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PubMed2026

The Fragile Site Landscape of Induced Pluripotent Stem Cells: Hierarchy, Variability, Tissue Specificity, and Links to Culture-Acquired Rearrangements.

Induced pluripotent stem cells (iPSCs) are prone to genomic instability during prolonged culture, with recurrent chromosomal aberrations conferring selective advantages. Replication stress is a major driver of this instability, yet the repertoire of replication stress-sensitive loci in iPSCs remains largely unexplored. Here, we mapped aphidicolin-sensitive fragile sites (asFS) in three independent iPSC lines using classical cytogenetic break analysis combined with Monte Carlo simulation and MiDAS mapping directly on banded metaphase chromosomes. We identified 28 asFS, which segregated into a highly active Major cluster (8 sites, accounting for 59% of breaks among asFS) and a less active Minor cluster (20 sites). Five universal asFS (9p21, 6q25-26, 20p11-12, 10q22, Xq25) were present in all three lines, representing a fragility signature associated with the pluripotent state, with Xq25 shifting into the Major cluster after correction for X chromosome dosage. Minor asFS showed preferential co-localization with physical breakpoints or minimal overlapping regions of recurrent culture-acquired aberrations, including 20q11.21 (BCL2L1), 1q32 (MDM4), 8q24 (MYC), 17q21 (WNT3-WNT9B), and 18q21 (DCC/FRA18B). MiDAS mapping validated most asFS and revealed additional replication stress-sensitive loci in pericentromeric and subtelomeric regions that are difficult to score by conventional G-banding. Comparison with fragile site maps from other cell types revealed that the iPSC asFS repertoire is distinct in rank order and relative activity, characteristic of the pluripotent state. Collectively, our findings indicate that the asFS repertoire in iPSCs is hierarchically organized into a stable universal core and a variable peripheral component, and suggest that Minor asFS may contribute to, or be associated with, the genesis of culture-acquired rearrangements. This work provides a framework for understanding how replication stress and clonal selection shape the mutational landscape of pluripotent stem cells.

Induced Pluripotent Stem Cells↗
PubMed1999

Peptides that mimic the carboxy-terminal domain of SNAP-25 block acetylcholine release at an Aplysia synapse.

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release, presumably by cleaving SNAP-25, a protein involved in docking of synaptic vesicles with the presynaptic plasma membrane. Three excitation-secretion uncoupling peptides (ESUPs), which mimic the carboxy-terminal domain of SNAP-25 and span or adjoin the cleavage sites for BoNT/A and BoNT/E, also inhibit transmitter release from permeabilized bovine chromaffin cells. In this study, these peptides were tested for effects on acetylcholine (ACh) release at an identified cholinergic synapse in isolated buccal ganglia of Aplysia californica. The presynaptic neuron was stimulated electrically to elicit action potentials. The postsynaptic neuron was voltage-clamped, and evoked inhibitory postsynaptic currents (IPSCs) were recorded. The ESUPs were pressure-injected into the presynaptic neuron, and their effects on the amplitude of the IPSCs were studied. Acetylcholine release from presynaptic cells, as measured by IPSC amplitudes, was gradually inhibited by the ESUPs. All three peptides caused ca. 40% reduction in IPSC amplitude in 2 h. Random-sequence peptides of the same amino acid composition had no effect. Injection of BoNT/E, in contrast, caused ca. 50% reduction in IPSC amplitude in 30 min and almost complete inhibition in 2 h. These results are the first demonstration that ESUPs block neuronal cholinergic synaptic transmission. They are consistent with the concept that ESUPs compete with the intact SNAP-25 for binding with other fusion proteins, thus inhibiting stimulus-evoked exocytosis of neurotransmitter.

Acetylcholine↗
PubMed1994

Enhancement by serotonin of GABA-mediated inhibitory synaptic currents in rat cerebellar Purkinje cells.

