Light-evoked excitatory synaptic currents of X-type retinal ganglion cells

The excitatory amino acid receptor (EAAR) types involved in the generation of light-evoked excitatory postsynaptic currents (EPSCs) were examined in X-type retinal ganglion cells. Using isolated and sliced preparations of cat and ferret retina, the light-evoked EPSCs of X cells were isolated by adding picrotoxin and strychnine to the bath to remove synaptic inhibition. N-methyl-D-aspartate (NMDA) receptors contribute significantly to the light-evoked EPSCs of ON- and OFF-X cells at many different holding potentials. An NMDA receptor contribution to the EPSCs was observable when retinal synaptic inhibition was either normally present or pharmacologically blocked. NMDA receptors formed 80% of the peak light-evoked EPSC at a holding potential of -40 mV; however, even at -80 mV, 20% of the light-evoked EPSC was NMDA-mediated. An alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor-mediated component to the light-evoked EPSCs predominated at a holding potential of -80 mV. The light-evoked EPSC was blocked by the AMPA receptor-selective antagonist GYKI52466 (50-100 microM). The AMPA receptor-mediated EPSC component had a linear current-voltage relation. AMPA receptors form the main non-NMDA EAAR current on both ON- and OFF- X ganglion cell dendrites. When synaptic transmission was blocked by the addition of Cd(2+) to the Ringer, application of kainate directly to ganglion cells evoked excitatory currents that were strongly blocked by GYKI52466. Experiments using selective EAAR modulators showed the AMPA receptor-selective modulator cyclothiazide potentiated glutamate-evoked currents on X cells, while the kainate receptor-selective modulator concanavalin A (ConA) had no effect on kainate-evoked currents. Whereas the present study confirms the general notion that AMPA EAAR-mediated currents are transient and NMDA receptor-mediated currents are sustained, current-voltage relations of the light-evoked EPSC at different time points showed the contributions of these two receptor types significantly overlap. Both NMDA and AMPA EAARs can transmit transient and sustained visual signals in X ganglion cells, suggesting that much signal shaping occurs presynaptically in bipolar cells.     

GABAB receptor-mediated presynaptic inhibition of glutamatergic transmission in the inferior colliculus

Whole-cell patch clamp recordings were made from ICC neurons in brain slices of 9-16 day old rats. Postsynaptic currents were evoked by electrical stimulation of the lemniscal inputs. Excitatory postsynaptic currents (EPSCs) were isolated pharmacologically by blocking GABA(A) and glycine receptors. EPSCs were further dissected into AMPA and NMDA receptor-mediated responses by adding the receptor antagonists, APV and CNQX, respectively. The internal solution in the recording electrodes contained CsF and TEA to block K(+) channels that might be activated by postsynaptic GABA(B) receptors. The modulatory effects of GABA(B) receptors on EPSCs in ICC neurons were examined by bath application of the GABA(B) receptor agonist, baclofen, and the antagonist, CGP 35348. The amplitudes of EPSCs in ICC neurons were reduced to 34.4+/-3.2% of the control by baclofen (5-10 microM). The suppressive effect by baclofen was concentration-dependent. The reduction of the EPSC amplitude was reversed by CGP35348. The ratio of the 2nd to 1st EPSCs evoked by paired-pulse stimulation was significantly increased after application of baclofen. These results suggest that glutamatergic excitation in the ICC can be modulated by presynaptic GABA(B) receptors. In addition, baclofen reduced NMDA EPSCs more than AMPA EPSCs. The GABA(B) receptor-mediated modulation of glutamatergic excitation in the ICC provides a likely mechanism for preventing overstimulation and/or regulating the balance of excitation and inhibition involved in processing auditory information.     

Modulation of NMDA receptor properties and synaptic transmission by the NR3A subunit in mouse hippocampal and cerebrocortical neurons

Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-d-aspartate (NMDA)-induced currents and decreased Mg(2+) sensitivity and Ca(2+) permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg(2+) sensitivity, and decreased Ca(2+) permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg(2+) sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extrasynaptic receptors, likely composed of NR1, NR2, and NR3 subunits.     

