EPSPs in rat neocortical pyramidal neurones in vitro are prolonged by NMDA receptor-mediated currents.

We investigated the influence of 2-amino-5-phosphonovalerate (APV), a selective antagonist of the N-methyl-D-aspartate (NMDA) receptor, on the time course of small excitatory postsynaptic potentials (EPSPs) in pyramidal neurones in layer 2/3 of adult rat visual cortex in vitro. Time constants of the voltage decay following the EPSPs (T(s)) and after brief (2 ms) pulses of current injected at the soma (T(p)) were determined from semilogarithmic plots of averages of 100-250 trials. The mean T(s)/T(p) ratio decreased from 1.53 +/- 0.29 (S.D.) to 1.10 +/- 0.08 on addition of 50 microM APV to the bathing medium (P less than 0.001; n = 23), but there was no significant change in EPSP peak amplitude or rise-time. These results suggest that the time course of many small EPSPs, even at negative membrane potentials and in the presence of Mg2+, can be prolonged by NMDA receptor-mediated currents.     

Burst generation in neocortical neurons after GABA withdrawal in the rat.

1. gamma-Aminobutyric acid (GABA) withdrawal syndrome (GWS) represents a particular model of focal epilepsy consecutive to the interruption of a chronic intracortical GABA infusion and is characterized by the appearance of focal epileptic electroencephalographic (EEG) discharges and localized clinical signs on withdrawal of GABA. Effects of Ca2+ channel blockers and N-methyl-D-aspartate (NMDA) antagonists were evaluated in living rats presenting a GWS after interruption of a 5-day GABA infusion into the somatomotor cortex and in neocortical slices obtained from such rats. Bursting properties and morphology of neurons were also analyzed in slices. 2. In living rats, the noncompetitive NMDA antagonist phencyclidine [1-(1-phenylcyclohexyl)piperidine] and the Ca2+ antagonist flunarizine [E-1 (bis(4fluorophenyl)methyl)-4(3phenyl2-propenyl)-piperazine] were administered systemically to two groups of rats. Rats in the first group (n = 12) were injected with the drug 30-60 min before discontinuation of the GABA infusion. In this case, phencyclidine (10 mg/kg ip) prevented the development of GWS (n = 5), whereas flunarizine (40 mg/kg ip) had no consistent effect on the GWS appearance and characteristics (n = 7). Rats in the second group (n = 12) were injected 60-90 min after GABA discontinuation, i.e., during a fully developed GWS. In that case, neither drug suppressed GWS. 3. Neuronal activities in the epileptic focus were studied in slices with conventional intracellular recording and stimulation techniques. From the 65 neurons recorded, 29 responded with EPSPs and paroxysmal depolarization shifts (PDSs) to white matter stimulation (synaptic bursting or SB cells). Nineteen other neurons presented, in addition to synaptically induced PDSs, bursts of action potentials (APs) induced by intracellular depolarizing current injection (intrinsic bursting or IB cells). The remaining 17 neurons presented no bursting properties to either synaptic stimulation or depolarizing current injection (nonbursting or NB cells). 4. The recorded neurons were located 0.7-1.2 mm distant from the lesion because of the penetration of the GABA infusion cannula. Intracellular injection of neurons (n = 4) with biocytin or Lucifer yellow revealed that both SB and IB neurons were large, spiny pyramidal neurons localized in layer V of the sensorimotor cortex. 5. Bath application of the selective antagonist of NMDA receptors DL-2amino-5phosphonovalerate or DL-2amino-7phosphonoheptanoate (10-50 microM) reversibly reduced the amplitude (by 25-50%) and the duration (by 20-25%) of PDSs in all cases (n = 17).(ABSTRACT TRUNCATED AT 400 WORDS)     

Linear to supralinear summation of AMPA-mediated EPSPs in neocortical pyramidal neurons