The actions of serotonin (5-HT) on synaptic responses in Purkinje cells were examined using the whole-cell voltage-clamp recording from thin slices of the rat cerebellum. Focal stimulation within the molecular layer elicited synaptic responses: (1) inhibitory postsynaptic currents (IPSCs) that were completely blocked by bicuculline; (2) excitatory postsynaptic currents (EPSCs) that were antagonized by CNQX; and (3) combination of IPSC and EPSC. Application of 5-HT (3-30 microM) increased the amplitude of the stimulation-evoked IPSCs but did not significantly affect the EPSCs. The frequency and the mean amplitude of spontaneous IPSCs were increased in the presence of 5-HT. Neither the sensitivity to exogenously applied GABA nor the holding current of Purkinje cells were noticeably altered by 5-HT. The results indicate that 5-HT differentially enhances the GABA-mediated IPSCs in rat cerebellar Purkinje cells via a presynaptic mechanism. Serotonergic neurons thus appear to play a role in the motor control through selective presynaptic facilitation that modulates the output from the cerebellar cortex.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2004

Multiple metabotropic glutamate receptor subtypes modulate GABAergic neurotransmission in rat periaqueductal grey neurons in vitro.

The effect of metabotropic glutamate receptor (mGluR) activation on GABAergic synaptic transmission in rat periaqueductal grey (PAG) neurons was examined using whole-cell patch-clamp recordings in brain slices. The selective groups I, II and III mGluR agonists DHPG (10-30 microM), DCG-IV (1-3 microM) and L-AP4 (10-30 microM) inhibited electrically evoked GABA(A) mediated inhibitory postsynaptic currents (IPSCs) in all PAG neurons tested. DCG-IV and L-AP4 also reduced the frequency of spontaneous IPSCs, while DHPG produced both increases and decreases in spontaneous IPSC frequency in a dose dependent manner. In the presence of TTX, DHPG, DCG-IV and L-AP4 all reduced the frequency of spontaneous miniature IPSCs, but had no effect on their amplitudes. The DHPG, DCG-IV and L-AP4 effects on miniature IPSCs were dose dependent (EC(50)s=1.4, 0.055 and 0.52 microM, respectively) and were reduced by the selective mGluR antagonists MCPG, EGLU and MSOP, respectively. These results indicate that GABAergic synaptic transmission within the PAG is reduced by groups I, II and III mGluR activation via a presynaptic mechanism and is increased by group I mGluR activation via an action potential dependent mechanism. The finding of convergent groups I, II and III mGluR-mediated inhibition of synaptic transmission is novel and indicates that all groups of mGluRs have the potential to modulate the constellation of analgesic, behavioural and autonomic functions within the PAG.

Animals↗
PubMed1994

Variance analysis of gamma-aminobutyric acid (GABA)-ergic inhibitory postsynaptic currents from melanotropes of Xenopus laevis.

We have studied the variance in the decay of large spontaneous gamma-aminobutyric acid (GABA)-ergic inhibitory postsynaptic currents (IPSCs) in melanotropes of Xenopus laevis to obtain information about the number of GABAA receptor channels that bind GABA during the IPSCs. The average decay of the IPSCs is well described by the sum of two exponential functions. This suggests that a three-state Markov model is sufficient to describe the decay phase, with one of the three states being an absorbing state, entered when GABA dissociates from the GABAA receptor. We have compared the variance in the decay of large spontaneous IPSCs with the variance calculated for two different three-state models: a model with one open state, one closed state, and one absorbing state (I), and a model with two open states and one absorbing state (II). The data were better described by the more efficient model II. This suggests that the efficacy of GABA at synaptic GABAA receptor channels is high and that only a small number of channels are involved in generating the GABA-ergic IPSCs.

Analysis of Variance↗
PubMed1997

Vasoactive intestinal polypeptide enhances the GABAergic synaptic transmission in cultured hippocampal neurons.