Interaction of dopamine D1 and NMDA receptors mediates acute clozapine potentiation of glutamate EPSPs in rat prefrontal cortex

The atypical antipsychotic drug clozapine effectively alleviates both negative and positive symptoms of schizophrenia via unclear cellular mechanisms. Clozapine may modulate both glutamatergic and dopaminergic transmission in the prefrontal cortex (PFC) to achieve part of its therapeutic actions. Using whole cell patch-clamp techniques, current-clamp recordings in layers V-VI pyramidal neurons from rat PFC slices showed that stimulation of local afferents (in 2 microM bicuculline) evoked mixed [AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors] glutamate receptor-mediated excitatory postsynaptic potentials (EPSPs). Clozapine (1 microM) potentiated polysynaptically mediated evoked EPSPs (V(Hold) = -65 mV), or reversed EPSPs (rEPSP, V(Hold) = +20 mV) for >30 min. The potentiated EPSPs or rEPSPs were attenuated by elevating [Ca(2+)](O) (7 mM), by application of NMDA receptor antagonist 2-amino5-phosphonovaleric acid (50 microM), or by pretreatment with dopamine D1/D5 receptor antagonist SCH23390 (1 microM) but could be further enhanced by a dopamine reuptake inhibitor bupropion (1 microM). Clozapine had no significant effect on pharmacologically isolated evoked NMDA-rEPSP or AMPA-rEPSPs but increased spontaneous EPSPs without changing the steady-state resting membrane potential. Under voltage clamp, clozapine (1 microM) enhanced the frequency, and the number of low-amplitude (5-10 pA) AMPA receptor-mediated spontaneous EPSCs, while there was no such changes with the mini-EPSCs (in 1 microM TTX). Taken together these data suggest that acute clozapine can increase spike-dependent presynaptic release of glutamate and dopamine. The glutamate stimulates distal dendritic AMPA receptors to increase spontaneous EPSCs and enabled a voltage-dependent activation of neuronal NMDA receptors. The dopamine released stimulates postsynaptic D1 receptor to modulate a lasting potentiation of the NMDA receptor component of the glutamatergic synaptic responses in the PFC neuronal network. This sequence of early synaptic events induced by acute clozapine may comprise part of the activity that leads to later cognitive improvement in schizophrenia.     

Activity-dependent synaptic plasticity in the supraoptic nucleus of the rat hypothalamus

Activity-dependent long-term synaptic changes were investigated at glutamatergic synapses in the supraoptic nucleus (SON) of the rat hypothalamus. In acute hypothalamic slices, high frequency stimulation (HFS) of afferent fibres caused long-term potentiation (LTP) of the amplitude of AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) recorded with the whole-cell patch-clamp technique. LTP was also obtained in response to membrane depolarization paired with mild afferent stimulation. On the other hand, stimulating the inputs at 5 Hz for 3 min at resting membrane potential caused long-term depression (LTD) of excitatory transmission in the SON. These forms of synaptic plasticity required the activation of NMDA receptors since they were abolished in the presence of d -AP5 or ifenprodil, two selective blockers of these receptors. Analysis of paired-pulse facilitation and trial-to-trial variability indicated that LTP and LTD were not associated with changes in the probability of transmitter release, thereby suggesting that the locus of expression of these phenomena was postsynaptic. Using sharp microelectrode recordings in a hypothalamic explant preparation, we found that HFS also generates LTP at functionally defined glutamatergic synapses formed between the organum vasculosum lamina terminalis and SON neurons. Taken together, our findings indicate that glutamatergic synapses in the SON exhibit activity-dependent long-term synaptic changes similar to those prevailing in other brain areas. Such forms of plasticity could play an important role in the context of physiological responses, like dehydration or lactation, where the activity of presynaptic glutamatergic neurons is strongly increased.     

