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PubMed2006

Neuregulin effect on quantal content dissociated from effect on miniature endplate potential amplitude.

Members of the neuregulin family of signaling proteins increase transcription of acetylcholine receptor (AChR) subunit genes in muscle fibers and the number of AChRs in the muscle membrane. In adult mice heterozygous for targeted deletion of type I neuregulins (Ig-NRG(+/-)), postsynaptic AChR density was decreased and transmitter release was increased. We examined the relationship between functional AChR density and ACh release in postnatal day 7 (P7), P14, and adult NRG-deficient mice. Here we report that changes in postsynaptic sensitivity and transmitter release are not temporally coupled during postnatal development in Ig-NRG-deficient mice. Although miniature endplate potential (MEPP) amplitude was decreased compared with control in P7 Ig-NRG(+/-) mice, quantum content was not increased. Quantum content was increased in adult heterozygotes despite normal MEPP amplitudes. Thus, during postnatal maturation, both quantal size and quantum content were influenced by decreased Ig-NRG expression, although the effects were dissociated in time.

Animals↗
PubMed2002

Giant miniature EPSCs at the hippocampal mossy fiber to CA3 pyramidal cell synapse are monoquantal.

The mechanisms generating giant miniature excitatory postsynaptic currents (mEPSCs) were investigated at the hippocampal mossy fiber (MF) to CA3 pyramidal cell synapse in vitro. These giant mEPSCs have peak amplitudes as large as 1,700 pA (13.6 nS) with a mean maximal mEPSC amplitude of 366 +/- 20 pA (mean +/- SD; 5 nS; n = 25 cells). This is compared with maximal mEPSC amplitudes of <100 pA typically observed at other cortical synapses. We tested the hypothesis that giant mEPSCs are due to synchronized release of multiple vesicles across the release sites of single MF boutons by directly inducing vesicular release using secretagogues. If giant mEPSCs result from simultaneous multivesicular release, then secretagogues should increase the frequency of small mEPSCs selectively. We found that hypertonic sucrose and spermine increased the frequency of both small and giant mEPSCs. The peptide toxin secretagogues alpha-latrotoxin and pardaxin failed to increase the frequency of giant mEPSCs, but the possible lack of tissue penetration of the toxins make these results equivocal. Because a multiquantal release mechanism is likely to be mediated by a spontaneous increase in presynaptic calcium concentration, a monoquantal mechanism is further supported by results that giant mEPSCs were not affected by manipulations of extracellular or intracellular calcium concentrations. In addition, reducing the temperature of the bath to 15 degrees C failed to desynchronize the rising phases of giant mEPSCs. Together these data suggest that the giant mEPSCs are generated via a monovesicular mechanism. Three-dimensional analysis through serial electron microscopy of the MF boutons revealed large clear vesicles (50 to 160 nm diam) docked presynaptically at the MF synapse in sufficient numbers to account for the amplitude and frequency of giant mEPSCs recorded electrophysiologically. It is concluded that release of the contents of a single large clear vesicle generates giant mEPSCs at the MF to CA3 pyramidal cell synapse.

Animals↗
PubMed2003

Effect of adrenalectomy on miniature inhibitory postsynaptic currents in the paraventricular nucleus of the hypothalamus.