The whole-cell mode of patch-clamp techniques was used to investigate the effect of vasoactive intestinal polypeptide (VIP) on spontaneous gamma-aminobutyric acid (GABA)-mediated inhibitory postsynaptic currents (IPSCs) of cultured hippocampal neurons. Application of VIP caused a significant increase in the frequency of spontaneous IPSCs with a reversible and dose-dependent manner. VIP had no effect on the mean amplitude and kinetic parameters of spontaneous IPSCs. In the presence of tetrodotoxin, VIP increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs) without affecting their mean magnitude. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, mimicked the stimulatory effect of VIP on spontaneous IPSCs and mIPSCs. VIP and forskolin failed to modulate GABAergic IPSCs in the presence of Rp-cAMPs, a cell permeable antagonist of cAMP-dependent protein kinase (PKA). Calcium channel blocker CdCl2 did not prevent VIP and forskolin from increasing the frequency of mIPSCs. These results suggest that the activation of presynaptic VIP receptor enhances the GABAergic synaptic transmission in cultured hippocampal neurons through the cAMP-PKA pathway and that VIP is likely to increase GABA release by directly stimulating the vesicular release apparatus.

2-Amino-5-phosphonovalerate↗
PubMed2001

Modulation of miniature inhibitory postsynaptic currents by isoflurane in rat dissociated neurons with glycinergic synaptic boutons.

The effects of a volatile anesthetic, isoflurane, on glycinergic miniature inhibitory postsynaptic currents (IPSCs) were investigated in mechanically dissociated rat trigeminal nucleus neurons with intact glycinergic interneuronal presynaptic nerve terminals. The nystatin-perforated patch recording configuration was used to record the miniature IPSCs under voltage-clamp conditions. Isoflurane shifted in a parallel fashion the glycine (Gly) concentration-response curve of enzymatically dissociated neurons to the left without changing the maximum response. Isoflurane reversibly increased the frequency of the miniature IPSCs and prolonged the decay time constant without affecting the mean amplitude. The increase in the frequency of miniature IPSCs in the presence of isoflurane was also observed in Ca(2+)-free external solution. Thapsigargin prohibited the facilitatory effect of isoflurane on the miniature IPSC frequency. It is concluded that isoflurane increases the Ca(2+) concentration in the glycinergic presynaptic nerve terminal by enhancing the release and/or suppressing the uptake of Ca(2+) into stores.

Anesthetics, Inhalation↗
PubMed2002

Presynaptic mu-opioid receptors regulate a late step of the secretory process in rat ventral tegmental area GABAergic neurons.

Gamma-aminobutyric acid (GABA)-containing interneurons of the ventral tegmental area (VTA) regulate the activity of dopaminergic neurons. These GABAergic interneurons are known to be innervated by synaptic terminals containing enkephalin, an endogenous ligand of mu-opioid receptors. Bath application of mu-opioid receptor agonists inhibits the activity of VTA GABAergic neurons but the mechanism whereby mu-opioid receptors regulate synaptic GABA release from these neurons has not been directly identified. Using cultured VTA neurons we have confirmed that mu-opioid receptor agonists inhibit synaptic GABA release. DAMGO, a selective mu-opioid receptor agonist, had four distinct effects on GABAergic IPSCs: (1) it inhibited the frequency and amplitude of spontaneous IPSCs (sIPSCs), (2) it reduced the amplitude of IPSCs evoked by single action potentials, (3) it inhibited the frequency, but not the amplitude of miniature IPSCs (mIPSCs), and (4) DAMGO inhibited mIPSCs evoked by ionomycin, a Ca(2+) ionophore. The inhibition of action potential-evoked IPSCs and of spontaneous and ionomycin-evoked mIPSCs by DAMGO was prevented by the K(+) channel blocker, 4-aminopyridine (4-AP). In conclusion, our work shows that one of the mechanisms through which mu-opioid receptors inhibit GABA release by VTA neurons is through inhibition of the secretory process at the nerve terminal level. In addition, considering that ionomycin stimulates exocytosis through a mechanism that should be insensitive to membrane polarization, our experiments with 4-AP suggest that K(+) channels are implicated in the inhibition of the efficacy of the secretory process by mu-opioid receptors.