Different composition of glutamate receptors in corticothalamic and lemniscal synaptic responses and their roles in the firing responses of ventrobasal thalamic neurons in juvenile mice

Thalamic ventrobasal (VB) relay neurons receive information via two major types of glutamatergic synapses, that is, from the medial lemniscus (lemniscal synapses) and primary somatosensory cortex (corticothalamic synapses). These two synapses influence and coordinate firing responses of VB neurons, but their precise operational mechanisms are not yet well understood. In this study, we compared the composition of glutamate receptors and synaptic properties of corticothalamic and lemniscal synapses. We found that the relative contribution of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) to non-NMDA receptor-mediated EPSCs was significantly greater in corticothalamic synapses than in lemniscal synapses. Furthermore, NMDA receptor 2B-containing NMDA receptor- and kainate receptor-mediated currents were observed only in corticothalamic synapses, but not in lemniscal synapses. EPSCs in corticothalamic synapses displayed the postsynaptic summation in a frequency-dependent manner, in which the summation of the NMDA receptor-mediated component was largely involved. The summation of kainate receptor-mediated currents also partially contributed to the postsynaptic summation in corticothalamic synapses. In contrast, the contribution of NMDA receptor-mediated currents to the postsynaptic summation of lemniscal EPSCs was relatively minor. Furthermore, our results indicated that the prominent NMDA receptor-mediated component in corticothalamic synapses was the key determinant for the late-persistent firing of VB neurons in response to corticothalamic stimuli. In lemniscal synapses, in contrast, the onset-transient firing in response to lemniscal stimuli was regulated mainly by AMPA receptors.     

Fast NMDA receptor-mediated synaptic currents in neurons from mice lacking the epsilon2 (NR2B) subunit

The N-methyl-D-aspartate (NMDA) receptor has been implicated in the formation of synaptic connections. To investigate the role of the epsilon2 (NR2B) NMDA receptor subunit, which is prominently expressed during early development, we used neurons from mice lacking this subunit. Although epsilon2(-/-) mice die soon after birth, we examined whether NMDA receptor targeting to the postsynaptic membrane was dependent on the epsilon2 subunit by rescuing hippocampal neurons from these mice and studying them in autaptic cultures. In voltage-clamp recordings, excitatory postsynaptic currents (EPSCs) from epsilon2(-/-) neurons expressed an NMDA receptor-mediated EPSC that was apparent as soon as synaptic activity developed. However, compared with wild-type neurons, NMDA receptor-mediated EPSC deactivation kinetics were much faster and were less sensitive to glycine, but were blocked by Mg(2+) or AP5. Whole cell currents from epsilon2(-/-) neurons were also more sensitive to block by low concentrations of Zn(2+) and much less sensitive to the epsilon2-specific antagonist ifenprodil than wild-type currents. The rapid NMDA receptor-mediated EPSC deactivation kinetics and the pharmacological profile from epsilon2(-/-) neurons are consistent with the expression of zeta1/epsilon1 diheteromeric receptors in excitatory hippocampal neurons from mice lacking the epsilon2 subunit. Thus epsilon1 can substitute for the epsilon2 subunit at synapses and epsilon2 is not required for targeting of NMDA receptors to the postsynaptic membrane.     

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.     

AMPA receptor-mediated miniature synaptic calcium transients in GluR2 null mice

AMPA-type glutamate receptors are normally Ca(2+) impermeable due to the expression of the GluR2 receptor subunit. By using GluR2 null mice we were able to detect miniature synaptic Ca(2+) transients (MSCTs) associated with AMPA-type receptor-mediated miniature synaptic currents at single synapses in primary cortical cultures. MSCTs and associated Ca(2+) transients were monitored under conditions that isolated responses mediated by AMPA or N-methyl-D-aspartate (NMDA) receptors. As expected, addition of the antagonist 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 3 microM) blocked the AMPA receptor-mediated MSCTs. Voltage-gated Ca(2+) channels did not contribute to AMPA MSCTs because CdCl(2) (0.1-0.2 mM) did not significantly alter the frequency or the amplitude of the MSCTs. The amplitude of AMPA MSCTs appeared to be regulated independently from event frequency since the two measures were not correlated (R = 0.023). Synapses were identified that only expressed MSCTs attributed to either NMDA or AMPA receptors. At synapses with only NMDA responses, MSCT amplitude was significantly lower (by 40%) than synapses expressing both NMDA and AMPA responses. At synapses that showed MSCTs mediated by both AMPA and NMDA receptors, the amplitude of the transients in each condition was positively correlated (R = 0.94). Our results suggest that when AMPA and NMDA receptors are co-expressed at synapses, mechanisms exist to ensure proportional scaling of each receptor type that are distinct from the presynaptic factors controlling the frequency of miniature release.     