Within the rat paraventricular nucleus of the hypothalamus two types of neurons have been distinguished based on morphological appearance, i.e., parvocellular and magnocellular neurons. The parvocellular neurons play a key role in regulating the activity of the hypothalamo-pituitary-adrenal axis, which is activated, e.g., after stress exposure. These neurons receive humoral negative feedback via the adrenal hormone corticosterone but also neuronal inhibitory input, either directly or transsynaptically relayed via GABAergic interneurons. In the present study we examined to what extent the neuronal GABAergic input is influenced by the humoral signal. To this end, miniature inhibitory postsynaptic currents (mIPSCs) were recorded in parvo- and magnocellular neurons of adrenalectomized rats, which lack corticosterone, and in sham-operated controls. Under visual control neurons in coronal slices containing the paraventricular nucleus were designated as putative parvocellular or magnocellular neurons: the former were located in the medial part of the nucleus and displayed a small fusiform soma; the latter were mostly located in the lateral part and were recognized by their large round soma. Compared with putative magnocellular neurons, parvocellular neurons generally exhibited a lower membrane capacitance, lower mIPSC frequency, and smaller mIPSC amplitude. Following adrenalectomy, the mIPSC frequency was significantly enhanced in parvo- but not magnocellular neurons. Other properties of the cells were not affected. In a second series of experiments we examined whether the increase in mIPSC frequency was due to the absence of corticosterone or caused by other effects related to adrenalectomy. The data support the former explanation since implantation of a corticosterone releasing pellet after adrenalectomy fully prevented the change in mIPSC frequency. We conclude that, in the absence of humoral negative feedback, local GABAergic input of parvocellular neurons in the paraventricular nucleus is enhanced. This may provide a compensatory mechanism necessary for maintaining controllable network activity.

Adrenal Cortex Hormones↗
PubMed2006

Agonist-dependent postsynaptic effects of opioids on miniature excitatory postsynaptic currents in cultured hippocampal neurons.

Although chronic treatment with morphine is known to alter the function and morphology of excitatory synapses, the effects of other opioids on these synapses are not clear. Here we report distinct effects of several opioids (morphine, [d-ala(2),me-phe(4),gly(5)-ol]enkephalin (DAMGO), and etorphine) on miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons: 1) chronic treatment with morphine for >3 days decreased the amplitude, frequency, rise time and decay time of mEPSCs. In contrast, "internalizing" opioids such as etorphine and DAMGO increased the frequency of mEPSCs and had no significant effect on the amplitude and kinetics of mEPSCs. These results demonstrate that different opioids can have distinct effects on the function of excitatory synapses. 2) mu opioid receptor fused with green fluorescence protein (MOR-GFP) is clustered in dendritic spines in most hippocampal neurons but is concentrated in axon-like processes in striatal and corticostriatal nonspiny neurons. It suggests that MORs might mediate pre- or postsynaptic effects depending on cell types. 3) Neurons were cultured from MOR knock-out mice and were exogenously transfected with MOR-GFP. Chronic treatment with morphine suppressed mEPSCs only in neurons that contained postsynaptic MOR-GFP, indicating that opioids can modulate excitatory synaptic transmission postsynaptically. 4) Morphine acutely decreased mEPSC amplitude in neurons expressing exogenous MOR-GFP but had no effect on neurons expressing GFP. It indicates that the low level of endogenous MORs could only allow slow opioid-induced plasticity of excitatory synapses under normal conditions. 5) A theoretical model suggests that morphine might affect the function of spines by decreasing the electrotonic distance from synaptic inputs to the soma.

Analgesics, Opioid↗
PubMed2002

Prolongation of hippocampal miniature inhibitory postsynaptic currents in mice lacking the GABA(A) receptor alpha1 subunit.

GABA(A) receptors (GABA(A)-Rs) are pentameric structures consisting of two alpha, two beta, and one gamma subunit. The alpha subunit influences agonist efficacy, benzodiazepine pharmacology, and kinetics of activation/deactivation. To investigate the contribution of the alpha1 subunit to native GABA(A)-Rs, we analyzed miniature inhibitory postsynaptic currents (mIPSCs) in CA1 hippocampal pyramidal cells and interneurons from wild-type (WT) and alpha1 subunit knock-out (alpha1 KO) mice. mIPSCs recorded from interneurons and pyramidal cells obtained from alpha1 KO mice were detected less frequently, were smaller in amplitude, and decayed more slowly than mIPSCs recorded in neurons from WT mice. The effect of zolpidem was examined in view of its reported selectivity for receptors containing the alpha1 subunit. In interneurons and pyramidal cells from WT mice, zolpidem significantly increased mIPSC frequency, prolonged mIPSC decay, and increased mIPSC amplitude; those effects were diminished or absent in neurons from alpha1 KO mice. Nonstationary fluctuation analysis of mIPSCs indicated that the zolpidem-induced increase in mIPSC amplitude was associated with an increase in the number of open receptors rather than a change in the unitary conductance of individual channels. These data indicate that the alpha1 subunit is present at synapses on WT interneurons and pyramidal cells, although differences in mIPSC decay times and zolpidem sensitivity suggest that the degree to which the alpha1 subunit is functionally expressed at synapses on CA1 interneurons may be greater than that at synapses on CA1 pyramidal cells.