It has been hypothesized that voltage-sensitive conductances present on the dendrites of neurons can influence summation of excitatory postsynaptic potentials (EPSPs) and hence affect how neurons compile information. Greater than linear summation of EPSPs has been postulated to facilitate coincidence detection by cortical neurons. This study examined whether the summation of subthreshold AMPA-mediated EPSPs generated on layer V neocortical pyramidal neurons in vitro was linear and if any nonlinearities could be attributed to dendritic conductances. Evoked EPSPs (1-12 mV) were recorded somatically by means of intracellular sharp electrodes in the presence of 100 microM amino-5-phosphonopentanoic acid (AP-5) and 3 microM bicuculline. Two independent EPSPs were evoked by a stimulating electrode in layer I and another in layers III-V. The areas of stimulation were isolated from each other by a horizontal cut below layer I. By subtracting the algebraic sum of the individual EPSPs from the evoked response when both EPSPs were evoked simultaneously, we determined that they summed linearly to supralinearly. Supralinear summation was more likely when the soma was hyperpolarized by DC current injection. Summation was predominantly linear when postsynaptic conductances (i.e., Na(+) and Ca(2+)) were blocked with intracellular QX-314. The supralinear summation of EPSPs (without QX-314) decreased as the time between inputs was increased from 0 to 30 ms. To determine the role of dendrites in nonlinear summation, we substituted a current pulse (simulated EPSP) delivered at the soma for either or both of the evoked EPSPs. Simulated EPSPs combined with either an evoked EPSP or another simulated EPSP showed significantly less supralinear summation than two evoked EPSPs, indicating that the dendritic conductances were largely responsible for the observed supralinear summation.     

Electrophysiological properties of neocortical neurons in vitro

1. Intracellular recordings were obtained from neurons of the guinea pig sensorimotor cortical slice maintained in vitro. Under control recording conditions input resistances, time constants, and spiking characteristics of slice neurons were well within the ranges reported by other investigators for neocortical neurons in situ. However, resting potentials (mean of -75 mV) and spike amplitudes (mean of 93.5 mV) were 10-25 mV greater than has been observed in intact preparations. 2. Current-voltage relationships obtained under current clamp revealed a spectrum of membrane-rectifying properties at potentials that were subthreshold for spike generation. Ionic and pharmacologic analyses suggest that subthreshold membrane behavior is dominated by voltage-sensitive, very slowly inactivating conductances to K+ and Na+. 3. Action potentials were predominantly Na+ dependent under normal conditions but when outward K+ currents were reduced pharmacologically, it was possible, in most cells, to evoke a non-Na+-dependent, tetrodotoxin-(TTX) insensitive spike, which was followed by a prominent depolarizing after-potential. Both of these events were blocked by the Ca2+ current antagonists, Co2+ and Mn2+. 4. A small population of neurons generated intrinsic, all-or-none burst potentials when depolarized with current pulses or by synaptic activation. These cells were located at a narrow range of depths comprising layer IV and the more superficial parts of layer V. 5. Spontaneous excitatory synaptic potentials appeared in all neurons. Spontaneous inhibitory events were visible in only about 10% of the cells, and in those cases apparently reversed polarity at a level slightly positive to resting potential. Stimulation of the surface of the slice at low intensities evoked robust and usually concurrent excitatory and inhibitory synaptic potentials. Unitary inhibitory postsynaptic potentials (IPSPs) reversed at levels positive to rest. Stronger stimulation produced a labile, long-duration, hyperpolarizing IPSP with a reversal potential 15-20 mV negative to the resting level. 6. Neocortical neurons in vitro retain the basic membrane and synaptic properties ascribed to them in situ. However, the array of passive and active membrane behavior observed in the slice suggests that cortical neurons may be differentiated by specific functional properties as well as by their extensive morphological diversity.     

Involvement of N-methyl-D-aspartate receptors for the Ptychodiscus brevis toxin-induced depression of monosynaptic and polysynaptic reflexes in neonatal rat spinal cord in vitro