Substrates for coincidence detection and calcium signaling for induction of synaptic potentiation in the neonatal visual cortex

Regulation of the efficacy of synaptic transmission by activity-dependent processes has been implicated in learning and memory as well as in developmental processes. We previously described transient potentiation of excitatory synapses onto layer 2/3 pyramidal neurons in the visual cortex that is induced by coincident presynaptic stimulation and postsynaptic depolarization. In the adult visual cortex, activation of N-methyl-d-aspartate (NMDA) glutamate receptors is necessary to induce this plasticity. These receptors act as coincidence detectors, sensing presynaptic glutamate release and postsynaptic depolarization, and cause an influx of Ca(2+) that is necessary for the potentiation. In the neurons of the neonatal visual cortex, on the other hand, coincident presynaptic stimulation and postsynaptic depolarization induce stable long-term potentiation (LTP). In addition, reduced but significant LTP can be induced in many neurons in the presence of the NMDA receptor (NMDAR) antagonist, 2-amino-5-phosphonovaleric acid despite the Ca(2+) requirement. Therefore there must be an alternative postsynaptic Ca(2+) source and coincidence detection mechanism linked to the LTP induction mechanism in the neonatal cortex operating in addition to NMDARs. In this study, we find that in layer 2/3 pyramidal neurons, release of Ca(2+) from inositol trisphosphate (InsP(3)) receptor-mediated intracellular stores and influx through voltage-gated Ca(2+) channels (VGCCs) provide alternative postsynaptic Ca(2+) sources. We hypothesize that InsP(3)Rs are coincidence detectors, sensing presynaptic glutamate release through linkage with group I metabotropic glutamate receptors (mGluRs), and depolarization, through VGCCs. We also find that the downstream protein kinases, PKA and PKC, have a role in potentiation in layer 2/3 pyramidal neurons of the neonatal visual cortex.     

Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo

Although astrocytes are the most abundant cell type in the brain, evidence for their activation during physiological sensory activity is lacking. Here we show that whisker stimulation evokes increases in astrocytic cytosolic calcium (Ca(2+)) within the barrel cortex of adult mice. Increases in astrocytic Ca(2+) were a function of the frequency of stimulation, occurred within several seconds and were inhibited by metabotropic glutamate receptor antagonists. To distinguish between synaptic input and output, local synaptic activity in cortical layer 2 was silenced by iontophoresis of AMPA and NMDA receptor antagonists. The antagonists did not reduce astrocytic Ca(2+) responses despite a marked reduction in excitatory postsynaptic currents in response to whisker stimulation. These findings indicate that astrocytes respond to synaptic input, by means of spillover or ectopic release of glutamate, and that increases in astrocytic Ca(2+) occur independently of postsynaptic excitatory activity.     

Kainate receptor-mediated synaptic transmission in the adult anterior cingulate cortex

Kainate (KA) receptors are expressed widely in the CNS. However, little is known about their functional characterization, molecular identity, and role in synaptic transmission in the forebrain of adult mice. Patch-clamp recordings in genetically modified mice show that postsynaptic KA receptors contribute to fast synaptic transmission in pyramidal neurons in the anterior cingulate cortex (ACC), a forebrain region critical for higher-order cognitive brain functions such as memory and mental disorders. Single-shock stimulation could induce small KA receptor-mediated excitatory postsynaptic currents (KA EPSCs) in the presence of picrotoxin, D-2-amino-5-phosphono-pentanoic acid, and a selective AMPA receptor antagonist, GYKI 53655. KA EPSCs had a significantly slower rise time course and decay time constant compared with AMPA receptor-mediated EPSCs. High-frequency repetitive stimulation significantly facilitated the KA EPSCs. Genetic deletion of the GluR6 or GluR5 subunit significantly reduced, and GluR5 and 6 double knockout completely abolished, KA EPSCs and KA-activated currents in ACC pyramidal neurons. Our results show that KA receptors contribute to synaptic transmission in adult ACC pyramidal neurons and provide a synaptic basis for the physiology and pathology of KA receptors in ACC-related functions.     

