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PubMed2006

Major effects of sensory experiences on the neocortical inhibitory circuits.

During postnatal development, sensory experiences play critical roles in the refinement of cortical connections. However, both the process of postnatal experience-dependent maturation of neocortical inhibitory networks and its underlying mechanisms remain elusive. Here, we examined the differential properties of intracortical inhibitory networks of layer IV in "sensory-spared" and "sensory-deprived" cortices of glutamate acid decarboxylase 67 (GAD67)-green fluorescent protein (GFP) (delta neo) and wild-type mouse. Our results showed that row D whisker trimming (WT) begun at postnatal day 7 (P7), but not after P15, induced a robust reduction of parvalbumin (PV) expression, measured by the PV/GFP ratio and PV cell densities, in the deprived barrels. WT also induced a robust reduction in the number of inhibitory perisomatic varicosities and synaptic GAD65/67 immunoreactivities in spiny neurons of the deprived barrels. Although the GAD65/67 expressions in interneurons were also downregulated in the deprived barrels, the GFP expression remained unchanged. Patch-clamp recording from spiny cells showed a 1.5-fold reduction of intracortical evoked IPSCs (eIPSCs) in deprived versus spared cortices. The reduction in eIPSCs occurred via changes in presynaptic properties and unitary IPSC amplitudes. Miniature IPSCs showed subtle but significant differences between the two experimental conditions. In addition, properties of the IPSCs in deprived barrels resemble those of IPSCs recorded in immature brains (P7). Together, these results suggest that the properties of local intracortical inhibitory networks are modified by sensory experiences. Perisomatic inhibition mediated by PV-positive basket cells is pruned by sensory deprivation.

Animals↗
PubMed2000

Afferent regulation of inhibitory synaptic transmission in the developing auditory midbrain.

To determine whether afferent innervation regulates the strength of inhibitory connections in the gerbil auditory midbrain, both cochleas were surgically removed in postnatal day 7 animals, before sound-driven activity is first observed. Inhibitory synaptic currents were measured in a brain slice preparation 1-7 d after the ablations. Whole-cell and gramicidin-perforated patch recordings were obtained from inferior colliculus neurons, and IPSCs were evoked by stimulation of the commissure of the inferior colliculus (CIC) or the ipsilateral lateral lemniscus (LL) in the presence of kynurenic acid. Deafferentation led to a 24 mV depolarizing shift in the IPSC equilibrium potential within 1 d of deafferentation. As a consequence, there was a large reduction of IPSC amplitude at a holding potential of -20 mV in neurons from bilaterally ablated animals. Furthermore, both afferent pathways displayed a 50% reduction of the inhibitory synaptic conductance after deafferentation, indicating that driving force was not solely responsible for the decline in IPSC amplitude. When paired pulses were delivered to the LL or CIC pathway in control neurons, the evoked IPSCs exhibited facilitation. However, paired pulse facilitation was nearly eliminated after deafferentation. Thus, normal innervation affects inhibitory synaptic strength by regulating postsynaptic chloride homeostasis and presynaptic transmitter release properties.

Animals↗
PubMed2000

Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the hippocampus.

The localization of cannabinoid (CB) receptors to GABAergic interneurons in the hippocampus indicates that CBs may modulate GABAergic function and thereby mediate some of the disruptive effects of marijuana on spatial memory and sensory processing. To investigate the possible mechanisms through which CB receptors may modulate GABAergic neurotransmission in the hippocampus, whole-cell voltage-clamp recordings were performed on CA1 pyramidal neurons in rat brain slices. Stimulus-evoked GABA(A) receptor-mediated IPSCs were reduced in a concentration-dependent manner by the CB receptor agonist WIN 55,212-2 (EC(50) of 138 nM). This effect was blocked by the CB1 receptor antagonist SR141716A (1 microM) but not by the opioid antagonist naloxone. In contrast, evoked GABA(B)-mediated IPSCs were insensitive to the CB agonist. WIN 55,212-2 also reduced the frequency of spontaneous, action potential-dependent IPSCs (sIPSCs), without altering action potential-independent miniature IPSCs (mIPSCs), measured while sodium channels were blocked by tetrodotoxin (TTX). Blockade of voltage-dependent calcium channels (VDCCs) by cadmium also eliminated the effect of WIN 55,212-2 on sIPSCs. Depolarization of inhibitory terminals with elevated extracellular potassium caused a large increase in the frequency of mIPSCs that was inhibited by both cadmium and WIN 55,212-2. The presynaptic effect of WIN 55,212-2 was also investigated using the potassium channel blockers barium and 4-aminopyridine. Neither of these agents significantly altered the effect of WIN 55,212-2 on evoked IPSCs. Together, these data suggest that presynaptic CB1 receptors reduce GABA(A)- but not GABA(B)-mediated synaptic inhibition of CA1 pyramidal neurons by inhibiting VDCCs located on inhibitory nerve terminals.