The effects of Ptychodiscus brevis toxin (PbTx) on the Ia-alpha motoneuron synaptic transmission in neonatal rat spinal cord in vitro was examined. The stimulation of a dorsal root evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root in Mg2+-free medium. Superfusion with PbTx (2.8-84 microM) depressed the MSR and the PSR in a concentration-dependent manner. At 2.8 microM of PbTx, the depression of MSR and PSR was 24+/-8.3% and 37+/-9.7%, respectively. The maximal depression was seen at 84 microM of the toxin (78% for MSR and 96% for PSR). The concentration of toxin required to produce 50% depression was 28.3+/-6.4 microM for MSR and 5.5+/-1.1 microM for PSR. The PbTx (28 microM) did not alter the magnitude of the dorsal root or the ventral root potentials. Addition of MgSO4 (1.3 mM) or DL-2-amino-5-phosphonovaleric acid (APV; 10 microM) to the physiological solution abolished the PSR totally and decreased the MSR by about 30%. In both the conditions, the PbTx-induced depression of the MSR was attenuated significantly. The PbTx-induced depression was blocked completely in the presence of APV+6-cyano-7-nitroquinoxaline-2,3-dione (0.1 microM). NMDA (1 microM) by itself did not alter the magnitude of MSR or PSR but enhanced the PbTx-induced depression (28 microM) of PSR significantly. 7-Chlorokynurenic acid (3 microM; glycine(B) antagonist) did not block the PbTx-induced depression of MSR. D-serine (glycine(B) agonist) did not reverse the PbTx-induced depression of reflexes although it reversed the 7-chlorokynurenic acid-induced depression of PSR.The results indicate that the PbTx depressed the spinal reflexes without altering the magnitude of dorsal root or ventral root activity. The depression of the PSR involved NMDA receptors while that of the MSR involved NMDA and non-NMDA receptors. The PbTx actions did not involve the glycine(B) site of the NMDA receptor.     

D-baclofen does not antagonize the actions of L-baclofen on rat neocortical neurons in vitro

The ability of D-baclofen to antagonize the actions of L-baclofen on rat neocortical neurons was investigated. Intracellular recordings were made from neurons in cortical layers 2 and 3 in an in vitro slice preparation. Baclofen stereoisomers were applied at known concentrations in the superfusion medium. At a concentration of 3 microM, L-baclofen produced approximately 70% depressions of excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) that were evoked by stimulation of superficial cortical layers. L-baclofen also hyperpolarized neocortical neurons. These hyperpolarizations were accompanied by decreases in neuronal input resistance and in direct excitability. We have shown previously that these latter effects are secondary to the action of baclofen to increase the potassium conductance of neocortical neurons. D-baclofen, at concentrations of 1-100 microM, did not antagonize depressions by L-baclofen of EPSPs and IPSPs nor the action of L-baclofen to increase the potassium conductance of neocortical neurons. At concentrations of 50-100 microM, D-baclofen produced 20-30% effects when applied alone, thus suggesting that these concentrations of D-baclofen produced a significant degree of receptor occupancy. Our results demonstrate that D-baclofen is not an antagonist or high affinity partial agonist at the receptors through which baclofen exerts its effects on single neurons in the rat neocortex.     

Electrophysiological evidence for N-methyl-D-aspartate excitatory amino acid receptors in the rat supraoptic nucleus in vitro.

The responses of neurons in slices of the rat supraoptic nucleus (SON) to afferent stimulation were recorded under current-clamp conditions. In magnesium (Mg2+)-free incubation medium, synaptic responses were prolonged and were partially antagonized by the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist (+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). During blockade of non-NMDA excitatory amino acid (EAA) receptors, the synaptic responses in Mg(2+)-free medium were blocked by the competitive NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5). The results of these experiments provide electrophysiological evidence for the existence of NMDA receptors in the rat SON.     

N-methyl-D-aspartate receptors on neurons that synthesize nitric oxide in rat nucleus tractus solitarii

The aim of this study was to determine whether neuronal nitric oxide synthase and N-methyl-D-aspartate receptors are co-localized in the rat nucleus tractus solitarii. Such co-localization would support the hypothesis that nitric oxide participates in nucleus tractus solitarii-mediated functions, such as cardiovascular regulation, by a link to N-methyl-D-aspartate receptors. We used double fluorescent immunohistochemistry using antibodies against neuronal nitric oxide synthase and N-methyl-D-aspartate receptor subunit 1, the fundamental subunit for functional N-methyl-D-aspartate receptors. Labeled brainstem sections were examined with confocal laser scanning microscopy. Most of the N-methyl-D-aspartate receptor subunit 1 immunoreactivity was in cell bodies and proximal dendrites of the numerous labeled cells in the brainstem. High levels of N-methyl-D-aspartate receptor subunit 1 immunoreactivity were present in the dorsal motor nucleus of vagus, hypoglossal nucleus and nucleus ambiguus. All subnuclei of the nucleus tractus solitarii contained moderate levels of N-methyl-D-aspartate receptor subunit 1 immunoreactivity. The distribution of neuronal nitric oxide synthase immunoreactivity in the nucleus tractus solitarii was similar to that described in earlier reports. Superimposition of images revealed that almost all neuronal nitric oxide synthase immunoreactive neurons in the nucleus tractus solitarii contained N-methyl-D-aspartate receptor subunit 1 immunoreactivity, but a lesser portion of N-methyl-D-aspartate receptor subunit 1-immunoreactive cells contained neuronal nitric oxide synthase immunoreactivity. Although all nucleus tractus solitarii subnuclei contained double-labeled neurons, the central subnucleus exhibited the highest density of double-labeled neurons.Co-localization of neuronal nitric oxide synthase and N-methyl-D-aspartate receptor subunit 1 in the nucleus tractus solitarii provides anatomical support for the hypothesis that N-methyl-D-aspartate receptor activation can affect nucleus tractus solitarii-controlled functions via actions on neurons that synthesize nitric oxide.     