Axotomy-induced change in the properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor channels in rat motoneurons

Properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels were studied in fluorescence-labelled control and axotomized motoneurons in spinal cord slices using a patch-clamp technique. Axotomy performed on the third postnatal day resulted in motoneuron death. Application of AMPA or kainate induced large whole-cell currents, but outside-out patches isolated from control motoneurons were either unresponsive or displayed only single-channel activity in response to rapid application of AMPA. Measurement of AMPA receptor channel openings in outside-out patches revealed multiple single-channel conductance levels: 12.2+/-1.0, 21. 9+/-1.5 and 32.6+/-3.2pS. In control motoneurons dialysed with spermine, the current-voltage relationship of responses induced by activation of AMPA receptor channels exhibited various degrees of inward rectification. The rectification index, the ratio of responses at +40 and -60mV, was used to compare the degree of inward rectification. The mean values of rectification index of responses to focal application of AMPA and AMPA receptor-mediated excitatory postsynaptic currents induced by focal electric stimulation were 0. 64+/-0.17 and 0.50+/-0.27, respectively. In axotomized motoneurons, the degree of rectification was significantly less for both responses induced by application of AMPA and for excitatory postsynaptic currents (0.91+/-0.09 and 0.95+/-0.12, respectively). Deactivation of AMPA receptors assessed from motoneuron excitatory postsynaptic currents at -70 mV was independent of postnatal age, with tau(fast)=0.88+/-0.35ms (A(fast)=78.2+/-11.8%) and tau(slow)=6. 3+/-3.2ms. In axotomized motoneurons, the decay time constants of excitatory postsynaptic currents were similar, tau(fast)=0.91+/-0. 42ms (A(fast)=85.8+/-12.6%) and tau(slow)=5.9+/-3.4ms. However, the mean amplitude of excitatory postsynaptic currents was only 43% of the amplitude recorded in control motoneurons.The results show that the current induced by activation of AMPA receptors in neonatal motoneurons is mediated by opening of both Ca(2+)-permeable and Ca(2+)-impermeable channels. As a result of axotomy, an experimental model of neurodegeneration, AMPA receptor channels in injured motoneurons destined to die become predominantly Ca(2+) impermeable. These findings suggest phenotypic control of AMPA receptor channel properties, presumably by affecting their subunit composition.     

Postsynaptic currents in deep cerebellar nuclei

Postsynaptic currents were studied by whole cell recordings in visually identified large neurons of the deep cerebellar nuclei (DCN) in slices of 4- to 11-day-old mice. Spontaneous postsynaptic currents were abolished by the GABA(A) receptor antagonist bicuculline and had a single-exponential decay with a mean time constant of 13.6 +/- 3.2 (SD) ms. Excitatory postsynaptic currents (EPSCs) were evoked in 48/56 neurons recorded. The addition of AMPA and N-methyl-D-aspartate (NMDA) receptor antagonists together completely abolished all synaptic responses. In 1 mM [Mg(2+)](o) and at a holding potential of -60 mV, the peak amplitude of the NMDA component of the EPSC (NMDA-EPSC) was 83.2 +/- 21.2% of the AMPA component (AMPA-EPSC). This indicates that in DCN neurons, at a physiological [Mg(2+)](o) and at the resting membrane potential, NMDA receptors contribute to the synaptic signal. AMPA-EPSCs had a linear current-voltage relationship with a reversal potential of +2.3 +/- 0.4 mV and a single-exponential decay with a voltage-dependent time constant that at -60 mV was 7.1 +/- 3.3 ms. In 10 microM glycine and 1 mM [Mg(2+)](o), the I-V relationship of NMDA-EPSCs had a reversal potential of -0.5 +/- 3.3 mV and a maximal inward current at -33.4 +/- 5.8 mV. The apparent dissociation constant (K(D)) of Mg(2+) for the NMDA receptor-channel at -60 mV, measured by varying [Mg(2+)](o), was 135.5 +/- 55.3 microM, and when measured by fitting the I-V curves with a theoretical function, it was 169.9 +/- 119.5 microM. Thus in the DCN, NMDA receptors have a sensitivity to Mg(2+) that corresponds to subunits that are weakly blocked by this ion (epsilon 3 and epsilon 4) of which the DCN express epsilon 4. NMDA-EPSCs had a double-exponential decay with voltage-dependent time constants that at -60 mV were 20.2 +/- 8.9 and 136.4 +/- 62.8 ms. At positive voltages, the time constants were slower and their contributions were about equal, while in the negative slope conductance region of the I-V curve, the faster time constant became predominant, conferring faster kinetics to the EPSC. The weak sensitivity to Mg(2+) of NMDA receptors, together with a relatively fast kinetics, provide DCN neurons with strong excitatory inputs in which fast dynamic signals are relatively well preserved.     