Animals↗
PubMed2006

Development of spontaneous miniature EPSCs in mouse AVCN neurons during a critical period of afferent-dependent neuron survival.

During a critical period prior to hearing onset, cochlea ablation leads to massive neuronal death in the mouse anteroventral cochlear nucleus (AVCN), where cell survival is believed to depend on glutamatergic input. We investigated the development of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in AVCN neurons using whole cell patch-clamp techniques during [postnatal day 7 (P7)] and after (P14, P21) this critical period. We also examined the effects of unilateral cochlea ablation on mEPSC development. The two main AVCN neuron types, bushy and stellate cells, were distinguished electrophysiologically. Bushy cell mEPSCs became more frequent and faster between P7 and P14/P21 but with little change in amplitude. Dendritic filtering of mEPSCs was not detected as indicated by the lack of correlation between 10 and 90% rise times and decay time constants. Seven days after cochlea ablation at P7 or P14, mEPSCs in surviving bushy cells were similar to controls, except that rise and decay times were positively correlated (R = 0.31 and 0.14 for surgery at P7 and P14, respectively). Consistent with this evidence for a shift of synaptic activity from the somata to the dendrites, SV2 staining (a synaptic vesicle marker) forms a ring around somata of control but not experimental bushy cells. In contrast, mEPSCs of stellate cells showed few significant changes over these ages with or without cochlea ablation. Taken together, mEPSCs in mouse AVCN bushy cells show dramatic developmental changes across this critical period, and cochlea ablation may lead to the emergence of excitatory synaptic inputs impinging on bushy cell dendrites.

Action Potentials↗
PubMed1993

Miniature excitatory synaptic currents corrected for dendritic cable properties reveal quantal size and variance.

1. Non-NMDA (N-Methyl-D-aspartate) receptor mediated miniature excitatory synaptic currents (mEPSCs) were recorded from motoneurons in organotypic cultures of embryonic rat spinal cord. 2. Amplitude histograms of mEPSCs were unimodal and skewed toward larger events. The mean of the modes of the amplitude histograms was -18 pA with a maximal amplitude range of -4 to -160 pA for individual mEPSCs. 3. Current transients to a short voltage pulse were used to estimate the passive cable parameters of the motoneurons. The mean membrane time constant (tau) and the mean electrotonic length (L) were 20 and 0.96 ms, respectively. 4. The amplitudes of the mEPSCs were corrected for imperfect space and voltage clamp. The resulting amplitude histograms could be fitted by the sum of two Gaussian curves, revealing a mean quantal size of -48 pA with a coefficient of variation (cv) of 0.28. 5. Our data suggest that quantal size and its variance are masked by the cable properties of the neurons and that simultaneous release of elementary quanta occurs occasionally.

Animals↗
PubMed1993

NMDA receptor-mediated components of miniature excitatory synaptic currents in developing rat neocortex.