Excitatory amino acid receptors involved in primary afferent-evoked polysynaptic EPSPs of substantia gelatinosa neurons in the adult rat spinal cord slice.

Intracellular recordings were made from substantia gelatinosa (SG) neurons in spinal cord slices to determine a subclass of excitatory amino acid receptors involved in polysynaptic excitatory postsynaptic potentials (EPSPs). In the majority of neurons, polysynaptic EPSPs evoked by A delta fiber were not affected by 2-amino-5-phosphonovaleric acid (APV), while all EPSPs including monosynaptic EPSPs were depressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). All spontaneous EPSPs were blocked by CNQX, while spontaneous EPSPs in a few SG neurons were attenuated by APV. These observations suggest that polysynaptic EPSPs evoked through A delta fibers are predominantly mediated by activation of the non-N-methyl-D-aspartate (non-NMDA) receptor subclass.     

Activation of N-methyl-D-aspartate receptors evokes calcium spikes in the dendrites of rat hypothalamic paraventricular nucleus neurons

Activation of dendritic voltage-dependent calcium (Ca2+) conductances in neuroendocrine cells of the hypothalamus may underlie previously documented Ca2+ spikes in these cells. The present study, in which whole-cell recordings were obtained from paraventricular nucleus neurons in a hypothalamic slice preparation, addresses this issue by directly activating dendritic N-methyl-D-aspartate receptors in the presence of tetrodotoxin. Application of tetrodotoxin abolished spontaneous action potentials in all paraventricular nucleus neurons tested (n = 27). Following tetrodotoxin, spikes were evoked by depolarizing current pulses, in an all-or-none fashion in the majority of cells (n = 20). Removal of extracellular Ca2+ (n = 6) or addition of 500 microM CdCl2 (n = 4) abolished the spikes in response to pulses. Repetitive spiking activity (in tetrodotoxin) was also observed following N-methyl-D-aspartate agonist application in 75% of the cells tested (n = 15). The spikes, underscored by a slow membrane depolarization, were abolished by the administration of CdCl2 (n = 4). N-Methyl-D-aspartate agonist elicited a slow inward current in cells voltage-clamped at -60 mV (n = 5). Additionally, larger amplitude, transient inward currents were observed near the onset of the response. The activation threshold to elicit spikes following N-methyl-D-aspartate agonist application was significantly more negative (-54.6+/-3.6 mV) than the potential at which spikes were initiated as a result of depolarizing current injection (-32.3+/-1.8 mV; Student's t-test: P < 0.0001). In contrast to this, Na+ spikes in control solution had an invariable threshold (-49.6+/-0.7 mV vs -51.5+/-1.2 mV; P > 0.05), regardless of the stimulus used to initiate the spikes. These observations suggest that direct activation of N-methyl-D-aspartate receptors located on the dendrites of paraventricular nucleus neurons triggers Ca2+ spikes. Although the precise function of these spikes is unclear, previous data reporting dendritic neuropeptide release in the paraventricular nucleus raise the possibility that dendritically initiated spikes may serve as a local signal to trigger such release.     