Animals↗
PubMed2002

Furosemide reveals heterogeneous GABA(A) receptor expression at adult rat Golgi cell to granule cell synapses.

The contribution that alpha6 subunit-containing GABA(A) receptors make to inhibitory synaptic transmission to granule cells was investigated by making whole-cell patch clamp recordings from granule cells in adult rat cerebellar slices and applying furosemide, the specific alpha6 subunit-containing GABA(A) receptor antagonist. Endogenous, extracellular GABA continually activated GABA(A) receptors producing a tonic current. Since this current was markedly reduced by furosemide it was probably produced by alpha6 subunit-containing receptors. In contrast, furosemide had little effect on the amplitude or kinetics of fast spontaneous inhibitory postsynaptic currents (sIPSCs), although such sIPSCs were abolished by bicuculline and SR95331. However, the amplitude of evoked IPSCs with a very slow rise and decay were markedly reduced by furosemide. These IPSCs probably resulted from the spillover of GABA from neighbouring synapses activating high affinity alpha6 subunit-containing receptors. In the rest of the cells (40 out of 46), evoked IPSCs had rise and decay kinetics that lay in-between fast sIPSCs and slow 'spillover' IPSCs. Such IPSCs had variable kinetics and also exhibited considerable variation in the magnitude of furosemide block. Thus the GABA(A) receptors present at adult Golgi cell-granule cell synapses, at a developmental stage where receptor expression is complete, are highly heterogeneous.

Animals↗
PubMed2002

Pharmacological separation of cannabinoid sensitive receptors on hippocampal excitatory and inhibitory fibers.

Our earlier studies demonstrated that in the hippocampus, cannabinoids suppress inhibitory synaptic transmission via CB(1) cannabinoid receptors, whereas a novel cannabinoid-sensitive receptor modulates excitatory synapses (Katona, I. et al., Journal of Neuroscience 19 (1999) 4544; Hájos, N. et al., European Journal of Neuroscience 12 (2000) 3239; Hájos, N. et al., Neuroscience 106 (2001) 1). The novel receptor does not correspond to CB(2), since this receptor type is not expressed in the brain (Munro, S. et al., Nature 365 (1993) 61). Recent binding experiments revealed that the synthetic cannabinoid WIN 55,212-2 binds with lower affinity to brain membranes of CB(1) receptor-knockout mice indicating that pharmacological differences exist between these two types of cannabinoid receptors in the hippocampus (Breivogel et al., Molecular Pharmacology 60 (2001) 155). To analyze this difference in detail, we first determined the EC(50) values of WIN 55,212-2 for excitatory and inhibitory transmission in rat hippocampal slices using whole-cell patch-clamp recordings. The estimated EC(50) value for inhibitory postsynaptic currents (IPSC) evoked by electrical stimulation in CA1 pyramidal cells was 0.24 microM, whereas for excitatory postsynaptic currents (EPSC) it was 2.01 microM, respectively. The cannabinoid antagonist, AM251, blocked the WIN 55,212-2-induced inhibition of evoked IPSCs, but not of EPSCs, providing evidence for its selectivity for CB(1). We then tested the hypothesis of whether the cannabinoid effect on hippocampal excitatory neurotransmission is mediated via receptors with an affinity for vanilloid ligands. Co-application of the vanilloid receptor antagonist capsazepine (10 microM) with cannabinoids (WIN55,212-2 or CP55,940) prevented the reduction of EPSCs, but not of IPSCs. The amplitude of evoked EPSCs was also suppressed by superfusion of the vanilloid receptor agonist capsaicin (10 microM), an effect which could also be antagonized by capsazepine. In contrast, capsaicin did not change the amplitude of evoked IPSCs. These results demonstrate that WIN 55,212-2 is an order of magnitude more potent in reducing GABAergic currents via CB(1) than in inhibiting glutamatergic transmission via the new CB receptor. The sensitivity of the new CB receptor (and EPSCs) to vanilloid ligands, but not to the cannabinoid antagonist AM251, represents another pharmacological tool to distinguish the two receptors, since CB(1) (and its effect on IPSCs) is not modulated by vanilloids, but is antagonized by AM251.