1. In vitro slices of frontal neocortex were prepared from rat pups at various ages after birth: postnatal days (PN) 3-5, 6-8, and 9-14. Using whole-cell patch-clamp techniques, both spontaneous and evoked excitatory postsynaptic currents (EPSCs) were recorded from voltage-clamped layer II-III pyramidal neurons. Developmental changes in EPSCs were examined. 2. Four properties of miniature EPSCs (mEPSCs) were studied: rise time, amplitude, decay time constant (tau), and frequency. These parameters were not tetrodotoxin sensitive and did not exhibit significant developmental changes during the first two postnatal weeks. 3. mEPSCs occurred approximately every 2-3 s and had peak amplitudes of 25-30 pA. Within each age group, certain parameters of mEPSCs were voltage dependent. mEPSC rise time and decay tau were significantly increased at depolarized potentials (-30 to -45 mV) relative to hyperpolarized (-75 to -90 mV) or resting membrane potential (RMP) (-60 to -70 mV). 4. At threshold stimulation intensity, EPSCs were evoked in an "all-or-none" manner. The amplitude and decay tau of evoked unitary EPSCs and mEPSCs were not significantly different. As stimulation intensity was increased, a late EPSC component appeared that was not seen in mEPSCs. At suprathreshold stimulus intensities, EPSC duration was significantly longer in PN 3-5 than in PN 9-14 neurons. 5. The N-methyl-D-aspartate (NMDA) receptor antagonist D(-)2-amino-5-phosphonovaleric acid (APV, 10 microM) significantly decreased mEPSC decay tau and frequency only at depolarized membrane potentials. Likewise, EPSCs were depressed by APV to a greater extent at depolarized potentials, and the depression was mainly of the late component. mEPSCs recorded at RMP were blocked by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline- 2,3-dione (5 microM). 6. Removal of extracellular Mg2+ reversibly increased the decay tau of mEPSCs at RMP but not at depolarized membrane potentials. The decay tau and duration of evoked EPSCs were also increased in zero Mg2+. These effects were reversible with application of APV. All effects of zero Mg2+ on mEPSCs and EPSCs were observed as early as PN 3-5. 7. These results indicate that the basic kinetic properties of mEPSCs are present by PN 3-5 and do not change significantly over the first two postnatal weeks. NMDA receptor activation contributes to mEPSCs and sensitivity to Mg2+ appears as early as PN 3-5. Unitary EPSCs resemble mEPSCs, but a late NMDA receptor-mediated component appears in EPSCs as stimulus intensity is increased.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1994

Noise analysis of miniature IPSCs in adult rat brain slices: properties and modulation of synaptic GABAA receptor channels.

1. The properties of synaptic gamma-aminobutyric acid (GABA)A receptor channels were resolved by using tight-seal, whole-cell recordings from granule cells of the dentate gyrus in adult rat hippocampal slices and by applying the technique of nonstationary noise analysis to study miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of tetrodotoxin (TTX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and D-2-amino-5-phosphonovaleric acid (D-AP5). This technique allowed us to extract information about the conductance, the number, and the kinetics of ligand gated channels underlying elementary synaptic currents. 2. To ascertain the validity of the nonstationary noise analysis method we have first tested it on computer simulated mIPSCs with different channel activation, lifetime kinetics, and opening probabilities. Using intraburst mean open times, shorter than the time to the first opening following activation, caused a large variance at the peak due to the stochastic channel properties. This resulted in a skewed mean current-variance relationship, which precluded proper estimation of unit conductance and especially the number of channels open at the peak of mIPSCs. Regardless of the probability of channel opening, accurate estimates of the unit conductance and the number of channels underlying each simulated mIPSC were obtained when channels had mean open times longer than the time to first opening. 3. Once the validity of the nonstationary analysis had been ascertained, it was used on mIPSCs recorded at 35 degrees C. The unit conductance of the synaptic GABAA channels was 28 +/- 1 (SE) pS and the average number of channels underlying mIPSCs was 46 +/- 4. The mean current-variance relationship was not skewed at higher amplitudes, suggesting that the intrinsic variance at the peak of the GABAA mIPSCs is low and that the open time of the channels is longer than the time to first opening. The estimated unit conductance of the channels was constant over a wide range of holding potentials. 4. The amplitude distribution of mIPSCs with rapid 10-90% rise times (290 +/- 20 microseconds) was clearly skewed towards low values. This skew was not due to filtering of electrotonically distant currents. Current-variance analysis revealed that the skewness resulted from differences in the number of GABAA receptor channels and not from the heterogeneity of unitary conductances at various synapses. Selection of mIPSCs with slower rise times yielded smaller unit conductance estimates.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Ca(2+)-dependent plasticity of miniature inhibitory postsynaptic currents after amputation of dendrites in central neurons.