Confocal imaging of N-methyl-D-aspartate receptors in living cortical neurons

The fluorescence-conjugated N-methyl-D-aspartate receptor-selective antagonist, BODIPY-conantokin-G, was employed to label N-methyl-D-aspartate receptors in living neurons derived from the visual cortex of embryonic rats. The fluorescent labeling was visualized and analysed using confocal microscopy and digital imaging techniques. BODIPY-conantokin-G binding sites were homogeneously distributed across somata four days after neurons (E17-20) were placed in culture. In five-day-old cultures, BODIPY-conantokin-G binding sites became clusters of fluorescently labeled spots which were arranged irregularly on somata and proximal neurites. Distal neurites displayed fluorescent labeling after 10-15 days in culture. Displacement experiments showed that spermine and unlabeled conantokin-G compete with BODIPY-conantokin-G labeling at the N-methyl-D-aspartate receptor-associated polyamine site. The N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid also depressed the labeling but with a weaker effect, probably due to interactions occurring between the N-methyl-D-aspartate receptor agonist binding site and the polyamine modulatory site. The fluorescent dyes FM 1-43 and FM 4-64 were used in double-labeling studies to compare the distribution of nerve terminals with that of BODIPY-conantokin-G binding sites. BODIPY-conantokin-G binding clusters were associated with presynaptic nerve terminals while isolated BODIPY-conantokin-G binding sites were not always opposed to terminals. The aggregation of receptors to form clusters may lead to the functional formation of excitatory synapses. To investigate whether modulation of membrane potentials affected the formation of N-methyl-D-aspartate receptor clusters, cultured neurons were chronically treated for a week with either tetrodotoxin (to block membrane action potentials) or a high concentration of potassium to depolarize the membrane. While neurons in the tetrodotoxin-treated group showed a similar number of fluorescently labeled clusters compared with the control group, neurons in the high potassium group exhibited a higher number of fluorescently labeled receptor clusters. These results suggest that more active neurons may tend to form more N-methyl-D-aspartate synapses during early development.     

Excitatory postsynaptic potentials in rat neocortical neurons in vitro. III. Effects of a quinoxalinedione non-NMDA receptor antagonist

1. Intracellular microelectrodes were used to obtain recordings from neurons in layer II/III of rat frontal cortex. A bipolar electrode positioned in layer IV of the neocortex was used to evoke postsynaptic potentials. Graded series of stimulation were employed to selectively activate different classes of postsynaptic responses. The sensitivity of postsynaptic potentials and iontophoretically applied neurotransmitters to the non-N-methyl-D-asparate (NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was examined. 2. As reported previously, low-intensity electrical stimulation of cortical layer IV evoked short-latency early excitatory postsynaptic potentials (eEPSPs) in layer II/III neurons. CNQX reversibly antagonized eEPSPs in a dose-dependent manner. Stimulation at intensities just subthreshold for activation of inhibitory postsynaptic potentials (IPSPs) produced long-latency (10 to 40-ms) EPSPs (late EPSPs or 1EPSPs). CNQX was effective in blocking 1EPSPs. 3. With the use of stimulus intensities at or just below threshold for evoking an action potential, complex synaptic potentials consisting of EPSP-IPSP sequences were observed. Both early, Cl(-)-dependent and late, K(+)-dependent IPSPs were reduced by CNQX. This effect was reversible on washing. This disinhibition could lead to enhanced excitability in the presence of CNQX. 4. Iontophoretic application of quisqualate produced a membrane depolarization with superimposed action potentials, whereas NMDA depolarized the membrane potential and evoked bursts of action potentials. At concentrations up to 5 microM, CNQX selectively antagonized quisqualate responses. NMDA responses were reduced by 10 microM CNQX. D-Serine (0.5-2 mM), an agonist at the glycine regulatory site on the NMDA receptor, reversed the CNQX depression of NMDA responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quinolinate activation of N-methyl-D-aspartate ion channels in rat hippocampal neurons.

The cell attached configuration of the patch clamp method has been used to determine the single channel properties of the ion channel coupled to activation of the N-methyl-D-aspartate (NMDA) receptor by the endogenous NMDA agonist quinolinate. Openings of the NMDA channel were recorded from cultured CA1 hippocampal neurons over a hyperpolarizing potential range from cell resting potential. The slope conductance of the channel was 39 pS with 75 microM quinolinate, 1.8 mM Ca2+ and no Mg2+ in the patch pipette. The mean channel open times were decreased with hyperpolarization in an exponential manner with a mean slope of 0.6 ms/20 mV. Addition of Mg2+ to the pipette (at 30 microM) caused the mean open time, at a potential of -100 mV, to be decreased to a value about one-third that of control. The mean open times with quinolinate as the agonist were shorter for all potentials studied compared with activation of the NMDA receptor with NMDA or D-cis-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD). Both the mean open times and the channel amplitudes were significantly altered when the bath temperature was decreased; the Q10 values for both quantities were in excess of 2.8.