Animals↗
PubMed2002

Pre- and postsynaptic properties of somatic and dendritic inhibition in dentate gyrus.

We compared somatic and dendritic inhibition in paired recordings from two classes of anatomically identified interneurons and granule cells of the dentate gyrus. Inhibitory postsynaptic current (IPSC) amplitude and decay were remarkably similar at somatic and dendritic synapses. Slower IPSC rise times and longer latencies at dendritic synapses were consistent with their distal location, without requiring differences in postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptor properties. In contrast, higher transmission failure rate and greater paired-pulse depression at dendritic synapses suggest that somatic and dendritic inhibition differ in presynaptic properties. Cholinergic input has been suggested to modulate hippocampal rhythmicity as well as episodic memory function. We therefore tested the effects of acetylcholine (ACh) on paired IPSCs and on spontaneous synaptic activity in interneurons and granule cells. We found no effect of ACh on paired IPSCs; however, spontaneous IPSCs recorded in granule cells were enhanced in amplitude and frequency. ACh potentiated spontaneous excitatory postsynaptic potentials (sEPSPs) and induced spiking in both types of interneuron, and preferentially increased sEPSP frequency in dendritic interneurons. Our findings suggest that patterns of activity in the two classes of interneurons, coupled with differences in their presynaptic properties, are likely to determine the roles of somatic and dendritic inhibition in network function.

Acetylcholine↗
PubMed1998

Morphological and electrical characteristics of postnatal hippocampal neurons in culture: the presence of bicuculline- and strychnine-sensitive IPSPs.

A modified method was developed for tissue-culturing postnatal hippocampal neurons using simple mechanical trituration for cell isolation and not including any hydrolysing enzymes, nerve growth factors or antiproliferating agents. The morphological properties of such neurons were characterized with light and interference polarizing microscopy, which revealed the appearance of growth cones from peripheral neurons and the presence of different types of neurons, including bipolar, stellate and pyramidal-like cells (i.e., pyramidal and dentate gyrus granule cells), which could be related to their putative counterparts in intact brain. The whole-cell configuration of the patch-clamp method was used for electrophysiological recordings of inhibitory synapses between these dissociated cultured neurons from the early postnatal rat hippocampus. This study indicated the presence of tetrodotoxin (TTX)-sensitive and TTX-resistant inhibitory postsynaptic potential (IPSPs) and inhibitory postsynaptic currents (IPSCs) in current-clamp and voltage clamp modes respectively. The coincident reversal potentials for IPSCs and for GABAA and glycine-evoked currents, and the sensitivity of the IPSCs to bicuculline or strychnine, indicated that these IPSCs were Cl-(-)dependent and mediated by either GABAA or glycine receptors. Inhibitory postsynaptic currents recorded under voltage-clamp conditions decayed with a time course that could be fitted by a single exponential with a value of 26 ms. An average quantal content of 2.5 was responsible for a typical GABA and glycine-activated IPSC and a single quantum for GABAergic input was inferred to activate about 160, and for glycinergic, about 200 Cl-, channels.

Action Potentials↗
PubMed2001

Spontaneously active GABAergic interneurons in the subfornical organ of rat slice preparations.

Inhibitory postsynaptic currents (IPSCs) were recorded from subfornical organ (SFO) neurons in slice preparations of rats, using whole-cell voltage clamp techniques. Some SFO neurons showed bimodal distributions in amplitude with the large and small IPSCs. The large IPSCs vanished in the tetrodotoxin perfusion medium, but the small did not. Both sizes of the IPSCs were completely abolished by application of bicuculline and picrotoxin. Further subpopulation of SFO neurons with the bimodal distributions showed intermittent bursts of the large IPSCs. Immunohistochemical approach revealed existence of gamma-aminobutyric acid (GABA)-immunoreactive neurons and axons in the SFO. These suggest that spontaneously-active and intermittently-burst-firing GABA interneurons affect other SFO neurons in slice preparations of rats.