Animals↗
PubMed2000

The in vitro ethanol sensitivity of hippocampal synaptic gamma-aminobutyric acid(A) responses differs in lines of mice and rats genetically selected for behavioral sensitivity or insensitivity to ethanol.

Previous work has demonstrated that in the hippocampal CA1 region of Sprague-Dawley rats, there are ethanol-sensitive and ethanol-insensitive populations of GABAergic synapses on pyramidal neurons. The present experiments characterized the ethanol sensitivity of these pathways in lines of rats and mice genetically selected for sensitivity or insensitivity to the behavioral effects of ethanol. In ethanol-sensitive inbred long sleep mice, GABA(A) IPSCs induced by stimulation of proximal (probably somatic) synapses were enhanced by 80 mM ethanol, whereas the distal (i.e., dendritic) pathway was unaffected. Thus, the relative sensitivity of these pathways (proximal > distal) is the same in both Sprague-Dawley rats and in inbred long sleep mice. However, in the ethanol-insensitive inbred short sleep mice, neither proximal nor distal IPSCs were affected by 80 mM ethanol. The ethanol sensitivity of the proximal pathway was also examined in replicate lines of rats selected for either high ethanol sensitivity or low ethanol sensitivity. GABA(A) IPSCs in the high ethanol sensitivity lines were significantly enhanced by 80 mM ethanol, whereas IPSCs in the low ethanol sensitivity lines were unaffected. Thus, IPSCs evoked via the proximal pathway were enhanced by ethanol in all the sensitive mouse and rat lines, and unaffected in all the insensitive lines. These experiments demonstrate that GABA(A) synapses in brain differ in their sensitivity to enhancement by ethanol, and the sensitivity to such enhancement is under the control of genes that can be selected for using classical genetic selective breeding based on a behavioral phenotype.

Animals↗
PubMed2002

Heterotopic neurons with altered inhibitory synaptic function in an animal model of malformation-associated epilepsy.

Children with brain malformations often exhibit an intractable form of epilepsy. Although alterations in cellular physiology and abnormal histology associated with brain malformations has been studied extensively, synaptic function in malformed brain regions remains poorly understood. We used an animal model, rats exposed to methylazoxymethanol (MAM) in utero, featuring loss of lamination and distinct nodular heterotopia to examine inhibitory synaptic function in the malformed brain. Previous in vitro and in vivo studies demonstrated an enhanced susceptibility to seizure activity and neuronal hyperexcitability in these animals. Here we demonstrate that inhibitory synaptic function is enhanced in rats exposed to MAM in utero. Using in vitro hippocampal slices and whole-cell voltage-clamp recordings from visualized neurons, we observed a dramatic prolongation of GABAergic IPSCs onto heterotopic neurons. Spontaneous IPSC decay time constants were increased by 195% and evoked IPSC decay time constants by 220% compared with age-matched control CA1 pyramidal cells; no change in IPSC amplitude or rise time was observed. GABA transport inhibitors (tiagabine and NO-711) prolonged evoked IPSC decay kinetics of control CA1 pyramidal cells (or normotopic cells) but had no effect on heterotopic neurons. Immunohistochemical staining for GABA transporters (GAT-1 and GAT-3) revealed a low level of expression in heterotopic cell regions, suggesting a reduced ability for GABA reuptake at these synapses. Together, our data demonstrate that GABA-mediated synaptic function at heterotopic synapses is altered and suggests that inhibitory systems are enhanced in the malformed brain.

Animals↗
PubMed2002

Depression of inhibitory synaptic transmission between Purkinje cells and neurons of the cerebellar nuclei.