Cortical injury affects short-term plasticity of evoked excitatory synaptic currents

The hypothesis that plastic changes in the efficacy of excitatory neurotransmission occur in areas of chronic cortical injury was tested by assessing short-term plasticity of evoked excitatory synaptic currents (EPSCs) in neurons of partially isolated neocortical islands (undercut cortex). Whole cell recordings were obtained from layer V pyramidal neurons of sensorimotor cortical slices prepared from P36-P43 control and undercut rats. AMPA/kainate receptor-mediated EPSCs elicited by stimuli delivered at 40 to 66.7 Hz exhibited more paired-pulse depression (PPD) in undercut cortex than control, the time constant of depression evoked by trains of 20- to 66.7-Hz stimuli was faster, and the steady-state amplitude of EPSCs reached after five to seven EPSCs was lower. An antagonist of the glutamate autoreceptor, group II mGluR, increased the steady-state amplitude of EPSCs from undercut but not control cortex, suggesting that activation of presynaptic receptors by released glutamate is more prominent in undercut cortex. In contrast, the GABA(B) receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid had no effect. Increasing [Ca(2+)](o) from 2 to 4 mM increased PPD, with a smaller effect in neurons of the undercut. The I-V relationship of AMPA/kainate receptor-mediated EPSCs was close to linear in both control and undercut neurons, and spermine had no significant effect on the EPSCs, suggesting that decreases in postsynaptic glutamate receptors containing the GluR2 subunit were not involved in the alterations in short-term plasticity. Results are compatible with an increase in the probability of transmitter release at excitatory synapses in undercut cortex due to functional changes in presynaptic terminals.     

Effects of gamma hydroxybutyric acid on inhibition and excitation in rat neocortex

The mechanism by which the sedative and amnestic recreational drug gamma hydroxybutyric acid (GHB) acts is controversial. Some studies indicate that it acts at its unique receptor, while others demonstrate effects mediated through the GABAB receptor. We examined the effect of GHB on evoked GABAA receptor-mediated mono- and polysynaptic inhibitory postsynaptic currents (IPSCs) as well as on N-methyl-d-aspartate (NMDA) and AMPA-mediated excitatory postsynaptic currents (EPSCs) in layers II/III pyramidal cells of the frontal cortex of rat brain. One millimolar (mM) GHB suppressed monosynaptic IPSCs by 20%, whereas polysynaptic IPSCs were reduced by 56%. GHB (1 mM) also produced a significant suppression of NMDA-mediated EPSCs by 53% compared with 27% suppression of AMPA-mediated EPSCs. All effects of GHB on IPSCs and EPSCs were reversed by the specific GABAB antagonist CGP 62349, but not by the GHB receptor antagonist (2E)-5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene ethanoic acid. Consistent with a presynaptic site of action, GHB reduced the frequency but not the amplitude of AMPA receptor-mediated mEPSCs and had no effect on postsynaptic currents evoked by direct application of NMDA. Finally, even though GHB appeared to be acting at presynaptic GABAB receptors, GHB and the GABAB agonist baclofen appeared to have opposite potencies for depression of NMDA- vs. AMPA-mediated EPSCs. GHB showed a preference for depressing NMDA responses while baclofen more potently suppressed AMPA responses. The suppression of NMDA more than AMPA responses by GHB at intoxicating doses may make it attractive as a recreational drug and may explain why GHB is abused and baclofen is not.     

