ear. Lateral lemniscal and commissure of the IC-evoked inhibitory postsynaptic currents (IPSCs) were recorded in an IC brain slice preparation using whole-cell and gramicidin perforated-patch electrodes in the presence of kynurenic acid. Unilateral cochlear ablation led to a 23 mV depolarizing shift in the IPSC equilibrium potential for IC neurons contralateral to the deafened ear, but only a 10 mV depolarization in the ipsilateral IC. Lateral lemniscal-evoked inhibitory synaptic conductance declined significantly in the ipsilateral and contralateral IC, whereas commissural-evoked inhibitory synaptic conductance declined only contralateral to the ablated cochlea. An analysis of paired-pulse facilitation showed that inhibitory transmitter release was more affected ipsilateral to the ablated cochlea. Thus, unilateral cochlear ablation modifies inhibitory synapses in the inferior colliculus, but these changes appear to be dominated by postsynaptic alterations in the contralateral IC, and by presynaptic changes in the ipsilateral IC.

Animals↗
PubMed2005

Nicotine-induced switch in the nicotinic cholinergic mechanisms of facilitation of long-term potentiation induction.

Nicotine facilitates the induction of long-term potentiation (LTP) in the hippocampal CA1 region. The present study reveals the potential mechanisms underlying this effect of nicotine. Timed ACh-mediated activation of alpha7 nicotinic acetylcholine receptors (nAChRs) on pyramidal cells is known to promote LTP induction. Nicotine could suppress this timing-dependent mechanism by desensitizing nAChRs. Timed ACh-mediated activation of alpha7 nAChRs on feedforward interneurons can prevent LTP induction by inhibiting pyramidal cells. Nicotine diminished this ACh-mediated inhibition by desensitizing alpha7 nAChRs, thereby reducing the inhibitory influence on pyramidal cells. In addition to these desensitizing effects, nicotine activated presynaptic non-alpha7 nAChRs on feedforward interneurons to decrease the evoked release of gamma-aminobutyric acid (GABA) onto pyramidal cells. Furthermore, nicotine increased the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) in pyramidal cells, and concomitantly caused a reduction in the size of responses to focal GABA application onto the dendrites of pyramidal cells, suggesting that the nicotine-induced increase in interneuronal activity leads ultimately to a use-dependent depression of evoked IPSCs in pyramidal cells. These nicotine-induced suppressions of inhibition of pyramidal cells were accompanied by enhanced N-methyl-D-aspartate (NMDA) responses in pyramidal cells. Thus, our results suggest that nicotine promotes the induction of LTP by diminishing inhibitory influences on NMDA responses while suppressing the ACh-mediated mechanisms. These ACh-independent mechanisms probably contribute to the nicotine-induced cognitive enhancement observed in the presence of cholinergic deficits, such as those in Alzheimer's disease patients.

Acetylcholinesterase↗
PubMed2006

Selective 5-HT receptor inhibition of glutamatergic and GABAergic synaptic activity in the rat dorsal and median raphe.

The dorsal (DR) and median (MR) raphe nuclei contain 5-hydroxytryptamine (5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain. The DR and MR have differential roles in mediating stress, anxiety and depression. Glutamate and GABA activity sculpt putative 5-HT neuronal firing and 5-HT release in a seemingly differential manner in the MR and DR, yet isolated glutamate and GABA activity within the DR and MR has not been systematically characterized. Visualized whole-cell voltage-clamp techniques were used to record excitatory and inhibitory postsynaptic currents (EPSC and IPSC) in 5-HT-containing neurons. There was a regional variation in action potential-dependent (spontaneous) and basal [miniature (m)] glutamate and GABAergic activity. mEPSC activity was greater than mIPSC activity in the DR, whereas in the MR the mIPSC activity was greater. These differences in EPSC and IPSC frequency indicate that glutamatergic and GABAergic input have distinct cytoarchitectures in the DR and MR. 5-HT(1B) receptor activation decreased mEPSC frequency in the DR and the MR, but selectively inhibited mIPSC activity only in the MR. This finding, in concert with its previously described function as an autoreceptor, suggests that 5-HT(1B) receptors influence the ascending 5-HT system through multiple mechanisms. The disparity in organization and integration of glutamatergic and GABAergic input to DR and MR neurons and their regulation by 5-HT(1B) receptors may contribute to the distinction in MR and DR regulation of forebrain regions and their differential function in the aetiology and pharmacological treatment of psychiatric disease states.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

Frequency-dependent actions of benzodiazepines on GABAA receptors in cultured murine cerebellar granule cells.