1. The effects of cutting off the bulk (> 2/3) of the dendritic tree (dendrotomy) on GABAergic miniature inhibitory postsynaptic currents (mIPCSs) were studied in granule cells of the adult rat dentate gyrus in 400-microns-thick slices in vitro. 2. After dendrotomy carried out in warm (32 degrees C) control artificial cerebrospinal fluid (ACSF), only small antidromic population spikes could be evoked in the granule cell layer, and no viable whole cell recordings could be obtained. However, when dendrotomy was performed in cold (8-10 degrees C) control ACSF, the amplitude of the antidromic population spikes increased, and stable whole cell recordings became possible. 3. Whole cell recordings, with CsCl-filled pipettes, from granule cells dendrotomized in cold control ACSF, revealed significant alterations, lasting > 10 h, in the decay kinetics of mIPSACs. The change consisted of a calcium-dependent transformation of the normal, single exponential decay into a prolonged double exponential that effectively increased the charge transferred by the synaptic events (the total area of the currents) by 67%. When 30 mM 1,2 bis-(2-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) was included in the pipette, the changes in the mIPSCs decay kinetics could still be observed after dendrotomy, indicating that the maintenance phase of this plasticity did not depend on elevated intracellular calcium levels. 4. Viable whole cell recordings could also be obtained in dendrotomized granule cells when the amputation of dendrites was carried out at 32 degrees C after incubation for 2 h with the cell-permeant Ca2+ chelator, BAPTA-AM (50 microM), or the cutting process was done in an ACSF containing either a combination of excitatory amino acid receptor antagonists 2-amino-5-phosphonovaleric acid (APV; 25 microM) + 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM), a blocker of intracellular Ca2+ release dantrolene-Na (20 microM), or the voltage-gated Na+ channel blocker tetrodotoxin (TTX; 1 microM). 5. After dendrotomy in BAPTA-AM, APV + CNQX, APV + CNQX + TTX, and/or dantrolene, the changes in decay kinetics were prevented, indicating that a rise in intracellular Ca2+ concentration plays a pivotal role in this plasticity. 6. Computer simulations of mIPSCs suggested that changes in single channel kinetics alone can, in principle, account for the Ca(2+)-dependent changes in mIPSC decay kinetics. 7. These findings are consistent with a lasting Ca(2+)-dependent increase in gamma-aminobutyric acid-A (GABAA) receptor function in cells that survive physical injury to their dendrites.

Animals↗
PubMed1996

Cable properties of cultured hippocampal neurons determined from sucrose-evoked miniature EPSCs.

1. The passive cable properties of rat hippocampal neurons in dissociated culture were studied using focal application of hypertonic solution to locally elicit miniature excitatory postsynaptic currents (mEPSCs) on the soma and dendrites. Neurons were filled with Lucifer yellow and portions of their dendritic trees were measured. 2. The average mEPSC measured at the soma appeared smaller and slower as the site of sucrose application was made more distal. Normalizing to a 1-micron diam dendrite, the mean mEPSC peak amplitude and charge was reduced e-fold in 170 and 1,000 microns, respectively, and the mean mEPSC decay time constant was increased e-fold in 150 microns. However, for any particular sucrose site, individual mEPSCs varied widely in their amplitudes and time courses. Plots of individual peak amplitudes versus half-width or rise time showed much overlap for mEPSCs originating from sites as much as 100 microns apart. This suggests that use of such plots to estimate the electrotonic location of synaptic currents is highly prone to error. 3. Averaged mEPSCs recorded when applying sucrose at the soma were poorly fitted by an alpha function but were well-described by an equation of the form mxh, where m incorporates a rise-time constant tau 1 and h a decay time constant tau 2. Averaged fits to mean mEPSCs elicited at the somas of five cells gave (mean +/- SE): peak conductance = 832 +/- 126 pS, tau 1 = 0.29 +/- 0.06 ms, tau 2 = 3.03 +/- 0.24 ms, x = 4.7 +/- 0.7. 4. For three cells, the entire dendritic branch to which sucrose was applied was measured and used to construct a passive cable model. The specific membrane resistance (Rm) and intracellular resistivity (Ri) were varied systematically in the model (assuming membrane capacitance Cm = 1 microF/cm2) to search for the best agreement between the mean mEPSCs and the model. Optimal Rm was found to lie in the range 20-30 k omega cm2, Ri in the range 100-200 omega cm. 5. These results confirm those obtained by other methods and emphasize the considerable cable filtering of fast electrical events in cultured hippocampal neurons.

