Presynaptic suppression of dorsal horn inhibitory transmission by mu-opioid receptors

Opioids modify sensory experience at many levels in the CNS. The mechanisms of this action, including the ways opioid receptors affect synaptic transmission, are not yet fully understood. Here we show that the selective activation of mu-opioid receptors suppressed inhibitory transmission between spinal cord dorsal horn neurons in vitro. mu-Opioid receptor activation reduced evoked inhibitory postsynaptic current (eIPSC) amplitude by acting presynaptically, because it altered the paired-pulse ratio, did not affect GABA-evoked currents, and decreased miniature IPSC (mIPSC) frequency. The mechanism of this effect was independent both of presynaptic Ca(2+) entry and of the pathway linking presynaptic kainate (KA) receptors to suppression of inhibitory transmission in the same cells. These data identify mu-opioid receptors as important presynaptic modulators of dorsal horn inhibitory transmission.     

Excitatory mechanisms in the suprachiasmatic nucleus: the role of AMPA/KA glutamate receptors

A variety of evidence suggests that the effects of light on the mammalian circadian system are mediated by direct retinal ganglion cell projection to the suprachiasmatic nucleus (SCN). This synaptic connection is glutamatergic and the release of glutamate is detected by both N-methyl-D-asparate (NMDA) and amino-methyl proprionic acid/kainate (AMPA/KA) iontotropic glutamate receptors (GluRs). It is well established that NMDA GluRs play a critical role in mediating the effects of light on the circadian system; however, the role of AMPA/KA GluRs has received less attention. In the present study, we sought to better understand the contribution of AMPA/KA-mediated currents in the circadian system based in the SCN. First, whole cell patch-clamp electrophysiological techniques were utilized to measure spontaneous excitatory postsynaptic currents (sEPSCs) from SCN neurons. These currents were widespread in the SCN and not just restricted to the retino-recipient region. The sEPSC frequency and amplitude did not vary with the daily cycle. Similarly, currents evoked by the exogenous application of AMPA onto SCN neurons were widespread within the SCN and did not exhibit a diurnal rhythm in their magnitude. Fluorometric techniques were utilized to estimate AMPA-induced calcium (Ca(2+)) concentration changes in SCN neurons. The resulting data indicate that AMPA-evoked Ca(2+) transients were widespread in the SCN and that there was a daily rhythm in the magnitude of AMPA-induced Ca(2+) transients that peaked during the night. By itself, blocking AMPA/KA GluRs with a receptor blocker decreased the spontaneous firing of some SCN neurons as well as reduced resting Ca(2+) levels, suggesting tonic glutamatergic excitation. Finally, immunohistochemical techniques were used to describe expression of the AMPA-preferring GluR subunits GluR1 and GluR2/3s within the SCN. Overall, our data suggest that glutamatergic synaptic transmission mediated by AMPA/KA GluRs play an important role throughout the SCN synaptic circuitry.     

Possible roles of kainate receptors on GABAergic nerve terminals projecting to rat substantia nigra dopaminergic neurons

GABAergic afferent inputs are thought to play an important role in the control of the firing pattern of substantia nigra pars compacta (SNc) dopaminergic neurons. We report here the actions of presynaptic kainite (KA) receptors in GABAergic transmission of rat SNc dopaminergic neurons. In mechanically dissociated rat SNc dopaminergic neurons attached with native presynaptic nerve terminals, GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded by use of conventional whole cell patch recording mode. In the voltage-clamp condition, KA (3 microM) significantly increased GABAergic mIPSC frequency without affecting the current amplitude. This facilitatory effect of KA was not affected in the presence of 20 microM GYKI52466, a selective AMPA receptor antagonist, but was completely inhibited in the presence of 20 microM CNQX, an AMPA/KA receptor antagonist. Presynaptic KA receptors on GABAergic terminals were mainly permeable to Na+ but impermeable to Ca2+ because KA-induced facilitation of mIPSC frequency was completely suppressed in either Na+-free or Ca2+-free external solutions, and in the presence of 200 microM Cd2+, a general voltage-dependent Ca2+ channel blocker. In the slice preparation, KA increased GABAergic spontaneous mIPSC frequency, but significantly suppressed evoked IPSC (eIPSC) amplitude. However, this inhibitory action on eIPSCs was reversed by 10 microM CGP55845, a selective GABAB receptor antagonist, implicating the possible involvement of GABAB autoreceptors in KA-induced modulation of GABAergic transmission. Thus presynaptic KA receptors on GABAergic nerve terminals synapsing onto SNc neurons may play functional roles contributing the fine control of neuronal excitability and firing pattern of SNc.     