Neurons of the cerebellar nuclei have basal firing rates of 10-20 Hz, despite the convergence of many GABAergic Purkinje terminals onto cerebellar nuclear somata and the high spontaneous firing rate of Purkinje neurons. This persistence of firing during a constant barrage of inhibition raises the question of what patterns of Purkinje cell input inhibit nuclear cells most effectively. To explore the hypothesis that synaptic depression moderates inhibition at this synapse, we made whole-cell recordings from cerebellar nuclear neurons in mouse brain slices. IPSCs and IPSPs were elicited by electrically stimulating the corticonuclear tract at 10, 50, and 100 Hz. IPSCs evoked at the mean spontaneous firing rate of Purkinje cells (50 Hz) depressed by approximately 60%. The onset of depression had a fast, frequency-dependent component, from which recovery was rapid (approximately 100 msec), and a slower, frequency-independent component, from which recovery was slow (approximately 10 sec). As stimulation rate increased, steady-state depression increased, but each IPSC decayed less completely between stimuli, producing a "tonic" IPSC. Changes in stimulation rate produced rapid changes in the level of depression. Under current clamp, cerebellar nuclear neurons fired spontaneously. During 50 Hz trains of IPSPs, firing was initially interrupted, but resumed coincident with the onset of depression. Low-frequency trains entrained postsynaptic firing, and high-frequency trains greatly slowed firing, primarily because of the tonic IPSC. Thus, the properties of depression at this synapse appear to limit the sensitivity of nuclear cells to basal inhibition, while allowing the cells to respond to increases and decreases in Purkinje cell activity.

Animals↗
PubMed2003

Major differences in inhibitory synaptic transmission onto two neocortical interneuron subclasses.

Locally projecting GABAergic interneurons are the major providers of inhibition in the neocortex and play a crucial role in several brain functions. Neocortical interneurons are connected via electrical and chemical synapses that may be crucial in modulating complex network oscillations. We investigated the properties of spontaneous and evoked IPSCs in two morphologically and physiologically identified interneuron subtypes, the fast-spiking (FS) and low threshold-spiking (LTS) cells in layer V of rodent sensorimotor cortex. We found that IPSCs recorded in FS cells were several orders of magnitude more frequent, larger in amplitude, and had faster kinetics than IPSCs recorded in LTS cells. GABA(A) receptor alpha- and beta-subunit selective modulators, zolpidem and loreclezole, had different effects on IPSCs in FS and LTS interneurons, suggesting differential expression of GABA(A) receptor subunit subtypes. These pharmacological data indicated that the alpha1 subunit subtype is poorly expressed by LTS cells but makes a large contribution to GABA(A) receptors on FS cells. This was confirmed by experiments performed in genetically modified mice in which the alpha1 subunit had been made insensitive to benzodiazepine-like agonists. These results suggest that differences in IPSC waveform are likely attributable to distinctive expression of GABA(A) receptor subunits in FS and LTS cells. The particular properties of GABAergic input on different interneuronal subtypes might have important consequences for generation and pacing of cortical rhythms underlying several brain functions. Moreover, selective pharmacological manipulation of distinct inhibitory circuits might allow regulation of pyramidal cell activities under specific physiological and pathophysiological conditions.

Action Potentials↗
PubMed2004

Activation of ryanodine receptors influences the paired-pulse depression in cultured rat hippocampal neurons.

Role of intraterminal calcium stores in modulation of short-term plasticity of evoked inhibitory postsynaptic currents (IPSCs) was studied in synaptically connected cultured hippocampal neurons using patch-clamp technique in whole-cell configuration. Currents were induced by voltage stimulation which were applied externally to presynaptic fiber. Paired stimuli resulted in paired-pulse depression (n=18) or facilitation (n=7) of the second IPSC at interpulse intervals 150 and 500 ms. Calcium release from intracellular calcium stores was activated by local application of caffeine and ryanodine, ryanodine receptor agonists. One of the characteristics of short-term plasticity, the pair-pulsed ratio (ratio of amplitudes of second IPSC to first IPSC), decreased during addition of ryanodine (50 nM) from 0.79 +/- 0.02 to 0.71 +/- 0.04 (n=10). This change was observed only for cells that demonstrated pair-pulsed depression. We also studied the influence of caffeine and ryanodine on spontaneous currents. Attenuation of the mean amplitude to 0.71 +/- 0.06 and frequency to 0.42 +/- 0.08 (n=7) of spontaneous IPSCs was observed during application of caffeine (10 mM). Upon ryanodine application the mean amplitude did not change but frequency of spontaneous events decreased to 0.74 +/- 0.09 (n=12). The amplitudes of currents evoked by fast local application of gamma-aminobutyric acid (GABA 100 mM) were diminished in the presence of caffeine (10 mM) to 0.59 +/- 0.03 (n=5) and in the presence of ryanodine (50 nM) to 0.56 +/- 0.11 (n=7). Thus we conclude that endoplasmic reticular calcium stores are able to modulate synaptic transmission from both presynaptic (in assumption that short-term plasticity and spontaneous activity are believed to have presynaptic nature) and postsynaptic (since GABA-receptors are situated on postsynaptic cell) sides.