Animals↗
PubMed1996

Neuropeptide Y2 receptors inhibit the frequency of spontaneous but not miniature EPSCs in CA3 pyramidal cells of rat hippocampus.

1. Neuropeptide Y (NPY) inhibits synaptic excitation in hippocampal area CA3. We studied its site of action with the use of whole cell patch-clamp recordings from CA3 pyramidal cells of rat hippocampal slices in vitro. 2. Spontaneous excitatory postsynaptic currents (sEPSCs) were isolated with picrotoxin, to block gamma-aminobutyric acid-A receptors, whereas miniature excitatory postsynaptic currents (mEPSCs) were isolated by additionally treating the slice with tetrodotoxin (TTX) and/or Cd2+, sEPSCs and mEPSCs were eliminated by the excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and DL-2-amino-5-phosphonovaleric acid (50 microM), and were thus solely attributable to glutamate release. 3. The interval and amplitude distributions of sEPSCS and (TTX-isolated) mEPSCS were analyzed. Either NPY or the rapidly reversible, Y2-receptor-selective agonist [6-aminohexanoic5-24] NPY, ([ahx5-24]NPY) sharply increased the inter-sEPSC intervals in 16 of 16 neurons tested. In 11 of these cells, these agonists also simultaneously shifted the sEPSC amplitude distribution to somewhat smaller amplitudes, whereas in the remaining 5 cells, no concurrent effect on amplitudes was observed. By contrast, in 15 separate neurons treated with 1 microM TTX, neither NPY nor [ahx5-24]NPY altered either mEPSC amplitude or interval distributions of the mEPSCs. 4. To directly compare the effects of Y2 receptor activation on sEPSC and mEPSC properties, we applied [ahx5-24]NPY to the same cell in the absence and presence of TTX (n = 7). sEPSC intervals were characteristically increased by the Y2 agonist in all cells; in six of seven cells the sEPSC distribution was also shifted to smaller amplitudes. TTX application reduced the mean amplitude of the synaptic events more than did [ahx5-24]NPY, while increasing their intervals. [ahx5-24]NPY had no effect in TTX. 5. NPY, acting on a Y2 receptor, inhibits impulse-dependent synaptic excitation of CA3 pyramidal cells of the rat hippocampus by an entirely presynaptic action.

Animals↗
PubMed1997

Large amplitude miniature excitatory postsynaptic currents in hippocampal CA3 pyramidal neurons are of mossy fiber origin.