A comparison of release kinetics and glutamate receptor properties in shaping rod–cone differences in EPSC kinetics in the salamander retina

Synaptic transmission from cones is faster than transmission from rods. Using paired simultaneous recordings from photoreceptors and second-order neurones in the salamander retina, we studied the contributions of rod–cone differences in glutamate receptor properties and synaptic release rates to shaping postsynaptic responses. Depolarizing steps evoked sustained calcium currents in rods and cones that in turn produced transient excitatory postsynaptic currents (EPSCs) in horizontal and OFF bipolar cells. Cone-driven EPSCs rose and decayed faster than rod-driven EPSCs, even when comparing inputs from a rod and cone onto the same postsynaptic neurone. Thus, rod–cone differences in EPSCs reflect properties of individual rod and cone synapses. Experiments with selective AMPA and KA agonists and antagonists showed that rods and cones both contact pharmacologically similar AMPA receptors. Spontaneous miniature EPSCs (mEPSCs) exhibited unimodal distributions of amplitude and half-amplitude time width and there were no rod–cone differences in mEPSC properties. To examine how release kinetics shape the EPSC, we convolved mEPSC waveforms with empirically determined release rate functions for rods and cones. The predicted EPSC waveform closely matched the actual EPSC evoked by cones, supporting a quantal release model at the photoreceptor synapse. Convolution with the rod release function also produced a good match in rod-driven cells, although the actual EPSC was often somewhat slower than the predicted EPSC, a discrepancy partly explained by rod–rod coupling. Rod–cone differences in the rates of exocytosis are thus a major factor in producing faster cone-driven responses in second-order retinal neurones.     

Presynaptic GABA(B) receptors regulate retinohypothalamic tract synaptic transmission by inhibiting voltage-gated Ca2+ channels

Presynaptic GABA(B) receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABA(B)-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by omega-conotoxin GVIA (< or = 72%), mibefradil (< or = 52%), and omega-agatoxin TK (< or = 15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP-sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.     

Potentiating effects of 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyaceta mide (PEPA) on excitatory synaptic transmission in dentate granule cells

A novel sulfonylamino compound, 4-[2-(phenylsulfonylamino)-ethylthio]-2,6-difluoro-phenoxyaceta mide (PEPA) has been shown to selectively potentiate glutamate-induced currents in Xenopus oocytes expressing recombinant AMPA receptor subunits, GluR1-GluR4, by attenuation of desensitization. Here, we examined the effects of PEPA on responses to excitatory amino acids as well as on excitatory synaptic transmission in dentate granule cells of rat hippocampal slices using the whole-cell patch clamp technique. PEPA at 100 microM produced a 3-4-fold increases in the peak amplitude of current responses to AMPA and glutamate applied iontophoretically in the dentate granule cells, whereas it showed no effect on NMDA-induced currents. Excitatory postsynaptic currents (EPSCs) evoked in these neurons by stimulation of the perforant path had fast and slow components mediated by AMPA and NMDA receptors, respectively. PEPA at concentrations between 10 and 100 microM potentiated only the AMPA component of the EPSC (AMPA EPSC) in a dose-dependent manner without affecting the NMDA component. Although the potentiating effect of PEPA on the amplitude of the AMPA EPSC was weaker than that on the AMPA-induced current, it clearly prolonged the duration of the EPSC. PEPA at 100 microM increased the peak amplitude of the AMPA EPSC by 17%, and increased the area enclosed by the AMPA EPSC by 72%.     