Animals↗
PubMed1983

Physostigmine prolongs the elementary event underlying decay of inhibitory postsynaptic currents in Aplysia.

Inhibitory postsynaptic currents (IPSCs) recorded at cholinergic synapses between identified neurons of the buccal ganglia of Aplysia decay exponentially. IPSC decay has been ascribed to a receptor-channel-dependent kinetic process, rather than to changes in subsynaptic acetylcholine (ACh) concentration, but is prolonged by the anticholinesterase eserine. Our data are consistent with a direct effect of 1.6 X 10(-4) M eserine on an elementary channel process. IPSC decay remains single exponential during eserine prolongation, suggesting that eserine slows the kinetic process responsible for decay, rather than substituting an additional kinetic process. In either control sea water or eserine, noise spectra computed from currents induced by pressure ejection of ACh are fitted by the sum of two Lorentzians. The slow corner frequency f1 is reduced from 6.3 to 3.5 Hz by eserine, consistent with a direct eserine action on receptor-channel kinetics, whereas apparent single-channel conductance was unchanged. Both IPSCs and ACh noise recorded in the same cells were comparably slowed by eserine. In eserine, decay time constant tau agreed with noise f1; however, tau and f1 in control sea water differed by 31%. The discrepancy may be accounted for by invoking an additional component to the recorded noise spectra, perhaps produced by synaptically active choline. In addition to the direct effect on kinetics, prolonged exposure to eserine produces a slow extra component to the IPSC decay tail.

Acetylcholine↗
PubMed1993

Potentiation of gamma-aminobutyric acid type A receptor-mediated synaptic currents by pentobarbital and diazepam in immature hippocampal CA1 neurons.

Previous studies have demonstrated age-dependent changes in the expression of gamma-aminobutyric acid type A (GABAA) benzodiazepine receptor binding sites and mRNAs encoding GABAA receptor subunits during postnatal development. However, little is known about the pharmacology of GABAA-mediated synaptic events in immature brain neurons. The effects of pentobarbital and diazepam were examined on the GABAA-mediated inhibitory postsynaptic current (IPSC) in postnatal 2- to 8-day-old (PN 2-8) and 18- to 30-day-old (PN 18-30) hippocampal CA1 neurons, using whole-cell recordings in brain slices. In both age groups of immature neurons recorded with an internal solution containing 2 mM ATP, application of diazepam at a concentration as low as 10 nM consistently potentiated the IPSC and Cl- currents evoked by local ejection of GABA (GABA currents). Pretreatment with the benzodiazepine antagonist, flumazenil, blocked the diazepam-induced potentiation of the IPSC, which suggested a direct action of diazepam on the GABAA/benzodiazepine receptor complex. With a patch pipette solution containing no added ATP, similar application of diazepam caused consistent potentiation of the IPSC in PN 18-30 neurons but not in PN 2-8 neurons. In contrast, pentobarbital potentiated the IPSCs with or without internally applied ATP in the neurons of both age groups. The authors suggest that functional GABAA/benzodiazepine receptors are expressed in neonatal CA1 hippocampal neurons. However, their sensitivity to benzodiazepines is altered by intracellular ATP.

Adenosine Triphosphate↗
PubMed1996

Excitatory actions of norepinephrine on multiple classes of hippocampal CA1 interneurons.