Neonatal (P0) gamma-irradiation was used to lesion selectively the mossy fiber (MF) synaptic input to CA3 pyramidal cells. This lesion caused a > 85% reduction in the MF input as determined by quantitative assessment of the number of dynorphin immunoreactive MF boutons. The gamma-irradiation lesion caused a reduction in the mean number of miniature excitatory postsynaptic currents (mEPSCs) recorded from CA3 pyramidal cells (2,292 vs. 1,429/3-min period; n = 10). The lesion also caused a reduction in the mean mEPSC peak amplitude from 19.1 +/- 0.45 to 14.6 +/- 0.49 pA (mean +/- SE; peak conductance 238.8 +/- 5.6 to 182.0 +/- 6.1 pS). Similarly, there was a reduction in the mean 10-85% rise time from 1.72 +/- 0.02 ms to 1.42 +/- 0.04 ms. The effects of the gamma-irradiation on both mEPSC amplitude and 10-85% rise time were significant at P < 0.002 and P < 0.005 (2-tailed Kolmogorov-Smirnov test). Based on the selectively of the gamma-irradiation, MF and non-MF mEPSC amplitude and 10-85% rise-time distributions were calculated. Both the amplitude and 10-85% rise-time distributions showed extensive overlap between the MF and non-MF mediated mEPSCs. The MF mEPSC distributions had a mean peak amplitude of 24.6 pA (307.5 pS) and a mean 10-85% rise time of 2.16 ms. THe non-MF mEPSC distributions had a mean peak amplitude of 12.2 pA (152.5 pS) and 10-85% rise time of 1.26 ms. The modes of the amplitude distributions were the same at 5 pA (62 pS). The MF and non-MF mEPSC amplitude and 10-85% rise-time distributions were significantly different at P << 0.001 (1-tailed, large sample Kolmogorov-Smirnov test). The data demonstrate that the removal of the MF synaptic input to CA3 pyramidal cells leads to the absence of the large amplitude mEPSCs that are present in control recordings.

Algorithms↗
PubMed1997

Rapid kinetics and inward rectification of miniature EPSCs in layer I neurons of rat neocortex.

With the use of the whole cell patch-clamp technique combined with visualization of neurons in brain slices, we studied the properties of miniature excitatory postsynaptic currents (mEPSCs) in rat neocortical layer I neurons. At holding potentials (-50 to -70 mV) near the resting membrane potential (RMP), mEPSCs had amplitudes of 5-100 pA and were mediated mostly by alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors. Amplitude histograms were skewed toward large events. An N-methyl-D-aspartate (NMDA) component was revealed by depolarization to -30 mV or by the use of a Mg2+-free bathing solution. At RMP, averaged AMPA mEPSCs had a 10-90% rise time of approximately 0.3 ms (uncorrected for instrument filtering). The decay of averaged mEPSCs was best fit by double-exponential functions in most cases. The fast, dominating component had a decay time constant of approximately 1.2 ms and comprised approximately 80% of the total amplitude. A small slow component had a decay time constant of approximately 4 ms. Positive correlations were found between rise and decay times of both individual and averaged mEPSCs, indicative of dendritic filtering. Some large-amplitude mEPSCs and spontaneous EPSCs (recorded in the absence of tetrodotoxin) had slower kinetics, suggesting a role of asynchronous transmitter release in shaping EPSCs. The amplitudes of mEPSCs were much smaller at +60 mV than at -60 mV, indicating that synaptic AMPA-receptor-mediated currents were inwardly rectifying. These results suggest that neocortical layer I neurons receive both NMDA- and AMPA-receptor-mediated synaptic inputs. The rapid decay of EPSCs appears to be largely determined by AMPA receptor deactivation. The observed rectification of synaptic responses suggests that synaptic AMPA receptors in layer I neurons may lack GluR-2 subunits and may be Ca2+ permeable.

Animals↗
PubMed1997

Differential impact of miniature synaptic potentials on the soma and dendrites of pyramidal neurons in vivo.

We studied the impact of transmitter release resistant to tetrodotoxin (TTX) in morphologically identified neocortical pyramidal neurons recorded intracellularly in barbiturate-anesthetized cats. It was observed that TTX-resistant release occurs in pyramidal neurons in vivo and at much higher frequencies than was previously reported in vitro. Further, in agreement with previous findings indicating that GABAergic and glutamatergic synapses are differentially distributed in the somata and dendrites of pyramidal cells, we found that most miniature synaptic potentials were sensitive to gamma-aminobutyric acid-A (GABA(A)) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonists in presumed somatic and dendritic impalements, respectively. Pharmacological blockage of spontaneous synaptic events produced large increases in input resistance that were more important in dendritic (approximately 50%) than somatic (approximately 10%) impalements. These findings imply that in the intact brain, pyramidal neurons are submitted to an intense spike-independent synaptic bombardment that decreases the space constant of the cells. These results should be taken into account when extrapolating in vitro findings to intact brains.