Layer-specific serotonergic facilitation of IPSC in layer 2/3 pyramidal neurons of the visual cortex

Serotonin (5-hydroxytryptamine, 5-HT) inhibits the induction of long-term synaptic plasticity in layer 2/3 of the visual cortex at the end of its critical period in rats. However, the cellular and molecular mechanisms remain unclear. Since inhibitory influence is crucial in the induction of synaptic plasticity, the effect of 5-HT on inhibitory transmission was investigated in layer 2/3 pyramidal neurons of the primary visual cortex. The amplitude of inhibitory postsynaptic current (IPSC), but not excitatory postsynaptic current, evoked by stimulation of the underlying layer 4, was increased by ～20% with a bath application of 5-HT. The amplitude of miniature IPSC was also increased by the application of 5-HT, while the paired-pulse ratio was not changed. The facilitating effect of 5-HT on IPSC was mediated by the activation of 5-HT(2) receptors. An increase in intracellular Ca(2+) via release from inositol 1,4,5-trisphosphate (IP(3))-sensitive stores, which was confirmed by confocal Ca(2+) imaging, and activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII) were involved in the facilitation of IPSC by 5-HT. However, 5-HT failed to facilitate IPSC evoked by the stimulation of layer 1. These results suggest that activation of 5-HT(2) receptors releases intracellular Ca(2+) via IP(3)-sensitive stores, which facilitates GABA(A)ergic transmission via the activation of CaMKII in layer 2/3 pyramidal neurons of the visual cortex in a layer-specific manner. Thus facilitation of inhibitory transmission by 5-HT might be involved in regulating the information flow and the induction of long-term synaptic plasticity, in a pathway-specific manner.     

Pre- and postsynaptic activation of M-channels by a novel opener dampens neuronal firing and transmitter release

The M-type K(+) current (M-current), encoded by Kv7.2/3 (KCNQ2/3) K(+) channels, plays a critical role in regulating neuronal excitability because it counteracts subthreshold depolarizations. Here we have characterized the functions of pre- and postsynaptic M-channels using a novel Kv7.2/3 channel opener, NH6, which we synthesized as a new derivative of N-phenylanthranilic acid. NH6 exhibits a good selectivity as it does not affect Kv7.1 and I(KS) K(+) currents as well as NR1/NR2B, AMPA, and GABA(A) receptor-mediated currents. Superfusion of NH6 increased recombinant Kv7.2/3 current amplitude (EC(50) = 18 muM) by causing a hyperpolarizing shift of the voltage activation curve and by markedly slowing the deactivation kinetics. Activation of native M-currents by NH6 robustly reduced the number of evoked and spontaneous action potentials in cultured cortical, hippocampal and dorsal root ganglion neurons. In hippocampal slices, NH6 decreased somatically evoked spike after depolarization of CA1 pyramidal neurons and induced regular firing in bursting neurons. Activation of M-channels by NH6, potently reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents. Activation of M-channels also decreased the frequency of miniature excitatory (mEPSC) and inhibitory (mIPSC) postsynaptic currents without affecting their amplitude and waveform, thus suggesting that M-channels presynaptically inhibit glutamate and GABA release. Our results suggest a role of presynaptic M-channels in the release of glutamate and GABA. They also indicate that M-channels act pre- and postsynaptically to dampen neuronal excitability.     

Effects of corticosteroids on synaptic transmission in rat dorsolateral septal nucleus

The effects of corticosteroids on synaptic transmission in the rat dorsolateral septal nucleus (DLSN) were examined, in vitro, by using intracellular and voltage-clamp recording methods. Prednisolone (100 microM) increased the amplitude of excitatory postsynaptic potential (EPSP) and depressed both fast and slow inhibitory postsynaptic potentials (IPSP). Under voltage-clamp conditions, prednisolone (100 microM) increased the amplitude of excitatory postsynaptic current (EPSC) and depressed the fast and slow inhibitory postsynaptic currents (IPSCs). Corticosterone (100 microM) mimicked the effects of prednisolone on the postsynaptic currents (PSCs). To examine the direct effects of prednisolone on the EPSC and slow IPSC, the fast IPSC was blocked by bicuculline (20 microM). Under these experimental conditions, prednisolone (100 microM) did not alter the isolated EPSC but depressed slow IPSC by 22 +/- 3% (n = 10). The fast IPSC was isolated by pretreatment with kynurenic acid and CGP55845A, where the EPSC and slow IPSC were blocked. Prednisolone (100 microM) depressed the isolated fast IPSC in DLSN neurons. Prednisolone (100 microM) did not change either the inward current produced by glutamate or the outward current produced by gamma-aminobutyric acid (GABA). The results suggest that corticosteroids facilitate excitatory synaptic transmission in the DLSN by reducing the release of GABA from the presynaptic nerve terminals of interneurons.     