1. Miniature IPSCs recorded from cultured murine cerebellar granule cells increased in half-width and amplitude following application of the benzodiazepine (BDZ) Flunitrazepam (Flu, 1 microM). The increase in the half-width was much greater than that in the amplitude. 2. Five-millisecond applications of 1 mM GABA to nucleated outside-out patches elicited rapidly rising biexponentially decaying responses that resembled IPSCs. Flu had no effect on the amplitude of such responses, but consistently slowed their deactivation by approximately 50%. This effect was reversed by Flu washout or application of the BDZ antagonist Ro15-1788. The partial inverse agonist. Ro15-4513 speeded deactivation and depressed peak current amplitude by 23 +/- 12%. 3. The EC50 for GABA was between 45 and 50 microM. At submaximally effective agonist concentrations, Flu increased response amplitude and slowed response deactivation. Both effects were present in all cells taken from young cultures (4-7 days in vitro) but the latter was absent in 55% of the neurones obtained from older cultures (14-27 days in vitro). 4. With 120 ms applications of 20 microM GABA, responses activated monoexponentially (time constant, 39.8 +/- 2.8 ms) and deactivated biexponentially (time constants, 40.4 +/- 2.1 and 251 +/- 15 ms). Application of Flu slowed both activation and deactivation. The latter effect arose from an increased contribution of the slower component of decay. 5. Desensitization of responses to 1 mM GABA was biexponential, with time constants of 47 +/- 11 and 479 +/- 49 ms. Flu speeded desensitization by decreasing both fast and slow time constants. GABAA receptor desensitization consistently slowed subsequent deactivation. No significant relationship between the level of desensitization and the amount of slowing of deactivation produced by Flu was found. 6. Responses to paired 5 ms applications of 1 mM GABA indicated that the slowing of deactivation and the speeding of desensitization produced by Flu combine to generate a marked frequency dependence in the actions of this BDZ. Thus when compared with control responses, GABA-induced charge transfer was only enhanced by Flu during the first of two successive agonist applications.

Animals↗
PubMed1998

Synaptic currents at individual connections among stellate cells in rat cerebellar slices.

1. Unitary inhibitory synaptic connections among stellate cells were studied in rat cerebellar slices. Presynaptic action potentials and inhibitory postsynaptic currents (IPSCs) were simultaneously recorded by loose cell-attached and tight-seal whole-cell recording, respectively. 2. Several types of synaptic connections were distinguished on the basis of the shape of the amplitude distribution of successfully evoked currents. For simple synapses, which presumably arise at single release sites, these histograms could be fitted to a single Gaussian (5 cases). In four additional cases a small amplitude component (< 50 pA) was superimposed to a single Gaussian peak. The small events had slow rise times and widely distributed amplitudes. Finally eleven histograms showed two or more Gaussian components and were classified as complex connections. 3. Failure rates ranged from 0.06 to 0.85 for unitary connections (n = 20) and from 0.59 to 0.78 for simple synapses (n = 5). 4. Coefficient of variation values derived from Gaussian fits to simple synapse histograms ranged between 0.20 and 0.38 (n = 5). 5. In simple synapses peak current amplitudes were positively correlated to both current rise time and decay half-width. 6. Intervals between presynaptic action potentials were widely distributed. During stationary periods there was no correlation between interspike interval and amplitude size, success rate or latency. In some experiments, episodes with shorter interspike intervals were observed. During these periods, amplitude and success rate decreased, and the latency increased. Thus, IPSC characteristics depend on the mean frequency of presynaptic spikes, but not on random fluctuations of interspike intervals during stationary periods.

Action Potentials↗
PubMed1999

Calcium dependence of depolarization-induced suppression of inhibition in rat hippocampal CA1 pyramidal neurons.