Muscarinic and nicotinic presynaptic modulation of EPSCs in the nucleus accumbens during postnatal development

We have studied the modulatory effects of cholinergic agonists on excitatory postsynaptic currents (EPSCs) in nucleus accumbens (nAcb) neurons during postnatal development. Recordings were obtained in slices from postnatal day 1 (P1) to P27 rats using the whole cell patch-clamp technique. EPSCs were evoked by local electrical stimulation, and all experiments were conducted in the presence of bicuculline methchloride in the bathing medium and with QX-314 in the recording pipette. Under these conditions, postsynaptic currents consisted of glutamatergic EPSCs typically consisting of two components mediated by AMPA/kainate (KA) and N-methyl-D-aspartate (NMDA) receptors. The addition of acetylcholine (ACh) or carbachol (CCh) to the superfusing medium resulted in a decrease of 30-60% of both AMPA/KA- and NMDA-mediated EPSCs. In contrast, ACh produced an increase ( approximately 35%) in both AMPA/KA and NMDA receptor-mediated EPSCs when administered in the presence of the muscarinic antagonist atropine. These excitatory effects were mimicked by the nicotinic receptor agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) and blocked by the nicotinic receptor antagonist mecamylamine, showing the presence of a cholinergic modulation mediated by nicotinic receptors in the nAcb. The antagonistic effects of atropine were mimicked by pirenzepine, suggesting that the muscarinic depression of the EPSCs was mediated by M(1)/M(4) receptors. In addition, the inhibitory effects of ACh on NMDA but not on AMPA/KA receptor-mediated EPSC significantly increased during the first two postnatal weeks. We found that, under our experimental conditions, cholinergic agonists produced no changes on membrane holding currents, on the decay time of the AMPA/KA EPSC, or on responses evoked by exogenous application of glutamate in the presence of tetrodotoxin, but they produced significant changes in paired pulse ratio, suggesting that their action was mediated by presynaptic mechanisms. In contrast, CCh produced consistent changes in the membrane and firing properties of medium spiny (MS) neurons when QX-314 was omitted from the recording pipette solution, suggesting that this substance actually blocked postsynaptic cholinergic modulation. Together, these results suggest that ACh can decrease or increase glutamatergic neurotransmission in the nAcb by, respectively, acting on muscarinic and nicotinic receptors located on excitatory terminals. The cholinergic modulation of AMPA/KA and NMDA receptor-mediated neurotransmission in the nAcb during postnatal development could play an important role in activity-dependent developmental processes in refining the excitatory drive on MS neurons by gating specific inputs.     

Removal of NMDA receptor Mg(2+) block extends the action of NT-3 on synaptic transmission in neonatal rat motoneurons.

NT-3 has previously been reported to enhance AMPA/kainate receptor-mediated synaptic responses in motoneurons via an effect on the N-methyl-D-aspartate (NMDA) receptor. To investigate neurotrophin-3 (NT-3) action further, we measured the NMDA receptor (NMDAR)-mediated synaptic response directly by intracellular recording in motoneurons after blocking AMPA/kainate, GABA(A), GABA(B) and glycine receptor-mediated responses pharmacologically. Two pathways were stimulated, the segmental dorsal root (DR) and the descending ventrolateral fasciculus (VLF). The DR-evoked NMDAR-mediated response in motoneurons of rats younger than 1 wk has two components, the initial one of which is generated monosynaptically. NT-3 strongly potentiated both NMDA components in a rapidly reversible manner. No NMDAR-mediated responses were present at VLF connections and at DR connections in older (1- to 2-wk-old) neonates. Bath-applied NT-3-induced potentiation of the AMPA/kainate receptor-mediated response occurred only at connections that exhibit a synaptic NMDA receptor-mediated response. Reducing Mg(2+) concentration in the bathing solution restored the NMDAR-mediated response elicited by DR stimulation in older neonates and by VLF throughout the neonatal period (0-2 wk). In low-Mg(2+), NT-3 enhanced AMPA/kainate receptor-mediated responses elicited by inputs normally not influenced by NT-3. Thus a major reason for the loss of NT-3 action on AMPA/kainate synaptic responses is the reduced activity of the NMDA receptor due to developing Mg(2+) block of NMDA receptor-channel complex as the animal matures, and both can be re-established by reducing Mg(2+) concentration in fluid bathing the spinal cord.     

A model of NMDA receptor-mediated activity in dendrites of hippocampal CA1 pyramidal neurons.