Acceleration of AMPA receptor kinetics underlies temperature-dependent changes in synaptic strength at the rat calyx of Held

It is well established that synaptic transmission declines at temperatures below physiological, but many in vitro studies are conducted at lower temperatures. Recent evidence suggests that temperature-dependent changes in presynaptic mechanisms remain in overall equilibrium and have little effect on transmitter release at low transmission frequencies. Our objective was to examine the postsynaptic effects of temperature. Whole-cell patch-clamp recordings from principal neurons in the medial nucleus of the trapezoid body showed that a rise from 25°C to 35°C increased miniature EPSC (mEPSC) amplitude from −33 ± 2.3 to −46 ± 5.7 pA ( n = 6) and accelerated mEPSC kinetics. Evoked EPSC amplitude increased from −3.14 ± 0.59 to −4.15 ± 0.73 nA with the fast decay time constant accelerating from 0.75 ± 0.09 ms at 25°C to 0.56 ± 0.08 ms at 35°C. Direct application of glutamate produced currents which similarly increased in amplitude from −0.76 ± 0.10 nA at 25°C to −1.11 ± 0.19 nA 35°C. Kinetic modelling of fast AMPA receptors showed that a temperature-dependent scaling of all reaction rate constants by a single multiplicative factor ( Q ₁₀= 2.4) drives AMPA channels with multiple subconductances into the higher-conducting states at higher temperature. Furthermore, Monte Carlo simulation and deconvolution analysis of transmission at the calyx of Held showed that this acceleration of the receptor kinetics explained the temperature dependence of both the mEPSC and evoked EPSC. We propose that acceleration in postsynaptic AMPA receptor kinetics, rather than altered presynaptic release, is the primary mechanism by which temperature changes alter synaptic responses at low frequencies.     

Restrictions on inhibitory circuits contribute to limited recruitment of fast inhibition in rat neocortical pyramidal cells.

To further define the operational boundaries on fast inhibition in neocortex, whole cell recordings were made from layer V pyramidal neurons in neocortical slices to evaluate evoked inhibitory postsynaptic currents (IPSCs) and spontaneous miniature IPSCs (mIPSCs). Stimulating electrodes were placed in layers VI and I/II to determine whether simultaneous stimulation of deep and superficial laminae could extend the magnitude of maximal IPSCs evoked by deep-layer stimulation alone. The addition of superficial-layer stimulation did not increase maximal IPSC amplitude, confirming the strict limit on fast inhibition. Spontaneous miniature IPSCs were recorded in the presence of tetrodotoxin. The frequency of spontaneous mIPSCs ranged from 10.0 to 33.1 Hz. mIPSC amplitude varied considerably, with a range of 5. 0-128.2 pA and a mean value of 20.7+/-4.1 pA (n = 12 cells). The decay phase of miniature IPSCs was best fit by a single exponential, similar to evoked IPSCs. The mean time constant of decay was 6.4+/-0.6 ms, with a range of 0.2-20.1 ms. The mean 10-90% rise time was 1.9+/-0.2 ms, ranging from 0.2 to 6.3 ms. Evaluation of mIPSC kinetics revealed no evidence of dendritic filtering. Amplitude histograms of mIPSCs exhibited skewed distributions with several discernable peaks that, when fit with Gaussian curves, appeared to be spaced equidistantly, suggesting that mIPSC amplitudes varied quantally. The mean separation of Gaussian peaks ranged from 6.1 to 7.8 pA. The quantal distributions did not appear to be artifacts of noise. Exposure to saline containing low Ca(2+) and high Mg(2+) concentrations reduced the number of histogram peaks, but did not affect the quantal size. Mean mIPSC amplitude and quantal size varied with cell holding potential in a near-linear manner. Statistical evaluation of amplitude histograms verified the multimodality of mIPSC amplitude distributions and corroborated the equidistant spacing of peaks. Comparison of mIPSC values with published data from single GABA channel recordings suggests that the mean mIPSC conductance corresponds to the activation of 10-20 GABA(A) receptor channels, and that the release of a single inhibitory quantum opens 3-6 channels. Further comparison of mIPSCs with evoked inhibitory events suggests that a single interneuron may form, on average, 4-12 functional synapses with a pyramidal cell, and that 10-12 individual interneurons are engaged during recruitment of maximal population IPSCs. This suggests that inhibitory circuits are much more restricted in both the size of the unit events and effective number of connections when compared with excitatory inputs.     