Norepinephrine (NE) causes an increase in the frequency of inhibitory postsynaptic potentials in CA1 pyramidal neurons in vitro. The possibility that this increase in tonic inhibition is caused by an excitatory effect on inhibitory interneurons was investigated through whole-cell recordings from pyramidal cells and both whole-cell and cell-attached patch recordings from visualized interneurons in acute slices of rat hippocampus. Adrenergic agonists caused a large increase in the frequency and amplitude of spontaneous IPSCs recorded from pyramidal cells in the presence of ionotropic glutamate receptor blockers, but they had no effect on either the frequency or the amplitude of action potential-independent miniature IPSCs recorded in tetrodotoxin. This effect was mediated primarily by an alpha adrenoceptor, although a slight beta adrenoceptor-dependent increase in IPSCs was also observed. NE caused interneurons located in all strata to depolarize and begin firing action potentials. Many of these cells had axons that ramified throughout the stratum pyramidale, suggesting that they are responsible for the IPSCs observed in pyramidal neurons. This depolarization was also mediated by an alpha adrenoceptor and was blocked by a selective alpha 1- but not a selective alpha 2-adrenoceptor antagonist. However, a slight beta adrenoceptor-dependent depolarization was detected in those interneurons that displayed time-dependent inward rectification. In the presence of a beta antagonist, NE induced an inward current that reversed near the predicted K+ equilibrium potential and was not affected by changes in intracellular Cl- concentration. In the presence of an alpha 1 antagonist, NE induced an inwardly rectifying current at potentials negative to approximately -70 mV that did not reverse (between -130 and -60 mV), characteristics similar to the hyperpolarization-activated current (lh). However, the depolarizing action of NE is attributable primarily to the alpha 1 adrenoceptor-mediated decrease in K+ conductance and not the beta adrenoceptor-dependent increase in lh. These results provide evidence that NE increases action potential-dependent IPSCs in pyramidal neurons by depolarizing surrounding inhibitory interneurons. This potent excitatory action of NE on multiple classes of hippocampal interneurons may contribute to the NE-induced decrease in the spontaneous activity of pyramidal neurons and the antiepileptic effects of NE observed in vivo.

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

Increased probability of GABA release during withdrawal from morphine.

Opioid receptors located on interneurons in the ventral tegmental area (VTA) inhibit GABA(A)-mediated synaptic transmission to dopamine projection neurons. The resulting disinhibition of dopamine cells in the VTA is thought to play a pivotal role in drug abuse; however, little is known about how this GABAA synapse is affected after chronic morphine treatment. The regulation of GABA release during acute withdrawal from morphine was studied in slices from animals treated for 6-7 d with morphine. Slices containing the VTA were prepared and maintained in morphine-free solutions, and GABAA IPSCs were recorded from dopamine cells. The amplitude of evoked IPSCs and the frequency of spontaneous miniature IPSCs measured in slices from morphine-treated guinea pigs were greater than placebo-treated controls. In addition, activation of adenylyl cyclase, with forskolin, and cAMP-dependent protein kinase, with Sp-cAMPS, caused a larger increase in IPSCs in slices from morphine-treated animals. Conversely, the kinase inhibitors staurosporine and Rp-CPT-cAMPS decreased GABA IPSCs to a greater extent after drug treatment. The results indicate that the probability of GABA release was increased during withdrawal from chronic morphine treatment and that this effect resulted from an upregulation of the cAMP-dependent cascade. Increased transmitter release from opioid-sensitive synapses during acute withdrawal may be one adaptive mechanism that results from prolonged morphine treatment.

2-Amino-5-phosphonovalerate↗
PubMed1997

Action of methylmercury on GABA(A) receptor-mediated inhibitory synaptic transmission is primarily responsible for its early stimulatory effects on hippocampal CA1 excitatory synaptic transmission.