Animals↗
PubMed2003

Spinal inhibitory synaptic transmission in the glycine receptor mouse mutant spastic.

Inhibitory glycine receptor (GlyR) and GABA(A) receptor (GABA(A)R)-mediated synaptic transmission was examined in two strains of the GlyR mutant mouse spastic and the respective wild types. The mutants display a mild and a severe neurological phenotype. Electrically evoked postsynaptic whole-cell currents were recorded from alpha-motoneurons in lumbar spinal cord slices. Amplitudes of GlyR-mediated IPSCs were significantly reduced in the severe phenotype in comparison to the respective wild type and the mild phenotype mutants. Surprisingly, amplitudes of GABA(A)R-mediated IPSCs were also significantly reduced in both mutants. Fast time constants of the decay phase of IPSCs were slightly reduced for the GlyR-mediated IPSCs and significantly larger for the GABA(A)R-mediated IPSCs in both mutant strains.

Animals↗
PubMed2000

Preferential potentiation by nitric oxide of spontaneous inhibitory postsynaptic currents in rat supraoptic neurones.

Magnocellular neurones in the supraoptic nucleus and paraventricular nucleus express mRNA for nitric oxide synthase (NOS) and the expression becomes more prominent when the release of vasopressin or oxytocin is stimulated. It has also been reported that NO donors inhibit the electrical activity of supraoptic nucleus neurones, but the mechanism involved in the inhibition remains unclear. In the present study, to know whether modulation of synaptic inputs into supraoptic neurones is involved in the inhibitory effect of NO, we measured spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) from rat supraoptic nucleus neurones in slice preparations identified under a microscope using the whole-cell mode of the slice-patch-clamp technique. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reversibly increased the frequency of spontaneous IPSCs mediated by GABAA receptors, without affecting the amplitude, indicating that NO potentiated IPSCs via a presynaptic mechanism. The NO scavenger, haemoglobin, suppressed the potentiation of IPSCs by SNAP. On the other hand, SNAP did not cause significant effects on EPSCs mediated by non-NMDA glutamate receptors. The membrane permeable analogue of cGMP, 8-bromo cGMP, caused a significant reduction in the frequency and amplitude of both IPSCs and EPSCs. The results suggest that NO preferentially potentiates the inhibitory synaptic inputs into supraoptic nucleus neurones by acting on GABA terminals in the supraoptic nucleus, possibly via a cGMP-independent mechanism. The potentiation may, at least in part, account for the inhibitory action of NO on the neural activity of supraoptic neurones.

Animals↗
PubMed2000

Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations.

Using a new antibody developed against the C-terminus of the cannabinoid receptor (CB1), the immunostaining in the hippocampus revealed additional axon terminals relative to the pattern reported previously with an N-terminus antibody. Due to a greater sensitivity of this antibody, a large proportion of boutons in the dendritic layers displaying symmetrical (GABAergic) synapses were also strongly immunoreactive for CB1 receptors, as were axon terminals of perisomatic inhibitory cells containing cholecystokinin. Asymmetrical (glutamatergic) synapses, however, were always negative for CB1. To investigate the effect of presynaptic CB1 receptor activation on hippocampal inhibition, we recorded inhibitory postsynaptic currents (IPSCs) from principal cells. Bath application of CB1 receptor agonists (WIN55,212-2 and CP55,940) suppressed IPSCs evoked by local electrical stimulation, which could be prevented or reversed by the CB1 receptor antagonist SR141716A. Action potential-driven IPSCs, evoked by pharmacological stimulation of a subset of interneurons, were also decreased by CB1 receptor activation. We also examined the effects of CB1 receptor agonists on Ca2+-independent miniature IPSCs (mIPSC). Both agonists were without significant effect on the frequency or amplitude of mIPSCs. Synchronous gamma oscillations induced by kainic acid in the CA3 region of hippocampal slices were reversibly reduced in amplitude by the CB1 receptor agonist CP 55,940, which is consistent with an action on IPSCs. We used CB1-/- knock-out mice to confirm the specificity of the antibody and of the agonist (WIN55,212-2) action. We conclude that activation of presynaptic CB1 receptors decreases Ca2+-dependent GABA release, and thereby reduces the power of hippocampal network oscillations.