cAMP-dependent presynaptic regulation of spontaneous glycinergic IPSCs in mechanically dissociated rat spinal cord neurons

Spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with intact glycinergic presynaptic nerve terminals and evoked IPSCs (eIPSCs) in the slice preparation were investigated using nystatin-perforated patch and conventional whole cell recording modes under the voltage-clamp conditions. Trans-ACPD (tACPD) reversibly reduced the mIPSC frequency without affecting the mean amplitude. The effect was mimicked by a specific metabotropic glutamate receptor (mGluR) II subtype agonist, (2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine (L-CCG-I), and a specific mGluRIII subtype agonist, 2-amino-4-phosphonobutyrate (L-AP4). These inhibitory effects on mIPSC frequency were blocked by the specific antagonists for mGluRII, alpha-methyl-1-(2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine and (RS)-alpha-cyclopropyl-4-phosphonophenylglycine. In the slice preparation, eIPSC amplitude and mIPSC frequency were decreased reversibly by L-CCG-I (10(-6) M) and L-AP4 (10(-6) M). In K(+)-free or K(+)-free external solution with Ba(2+) and Cs(+), Ca(2+)-free or Cd(2+) external solution, the inhibitory effect of tACPD on mIPSC frequency was unaltered. Forskolin and 8-Br-cAMP significantly increased presynaptic glycine release, and prevented the inhibitory action of tACPD on mIPSC frequency. Sp-cAMP, however, did not prevent the inhibitory action of tACPD on mIPSC frequency. It was concluded that the activation of mGluRs inhibits glycine release by reducing the action of cAMP/PKA pathway.     

Selective blockade of Ca2+ permeable AMPA receptors in CA1 area of rat hippocampus

Using whole cell patch-clamp recording from pyramidal cells and interneurons in the CA1 area of hippocampal slices, the effect of IEM-1460, a selective channel blocker of Ca2+ permeable AMPA receptors (AMPARs), on postsynaptic currents (PSCs) was studied. Excitatory postsynaptic currents (EPSCs) were evoked by stimulation of Schaffer collaterals (SCs) in the presence of APV and bicuculline to pharmacologically isolate the EPSCs mediated by AMPAR activation. IEM-1460 (50 microM) did not affect the amplitude of EPSCs in CA1 pyramidal cells but reversibly decreased their amplitude in interneurons of pyramidal layer (15 cells), radiatum (37 cells) and border radiatum-lacunosum-moleculare (R-LM) (55 cells) layers. The ability of IEM-1460 to decrease EPSC amplitude correlated with EPSC rectification properties in CA1 interneurons, providing evidence for synaptic localization of Ca2+ permeable AMPARs at the SC synaptic input. Independent of their localization, the majority of interneurons studied exhibited only modest sensitivity to IEM-1460 (EPSC amplitude decreased by less than 30%), while in 15% of interneurons IEM-1460 induced more than 50% reduction in EPSC amplitude. To reveal possible afferent-specific localization of Ca2+ permeable AMPARs on R-LM interneurons, the effect of IEM-1460 on EPSCs evoked by stimulation of SC was compared with that of perforant path (PP). Although average sensitivities did not differ significantly, in 61% of R-LM layer interneurons, the SC-evoked EPSCs exhibited higher sensitivity to IEM-1460 than the PP-evoked EPSCs. Moreover, in 54% of R-LM layer interneurons the EPSCs evoked by SC stimulation were complex, having an initial peak followed by one or several late components. Kinetics, latency distribution and reversal potential of late components suggest di- and polysynaptic origin of the late components. Late EPSCs were strongly and reversibly inhibited by IEM-1460 indicating that Ca2+ permeable AMPARs are involved in the indirect excitation of R-LM layer interneurons. Despite the ability to decrease the excitatory synaptic input to interneurons, IEM-1460 did not affect interneuron-mediated inhibitory postsynaptic currents (IPSCs) evoked in pyramidal neurons by SC stimulation. These data suggest that interneurons with a synaptic input highly sensitive to IEM-1460 do not contribute specifically to the feed-forward inhibition of hippocampal pyramidal neurons.     