Bath application of methylmercury (MeHg) causes an early stimulation before block of synaptic transmission in the CA1 region of hippocampal slices. Effects of MeHg and Hg++ on inhibitory postsynaptic potentials (IPSPs) or currents (IPSCs) and excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) were compared to test whether or not early block by MeHg of GABA(A)-mediated inhibitory synaptic transmission and MeHg-induced alterations of the resting membrane potentials of CA1 neurons contribute to this initial enhancement of excitability. MeHg affected IPSPs and IPSCs similarly, and more rapidly than EPSPs and EPSCs. In contrast, although Hg++ blocked IPSPs more rapidly than EPSPs, times to block of IPSCs and EPSCs by Hg++ were virtually identical when CA1 neurons were voltage-clamped at their resting membrane potential levels. MeHg increased EPSC amplitudes before their subsequent decrease even when CA1 neuronal membranes were voltage-clamped at their resting potentials. This suggests that effects of MeHg on CA1 cell membrane potentials are not a major factor for MeHg-induced early stimulation of hippocampal synaptic transmission. Effects of MeHg and Hg++ on the reversal potentials for IPSCs also differed. Both metals blocked all outward and inward currents generated at different holding potentials. However, MeHg shifted the current-voltage (I/V) relationship to more positive potentials, although Hg++ shifted the I/V curve to more negative potentials. Hg++ was a less potent blocker of on IPSCs and EPSPs or EPSCs than was MeHg. To determine if the early increase in amplitude of population spikes or EPSPs is due to an action of MeHg at GABA(A) receptors, extracellular recordings of population spikes and intracellular recordings of EPSPs were compared with or without pretreatment of hippocampal slices with bicuculline. After preincubation of slices with 10 microM bicuculline for 30 to 60 min, MeHg only decreased the amplitudes of population spikes and EPSPs to block; no early increase of synaptic transmission occurred. Pretreatment of slices with strychnine, did not prevent MeHg-induced early increase in population spikes. MeHg also blocked responses evoked by bath application of muscimol, a GABA(A) agonist. Thus, block by MeHg of GABA(A) receptor-mediated inhibitory synaptic transmission may result in disinhibition of excitatory hippocampal synaptic transmission, and appears to be primarily responsible for the initial excitatory effect of MeHg on hippocampal synaptic transmission.

Animals↗
PubMed1997

Chronic focal neocortical epileptogenesis: does disinhibition play a role?

Several lines of evidence have suggested that decreases in postsynaptic inhibition may have a role in epileptogenesis in cortical structures. However, other studies have suggested that GABAergic inhibition is spared, or even augmented in some forms of post-lesional epilepsy. In the studies described here, inhibitory events were recorded in two models of post-lesional chronic epileptogenesis. (i) As previously reported (D.A. Prince and G.-F. Tseng. J. Neurophysiol. 69: 1276-1291. 1993), epileptiform activity develops in slices from partially isolated rat neocortical islands 2-3 weeks after the initial in vivo lesion. In this model of post-traumatic epilepsy, large amplitude polyphasic inhibitory postsynaptic currents (IPSCs) in layer V pyramidal neurons are associated with each interictal epileptiform field potential. The frequency of spontaneous IPSCs as well as miniature IPSCs was significantly increased in neocortical slices from the epileptogenic chronically injured cortex versus controls. Immunocytochemical reactions for parvalbumin and calbindin, calcium binding proteins present in subgroups of GABAergic neurons, showed an increased staining of both neuropil and somata within the epileptogenic tissue. Immunoreactivity for glutamic acid decarboxylase (GAD) and GABA also appeared to be increased in the neuropil. (ii) Cortical microgyri resembling human malformations were produced by freeze lesions made transcranially in P0 rat cortex (K.M. Jacobs, M.J. Gutnick, and D.A. Prince. Cereb. Cortex, 6: 514-523. 1996). The boundary between the four-layered microgyrus and surrounding cortex become epileptogenic within about 2 weeks, as judged by evoked extracellular field potentials and cellular activities. Epileptogenesis in the surrounding cortex is not altered when the microgyrus itself is isolated by transcortical cuts. Patch-clamp recordings from layer V neurons in the epileptogenic zone showed that spontaneous IPSCs are larger and more dependent on glutamatergic synapses than in control neurons. The amplitudes of polysynaptic IPSCs evoked by threshold stimulation were also larger than in control cells. Although evaluation of inhibitory events in these models is still incomplete, results to date suggest that GABAergic inhibition may be enhanced in epileptogenic areas associated with chronic cortical injury. Sprouting of axonal arborizations of pyramidal cells onto interneurons, upregulation of GABAergic neurons, and perhaps sprouting of inhibitory axons that make increased numbers of contacts onto pyramidal cells may all contribute to the increased inhibitory drive. Results in these models do not support the disinhibitory hypothesis of chronic epileptogenesis.

Animals↗
PubMed1998

Evidence for metabotropic glutamate receptor activation in the induction of depolarization-induced suppression of inhibition in hippocampal CA1.