Anesthesia↗
PubMed1998

Effects of PKA and PKC on miniature excitatory postsynaptic currents in CA1 pyramidal cells.

Protein kinases play an important role in controlling synaptic strength at excitatory synapses on CA1 pyramidal cells. We examined the effects of activating cAMP-dependent protein kinase or protein kinase C (PKC) on the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) with perforated patch recording techniques. Both forskolin and phorbol-12,13-dibutryate (PDBu) caused a large increase in mEPSC frequency, but only PDBu increased mEPSC amplitude, an effect that was not observed when standard whole cell recording was performed. These results support biochemical observations indicating that PKC, similar to calcium/calmodulin-dependent protein kinase II, has an important role in controlling synaptic strength via modulation of AMPA receptor function, potentially through the direct phosphorylation of the GluR1 subunit.

Animals↗
PubMed1999

Glycinergic miniature synaptic currents and receptor cluster sizes differ between spinal cord interneurons.

The structural features of a synaptic connection between central neurons play an important role in determining the strength of the connection. In the present study, we have examined the relationship between the structural and functional properties of glycinergic synapses in the rat spinal cord. We have analyzed the structure of glycinergic receptor clusters on rat ventral horn interneurons using antibodies against the glycine receptor clustering protein, gephyrin. We have examined the properties of quantal glycinergic currents generated at these synapses using whole cell patch-clamp recordings of miniature postsynaptic inhibitory currents (mIPSCs) in rat spinal cord slices in vitro. Our immunolabeling results demonstrate that there is a considerable variability in the size of glycine receptor clusters within individual neurons. Furthermore there are large differences in the mean cluster size between neurons. These observations are paralleled closely by recordings of glycinergic mIPSCs. The mIPSC amplitude varies significantly within and between neurons. Results obtained using combined immunolabeling and electrophysiological recording on the same neurons show that cells with small glycine receptor clusters concurrently exhibit small mIPSCs. Our results suggest that the differences in the size of glycinergic receptor clusters may constitute an important factor contributing to the observed differences in mIPSC amplitude among spinal cord interneurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1999

Ca(2+)-induced Ca(2+) release activates spontaneous miniature outward currents (SMOCs) in parasympathetic cardiac neurons.

Mudpuppy parasympathetic cardiac neurons exhibit spontaneous miniature outward currents (SMOCs) that are thought to be due to the activation of clusters of large conductance Ca(2+)-activated K(+) channels (BK channels) by localized release of Ca(2+) from internal stores close to the plasma membrane. Perforated-patch whole cell recordings were used to determine whether Ca(2+)-induced Ca(2+) release (CICR) is involved in SMOC generation. We confirmed that BK channels are involved by showing that SMOCs are inhibited by 100 nM iberiotoxin or 500 microM tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is decreased in solutions that contain 0 Ca(2+)/3.6 mM Mg(2+), and also in the presence of 1 microM nifedipine and 3 microM omega-conotoxin GVIA, suggesting that SMOC activation is dependent on calcium influx. However, Ca(2+) influx alone is not sufficient; SMOC activation is also dependent on Ca(2+) release from the caffeine- and ryanodine-sensitive Ca(2+) store, because exposure to 2 mM caffeine consistently caused an increase in SMOC frequency, and 10-100 microM ryanodine altered the configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca(2+) stores by the Ca-ATPase inhibitor cyclopiazonic acid (10 microM) inhibited SMOC activity, even when Ca(2+) influx was not compromised. We also tested the effects of the membrane-permeable Ca(2+) chelators, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 microM) caused no inhibition of SMOC activation, whereas 10 microM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal of caffeine and suggests that the source of Ca(2+) that triggers the internal Ca(2+) release channel is different from the source of Ca(2+) that activates clusters of BK channels. We propose that influx of Ca(2+) through voltage-dependent Ca(2+) channels is required for SMOC generation, but that the influx of Ca(2+) triggers CICR from intracellular stores, which then activates the BK channels responsible for SMOC generation.

Animals↗