Cannabinoids depress inhibitory synaptic inputs received by layer 2/3 pyramidal neurons of the neocortex

Using whole cell voltage-clamp recordings we investigated the effects of a synthetic cannabinoid (WIN55,212-2) on inhibitory inputs received by layer 2/3 pyramidal neurons in slices of the mouse auditory cortex. Activation of the type 1 cannabinoid receptor (CB1R) with WIN55,212-2 reliably reduced the amplitude of GABAergic inhibitory postsynaptic currents evoked by extracellular stimulation within layer 2/3. The suppression of this inhibition was blocked and reversed by the highly selective CB1R antagonist AM251, confirming a CB1R-mediated inhibition. Pairing evoked inhibitory postsynaptic currents (IPSCs) at short interstimulus intervals while applying WIN55,212-2 resulted in an increase in paired-pulse facilitation suggesting that the probability of GABA release was reduced. A presynaptic site of cannabinoid action was verified by an observed decrease in the frequency with no change in the amplitude or kinetics of action potential-independent postsynaptic currents (mIPSCs). When Cd(2+) was added or Ca(2+) was omitted from the recording solution, the remaining fraction of Ca(2+)-independent mIPSCs did not respond to WIN55,212-2. These data suggest that cannabinoids are capable of suppressing the inhibition of neocortical pyramidal neurons by depressing Ca(2+)-dependent GABA release from local interneurons.     

NR2B-containing NMDA autoreceptors at synapses on entorhinal cortical neurons

We have previously shown that presynaptic N-methyl-D-aspartate receptors (NMDARs) can facilitate glutamate release onto principal neurons in the entorhinal cortex (EC). In the present study, we have investigated the subunit composition of these presynaptic NMDARs. We recorded miniature alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (mEPSCs), from visually identified neurons in layers II and V of the EC in vitro. In both layers, bath application of the NR2A/B subunit-selective agonist, homoquinolinic acid (HQA), resulted in a marked facilitation of mEPSC frequency. Blockade of presynaptic Ca(2+) entry through either NMDARs or voltage-gated Ca(2+) channels with Co(2+) prevented the effects of HQA, confirming that Ca(2+) entry to the terminal was required for facilitation. When the NR2B-selective antagonist, ifenprodil, was applied prior to HQA, the increase in mEPSC frequency was greatly reduced. In addition, we found that an NMDAR antagonist blocked frequency-dependent facilitation of evoked release and reduced mEPSC frequency in layer V. Thus we have demonstrated that NMDA autoreceptors in layer V of the EC bear the NR2B subunit, and that NMDARs are also present at terminals onto superficial neurons.     