1. The role of synaptic activation of NMDA (N-methyl-D-aspartate) receptor-mediated conductances on CA1 hippocampal pyramidal cells in short-term excitability changes was studied with the use of a computational model. Model parameters were based on experimental recordings from dendrites and somata and previous hippocampal simulations. Representation of CA1 neurons included NMDA and non-NMDA excitatory dendritic synapses, dendritic and somatic inhibition, five intrinsic membrane conductances, and provision for activity-dependent intracellular and extracellular ion concentration changes. 2. The model simulated somatic and dendritic potentials recorded experimentally. The characteristic CA1 spike afterdepolarization was a consequence of the longitudinal spread of dendritic charge, reactivation of slow Ca(2+)-dependent K+ conductances, slow synaptic processes (NMDA-dependent depolarizing and gamma-aminobutyric acid-mediated hyperpolarizing currents) and was sensitive to extracellular potassium accumulation. Calcium currents were found to be less important in generating the spike afterdepolarization. 3. Repetitive activity was influenced by the cumulative activation of the NMDA-mediated synaptic conductances, the frequency-dependent depression of inhibitory synaptic responses, and a shift in the potassium reversal potential. NMDA receptor activation produced a transient potentiation of the excitatory postsynaptic potential (EPSP). The frequency dependence of EPSP potentiation was similar to the experimental data, reaching a maximal value near 10 Hz. 4. Although the present model did not have compartments for dendritic spines, Ca2+ accumulation was simulated in a restricted space near the intracellular surface of the dendritic membrane. The simulations demonstrated that the Ca2+ component of the NMDA-operated synaptic current can be a significant factor in increasing the Ca2+ concentration at submembrane regions, even in the absence of Ca2+ spikes. 5. Elevation of the extracellular K+ concentration enhanced the dendritic synaptic response during repetitive activity and led to an increase in intracellular Ca2+ levels. This increase in dendritic excitability was partly mediated by NMDA receptor-mediated conductances. 6. Blockade of Ca(2+)-sensitive K+ conductances in the dendrites increased the size of EPSPs leading to a facilitation of dendritic and somatic spike activity and increased [Ca2+]i. NMDA receptor-mediated conductances appeared as an amplifying component in this mechanism, activated by the relatively depolarized membrane potential. 7. The results suggest that dendritic NMDA receptors, by virtue of their voltage-dependency, can interact with a number of voltage-sensitive conductances to increase the dendritic excitatory response during periods of repetitive synaptic activation. These findings support experimental results that implicate NMDA receptor-mediated conductances in the short-term response plasticity of the CA1 hippocampal pyramidal neuron.     

Redox modulation of synaptic responses and plasticity in rat CA1 hippocampal neurons

Effects of redox reagents on excitatory and inhibitory synaptic responses as well as on the bidirectional plasticity of α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) andN-methyl-d-aspartate (NMDA) receptor-mediated synaptic responses were studied in CA1 pyramidal neurons in rat hippocampal slices. The oxidizing agent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, 200 μM) did not affect AMPA, GABA_A or GABA_B receptor-mediated synaptic responses or the activation of presynaptic metabotropic receptors. However, DTNB irreversibly decreased (by approximately 50%) currents evoked by focal application of NMDA. DTNB also decreased the NMDA component of the EPSC. The reversal potential of NMDA currents and the Mg²⁺ block were not modified. In the presence of physiological concentrations of Mg²⁺ (1.3 mM), DTNB did not affect the NMDA receptor-dependent induction of long-term potentiation (LTP) or long-term depression (LTD) expressed by AMPA receptors. In contrast, DTNB fully prevented LTP and LTD induced and expressed by NMDA receptors. Plasticity of NMDA receptor-mediated synaptic responses could be reinstated by the reducing agenttris-(2-carboxyethyl) phosphine (TCEP, 200 μM). These results suggest that persistent, bidirectional changes in synaptic currents mediated by NMDA receptors cannot be evoked when these receptors are in an oxidized state, whereas NMDA-dependent LTP and LTD are still expressed by AMPA receptors. Our observations raise the possibility of developing therapeutic agents that would prevent persistent excitotoxic enhancement of NMDA receptor-mediated events without blocking long-term modifications of AMPA receptor-mediated synaptic responses, thought to underlie memory processes.