1. We made whole-cell recordings from CA1 pyramidal cells in the rat hippocampal slice preparation to study the calcium (Ca2+) dependence of depolarization-induced suppression of inhibition (DSI). DSI is a retrograde signalling process in which voltage-dependent Ca2+ influx into a pyramidal cell leads to a transient decrease in the release of GABA from interneurons. 2. To investigate the Ca2+ dependence of DSI without altering extracellular divalent cations, we varied the type and amount of Ca2+ chelator (EGTA or BAPTA) in the recording pipette (keeping the chelator : Ca2+ ratio constant). Evoked inhibitory postsynaptic currents (IPSCs) were induced in the presence of antagonists of ionotropic glutamate receptors. DSI was induced by depolarizing voltage steps, lasting from 0.025 to 5 s, to 0 mV. 3. DSI was directly dependent on the duration of the voltage step used to induce it, from threshold up to a maximal value of IPSC suppression, whether EGTA or BAPTA was used, and whether their concentrations were 0.1, 0.5 or 2 mM. For instance, a voltage step lasting 1.37 s produced half-maximal DSI with 2 mM BAPTA, but with 0. 1 mM BAPTA, half-maximal DSI was achieved with a step lasting 0.186 s. Peak DSI was the same in all cases, and DSI was blocked with either 10 mM EGTA or BAPTA in the pipette. Bath application of carbachol could overcome the block of DSI by 10 mM EGTA but not by 10 mM BAPTA. 4. We calculated that a voltage step lasting approximately 100 ms would be necessary to activate half-maximal DSI in the absence of exogenous Ca2+ buffers. 5. Log-log plots of calculated total Ca2+ influx, estimated from time integrals of Ca2+ currents, versus DSI yielded a straight line with a slope of approximately 1, and increasing extracellular [Ca2+] from 2.5 to 5 mM did not change the slope. 6. The time course of decay of DSI was well described by an exponential function with a time constant of approximately 20 s and was not affected by changes in either concentration or type of Ca2+ buffer. 7. The data suggest that, in its Ca2+ dependence, DSI more closely resembles the slow release of neuropeptides and hormones than it does the process of fast release of many neurotransmitters.

Animals↗
PubMed1999

Mechanism underlying increased neuronal activity in the rat ventrolateral periaqueductal grey by a mu-opioid.

1. The overall effect of the mu-opioid receptor agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) on ventrolateral periaqueductal grey (PAG) neurons in brain slices was studied using the whole-cell patch-clamp recording technique. 2. Under current-clamp conditions, DAMGO (1 microM) increased cell firing in many PAG neurons even though the opioid induced hyperpolarization and inhibited excitatory postsynaptic potentials (EPSPs) in these cells. 3. The increase in cell activity by DAMGO was observed in both transverse and horizontal slices. The increase persisted when the membrane potential was re-depolarized to the control level. Thus, different planes of sections or the removal of Na+ channel inactivation could not account for the observation. 4. The GABA antagonist bicuculline caused cell firing, mimicking the excitatory effect of DAMGO. Unlike DAMGO, however, bicuculline depolarized PAG cells. 5. Under voltage-clamp conditions, with the same driving force, the evoked inhibitory postsynaptic currents (IPSCs) in these neurons were 2.3 times larger than the evoked excitatory postsynaptic currents (EPSCs). Furthermore, DAMGO inhibited IPSCs by 60.7% while it inhibited EPSCs by 35.3%. 6. We propose that the overall effect of an opioid depends on the dynamic balance of its excitatory and inhibitory actions. In the PAG, the blockade of the inhibitory drive of GABAergic inputs by DAMGO is large. It overcomes the DAMGO-induced hyperpolarization and inhibition of EPSCs and thus results in the excitation of these neurons.

Analgesics, Opioid↗
PubMed1999

Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors.

1. Regional distinctions in GABA type A (GABAA) miniature IPSC responses are thought to be determined by postsynaptic receptor composition. The kinetics of receptor activation and deactivation were studied using rapid exchange (100 micros) of GABA at excised patches containing recombinant (alpha1beta1gamma2 or alpha2beta1gamma2) and native (cortical) GABAA receptors. 2. Receptors activated by brief (< 1 ms) pulses of GABA demonstrated a characteristic current response, hereby referred to as the 'receptor system response'. System response properties included agonist concentration-dependent peak amplitudes and concentration-independent maximal rates of activation and deactivation. Receptor subtypes were characterized functionally and phenotyped using the system response characteristics. 3. System responses obtained for alpha1beta1gamma2 receptors exhibited a single phenotype while alpha2beta1gamma2 receptors exhibited either a predominant slow deactivation (type I) or a relatively infrequent faster (type II) phenotype. Receptor system responses of alpha2beta1gamma2 receptors reached peak currents twice as fast as those of alpha1beta1gamma2 receptors (0.5 versus 1.0 ms) but decayed 2 or 6 times more slowly (taulong of approximately 190 and 62 ms for type I and II alpha2beta1gamma2, and approximately 34 ms for alpha1beta1gamma2 receptors). 4. Receptor system responses from cultured fetal mouse cortical neurons could be statistically separated and classified into five major types with little intragroup variability, primarily based on variations in the current deactivation phases. 5. Receptors subjected to pharmacological modulation exhibited alterations in system response properties consistent with known mechanisms of action, such that distinctions between binding and gating modulations were possible. 6. Brief agonist exposure places limits on receptor activation and deactivation response kinetics. Consequently, receptor system responses may be used to characterize and functionally phenotype an excised patch receptor population. Furthermore, since synaptic exposure to transmitter is postulated to be similarly brief, IPSC kinetics may reflect a functional fingerprint of synaptic receptors.

Animals↗