Cannabinoid receptor modulation of synapses received by cerebellar Purkinje cells

The high density of cannabinoid receptors in the cerebellum and the degradation of motor coordination produced by cannabinoid intoxication suggest that synaptic transmission in the cerebellum may be strongly regulated by cannabinoid receptors. Therefore the effects of exogenous cannabinoids on synapses received by Purkinje cells were investigated in rat cerebellar slices. Parallel fiber-evoked (PF) excitatory postsynaptic currents (EPSCs) were strongly inhibited by bath application of the cannabinoid receptor agonist WIN 55212-2 (5 microM, 12% of baseline EPSC amplitude). This effect was completely blocked by the cannabinoid CB1 receptor antagonist SR 141716. It is unlikely that this was the result of alterations in axonal excitability because fiber volley velocity and kinetics were unchanged and a cannabinoid-induced decrease in fiber volley amplitude was very minor (93% of baseline). WIN 55212-2 had no effect on the amplitude or frequency of spontaneously occurring miniature EPSCs (mEPSCs), suggesting that the effect of CB1 receptor activation on PF EPSCs was presynaptically expressed, but giving no evidence for modulation of release processes after Ca(2+) influx. EPSCs evoked by climbing fiber (CF) stimulation were less powerfully attenuated by WIN 55212-2 (5 microM, 74% of baseline). Large, action potential-dependent, spontaneously occurring inhibitory postsynaptic currents (sIPSCs) were either severely reduced in amplitude (<25% of baseline) or eliminated. Miniature IPSCs (mIPSCs) were reduced in frequency (52% of baseline) but not in amplitude, demonstrating suppression of presynaptic vesicle release processes after Ca(2+) influx and suggesting an absence of postsynaptic modulation. The decrease in mIPSC frequency was not large enough to account for the decrease in sIPSC amplitude, suggesting that presynaptic voltage-gated channel modulation was also involved. Thus, while CB1 receptor activation reduced neurotransmitter release at all major classes of Purkinje cell synapses, this was not accomplished by a single molecular mechanism. At excitatory synapses, cannabinoid suppression of neurotransmitter release was mediated by modulation of voltage-gated channels in the presynaptic axon terminal. At inhibitory synapses, in addition to modulation of presynaptic voltage-gated channels, suppression of the downstream vesicle release machinery also played a large role.     

Analysis of the Excitatory and Inhibitory Components of Postsynaptic Currents Recorded in Pyramidal Neurons and Interneurons in the Rat Hippocampus

Postsynaptic currents recorded from interneurons and pyramidal cells in hippocampal slices by local voltage clamping were found to be the sum of excitatory (EPSC) and inhibitory (IPSC) components. An approach allowing quantitative assessment of the amplitude and time course of EPSC and IPSC without pharmacological blockade of the major postsynaptic receptors involved in generating these currents was developed. The approach is based on the existence of a significant difference between reversion potentials of cationic and anionic currents and the presence of a linear zone in the voltage-current characteristics of responses to excitatory and inhibitory transmitters. Comparison of the results of this calculation-based method with those of classical pharmacological analysis of the excitatory and inhibitory components of postsynaptic currents showed them to be virtually identical, which allows synaptic currents in defined neurons to be studied without altering the state of synaptic connections throughout the brain slice. IPSC was found to make a smaller contribution to the total postsynaptic current recorded in interneurons as compared with pyramidal neurons in rat hippocampal field CA1.__________Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 90, No. 8, pp. 945–956, August, 2004.     

Ethanol alters the frequency, amplitude, and decay kinetics of Sr2+-supported, asynchronous NMDAR mEPSCs in rat hippocampal slices

To discriminate between pre- and postsynaptic effects of ethanol on N-methyl-d-aspartate receptor (NMDAR) signaling in hippocampus, we adapted the technique of Sr(2+) substitution to the hippocampal blind slice patch-clamp preparation. Hippocampal slices were isolated from 12- to 20-day-old rats that were killed in accordance with University of Texas Institutional Animal Care and Use Committee guidelines. NMDAR miniature excitatory postsynaptic currents (mEPSCs) were evoked from CA1 pyramidal neurons in the presence of Sr(2+) (4 mM), causing the synchronous EPSC observed in the presence of Ca(2+) to be supplanted by asynchronous mEPSCs. Amplitudes typically ranged from 5 to 40 pA and responded to the NMDAR antagonist (DL)-APV (50 microM), with a statistically significant reduction in mean amplitude. Ethanol (25, 50, and 75 mM) exerted dose-dependent effects on mEPSC amplitude and frequency. Peak amplitude inhibition was observed at 75 mM ethanol. Notably, ethanol significantly decreased event frequency at 50 and 75 mM ethanol. Ethanol (75 mM) also significantly increased the paired-pulse ratio of NMDAR EPSCs. Cumulative comparisons of decay time constants derived from single-exponential fitting of mEPSCs revealed significantly accelerated current decay kinetics in the presence of 75 mM ethanol. Taken together, these reductions in miniature event frequency and amplitude, concurrent with an increased rate of decay, suggest that the acute effects of ethanol on NMDAR signaling at hippocampal synapses are multifocal in nature. This finding of pre- and postsynaptic effects of ethanol on NMDAR signal strength in a brain region central to cognition is wholly consistent with previous reports of ethanol inhibition of NMDAR-long-term potentiation in vitro and with the profound cognitive deficits associated with binge-level intoxication in vivo.     