Action Potentials↗
PubMed2000

Oxytocin receptor agonists enhance inhibitory synaptic transmission in the rat hippocampus by activating interneurons in stratum pyramidale.

Oxytocin probably plays a role as a neurotransmitter/neuromodulator in the hippocampus of the rat. Oxytocin binding sites are present in the subiculum and CA1 region and oxytocin can excite a class of CA1 nonpyramidal neurons. In the present work we characterized the effect of oxytocin on hippocampal synaptic transmission. Whole-cell recordings were obtained from pyramidal neurons, in conditions of nearly symmetrical chloride concentrations. The selective oxytocin receptor agonist, [Thr4,Gly7]-oxytocin (TGOT), caused an increase in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in virtually all neurons. These peptide-enhanced IPSCs were blocked by bicuculline, but not by strychnine, and reversed near 0 mV, indicating that they were mediated by gamma-aminobutyric acid (GABA)A receptors. On average, TGOT caused a nearly threefold increase in the frequency and almost a doubling in the amplitude of spontaneous IPSCs. TGOT did not influence the frequency and the amplitude of miniature IPSCs or spontaneous excitatory postsynaptic currents (EPSCs), and had no effect on evoked IPSCs. The peptide did not affect the basic membrane properties of pyramidal neurons or their GABA sensitivity. Thus, TGOT facilitated inhibitory transmission by exerting an excitatory action on the soma and/or dendrites of GABAergic interneurons. Extracellular recordings were performed in interneurons located in various hippocampal strata. Their sensitivity to TGOT was compared to that of substance P (SP). Interneurons in stratum pyramidale were excited both by TGOT and by SP. By contrast, stratum radiatum interneurons responded to SP but not to TGOT. In stratum oriens, half of the interneurons responded to SP, but only a minority to TGOT. Thus, oxytocin-responsive interneurons appear to be preferentially located in close vicinity of pyramidal neurons.

Animals↗
PubMed2002

Potentiation of inhibitory glycinergic neurotransmission by Zn2+: a synergistic interplay between presynaptic P2X2 and postsynaptic glycine receptors.

The divalent cation zinc is known to modulate chloride currents carried by native and recombinant mammalian glycine receptors (GlyRs). To unravel the effect of Zn2+ on glycinergic neurotransmission, inhibitory postsynaptic currents (IPSC) of rat spinal neurons grown in culture were analysed in the absence and presence of Zn2+. Low concentrations of Zn2+ (0.5 and 5 micro m) augmented the mean amplitude of miniature IPSCs by approximately 40% over values obtained in the absence of zinc, whereas higher concentrations of Zn2+ (50 micro m) significantly decreased mean amplitude values. Remarkably, low concentrations of Zn2+ also significantly increased the mean frequency of miniature IPSCs. This effect was blocked by the P2X receptor antagonists PPADS and suramin, indicating the presence of Zn2+-sensitive presynaptic P2X receptors on glycinergic terminals. Immunostaining with antibodies against different P2X receptor subtypes revealed that P2X2 receptors partially colocalize with the GlyR. Potentiating concentrations of Zn2+ also affected the kinetics of miniature and evoked IPSCs by significantly prolonging their decay time constants. Electrophysiological analysis of heterologously expressed glycine transporters (GlyT) revealed for GlyT2 zero, and for GlyT1 a modest (< 20%), reduction of glycine uptake in the presence of 5 micro m Zn2+, indicating that prolongation of glycinergic IPSCs by Zn2+ is not due to inhibition of glycine removal from the synaptic cleft. Together, these results suggest that Zn2+ is a potent modulator of glycinergic synaptic transmission which increases in a synergistic manner the agonist affinity of both presynaptic P2X2 receptors and postsynaptic GlyRs.

Adenosine Triphosphate↗