Depolarization-induced suppression of inhibition (DSI) is a transient reduction of GABAA receptor-mediated IPSCs that is mediated by a retrograde signal from principal cells to interneurons. Using whole-cell recordings, we tested the hypothesis that mGluRs are involved in the DSI process in hippocampal CA1, as has been proposed for cerebellar DSI. Group II mGluR agonists failed to affect either evoked monosynaptic IPSCs or DSI, and forskolin, which blocks cerebellar DSI, did not affect CA1 DSI. Group I and group III mGluR agonists reduced IPSCs, but only group I agonists occluded DSI. (S)-MCPG blocked (1S,3R)-ACPD-induced IPSC suppression and markedly reduced DSI, whereas group III antagonists had no effect on DSI. Many other similarities between DSI and the (1S,3R)-ACPD-induced suppression of IPSCs also were found. Our data suggest that a glutamate-like substance released from pyramidal cells could mediate CA1 DSI by reducing GABA release from interneurons via the activation of group I mGluRs.

4-Aminopyridine↗
PubMed2024

Hepatitis B Virus-KMT2B Integration Drives Hepatic Oncogenic Processes in a Human Gene-edited Induced Pluripotent Stem Cells-derived Model.

BACKGROUND & AIMS: Hepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC; however, its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cell (iPSCs) model. METHODS: Based on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps). RESULTS: The reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation (H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function. CONCLUSIONS: Our disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.

Humans↗
PubMed1992

Development of GABAergic connections in vitro: increasing efficacy of synaptic transmission is not accompanied by changes in miniature currents.

Development of inhibitory synaptic transmission was studied using a dissociated cell culture from the superior colliculus of neonatal rat. Patch-clamp recordings in the whole-cell configuration were performed to measure evoked (single-cell-activated) inhibitory postsynaptic currents (IPSCs), miniature IPSCs and current responses to maximal concentrations of exogenous gamma-aminobutyric acid (GABA). Over a period of 3 weeks in vitro (DIV3-24), the fraction of synaptically coupled neurons raised from 0% to 76%. Evoked IPSCs were first observed at DIV5. They had an average amplitude of 33.9 pA during the first week (n = 13) and 129.7 pA during the fourth week (n = 48). This increase by a factor of 3.8 represents a significant rise in the efficacy of GABAergic transmission during in vitro development. However, no developmental change has been observed in the average amplitudes of miniature somatic IPSCs. The latter remained at an average level of about 9 pA (symmetrical chloride concentration and a driving force of 68 mV). No increase was found also in whole-cell current densities induced by saturating concentrations of exogenous GABA. Our results suggest that under the given conditions, synapse maturation was primarily the result of presynaptic sprouting. This conclusion is further supported by bouton counts in immunostained collicular cultures, where the number of axosomatic and axodendritic GABAergic contacts per neuron increased from 0.54 and 0.37, respectively, at DIV3, to 13.84 and greater than 23.1, at DIV24. The overall density of GABAergic neurons decreased during this period from about 41,000/cm2 to 15,600 cm2, indicating that a growing number of contacts is formed by a declining number of presynaptic neurons.

Animals↗
PubMed2005

Possible role of mitochondria in posttetanic potentiation of GABAergic synaptic transmission in rat neocortical cell cultures.

It has been previously demonstrated that mitochondria are of crucial importance for posttetanic potentiation (PTP) at neuromuscular junction. The aim of our study was to examine whether this may also be the case at a central synapse. To address this question, we studied possible mitochondrial involvement in PTP of GABAergic synaptic transmission in rat neocortical cultures, a preparation in which PTP has not been previously documented. Synaptic responses were evoked by local extracellular stimulation. Whole-cell patch-clamp technique was employed to record inhibitory postsynaptic currents (IPSCs) from postsynaptic neurons. Tetanic stimulation (30 Hz, 4 s) of the presynaptic neuron evoked an increase of IPSC amplitude, lasting for about 1 min. PTP was accompanied by a decrease of coefficient of variation of the IPSC and a decrease of paired-pulse (IPSC(2)/IPSC(1)) ratio, indicating involvement of presynaptic mechanism(s) in PTP. Possible role of mitochondria in PTP was addressed using drugs affecting Ca(2+) uptake and subsequent Ca(2+) efflux: carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and tetraphenylphosphonium ions (TPP(+)). It was found that both CCCP (1-2 microM) and TPP(+) (10 microM) either substantially decreased or eliminated PTP. These results further confirm presynaptic origin of PTP in neocortical neurons and suggest an important role of mitochondrial Ca(2+) turnover in this form of synaptic plasticity at the central synapse.

Animals↗