Activation of postsynaptic Ca(2+) stores modulates glutamate receptor cycling in hippocampal neurons

We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP(3)Rs) with the IP(3)R agonist adenophostin A (AdA) produces large increases in AMPA receptor (AMPAR) excitatory postsynaptic current (EPSC) amplitudes at hippocampal CA1 synapses. Co-perfusion of the Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid strongly inhibited AdA-enhanced increases in EPSC amplitudes. We examined the role of AMPAR insertion/anchoring in basal synaptic transmission. Perfusion of an inhibitor of synaptotagmin-soluble n-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor SNARE-mediated exocytosis depressed basal EPSC amplitudes, whereas a peptide that inhibits GluR2/3 interactions with postsynaptic density-95 (PDZ) domain proteins glutamate receptor interacting protein (GRIP)/protein interacting with C-kinase-1 (PICK1) enhanced basal synaptic transmission. These results suggest that constitutive trafficking and anchoring of AMPARs help maintain basal synaptic transmission. The regulation of postsynaptic AMPAR trafficking involves synaptotagmin-SNARE-mediated vesicle exocytosis and interactions between AMPARs and the PDZ domains in GRIP/PICK1. We show that inhibitors of synaptotagmin-SNARE-mediated exocytosis, or interactions between AMPARs and GRIP/PICK1, attenuated AdA-enhanced increases in EPSC amplitudes. These results suggest that IP(3)R-mediated Ca(2+) release can enhance AMPAR EPSC amplitudes through mechanisms that involve AMPAR-PDZ interactions and/or synaptotagmin-SNARE-mediated receptor trafficking.     

Adrenergic facilitation of GABAergic transmission in rat entorhinal cortex

Whereas the entorhinal cortex (EC) receives noradrenergic innervations from the locus coeruleus of the pons and expresses adrenergic receptors, the function of norepinephrine (NE) in the EC is still elusive. We examined the effects of NE on GABA(A) receptor-mediated synaptic transmission in the superficial layers of the EC. Application of NE dose-dependently increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from the principal neurons in layer II/III through activation of alpha(1) adrenergic receptors. NE increased the frequency and not the amplitude of miniature IPSCs (mIPSCs) recorded in the presence of TTX, suggesting that NE increases presynaptic GABA release with no effects on postsynaptic GABA(A) receptors. Application of Ca(2+) channel blockers (Cd(2+) and Ni(2+)), omission of Ca(2+) in the extracellular solution, or replacement of extracellular Na(+) with N-methyl-D-glucamine (NMDG) failed to alter NE-induced increase in mIPSC frequency, suggesting that Ca(2+) influx through voltage-gated Ca(2+) or other cationic channels is not required. Application of BAPTA-AM, thapsigargin, and ryanodine did not change NE-induced increase in mIPSC frequency, suggesting that Ca(2+) release from intracellular stores is not necessary for NE-induced increase in GABA release. Whereas alpha(1) receptors are coupled to G(q/11) resulting in activation of the phospholipase C (PLC) pathway, NE-mediated facilitation of GABAergic transmission was independent of PLC, protein kinase C, and tyrosine kinase activities. Our results suggest that NE-mediated facilitation of GABAergic function contributes to its antiepileptic effects in the EC.