EPSPs in rat neocortical neurons in vitro. II. Involvement of N-methyl-D-aspartate receptors in the generation of EPSPs

1. Intracellular recordings were obtained from neurons in layer II/III of rat frontal cortex. Single-electrode current- and voltage-clamp techniques were employed to compare the sensitivity of excitatory postsynaptic potentials (EPSPs) and iontophoretically evoked responses to N-methyl-D-aspartate (NMDA) to the selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-2-APV). The voltage dependence of the amplitudes of the EPSPs before and after pharmacologic changes in the neuron's current-voltage relationship was also examined. 2. NMDA depolarized the membrane potential, increased the neuron's apparent input resistance (RN), and evoked bursts of action potentials. The NMDA-induced membrane current (INMDA) gradually increased with depolarization from -80 to -40 mV. The relationship between INMDA and membrane potential displayed a region of negative slope conductance in the potential range between -70 and -40 mV which was sufficient to explain the apparent increase in RN and the burst discharges during the NMDA-induced depolarization. 3. Short-latency EPSPs (eEPSPs) were evoked by low-intensity electrical stimulation of cortical layer IV. Changes in the eEPSP waveform following membrane depolarization and hyperpolarization resembled those of NMDA-mediated responses. However, the eEPSP was insensitive to D-2-APV applied at concentrations (up to 20 microM) that blocked NMDA responses. 4. EPSPs with latencies between 10 and 40 ms [late EPSPs (lEPSPs)] were evoked by electrical stimulation using intensities just subthreshold to the activation of IPSPs. The amplitude of the lEPSP increased with hyperpolarization and decreased with depolarization. 5. The lidocaine derivative QX-314, injected intracellularly, suppressed sodium-dependent action potentials and depolarizing inward rectification. Simultaneously, the amplitude of the eEPSP significantly decreased with depolarization. Neither the amplitude of a long-latency EPSP nor the amplitude of inhibitory postsynaptic potentials (IPSPs) was significantly affected by QX-314. 6. Cesium ions (0.5-2.0 mM) added to the bathing solution reduced or blocked hyperpolarizing inward rectification. Under these conditions, the amplitude of the eEPSP increased with hyperpolarization. The amplitude of the lEPSP was unaltered or enhanced. 7. The lEPSP was reversibly blocked by D-2-APV (5-20 microM), although the voltage-dependence of its amplitude did not resemble the action of NMDA on neocortical neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excitatory transmission in the basolateral amygdala.

1. Intracellular current-clamp recordings obtained from neurons of the basolateral nucleus of the amygdala (BLA) were used to characterize postsynaptic potentials elicited through stimulation of the stria terminalis (ST) or the lateral amygdala (LA). The contribution of glutamatergic receptor subtypes to excitatory postsynaptic potentials (EPSPs) were analyzed by the use of the non N-methyl-D-aspartate (non-NMDA) antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), and the NMDA antagonist, (DL)-2-amino-5-phosphonovaleric acid (APV). 2. Basic membrane properties of BLA neurons determined from membrane responses to transient current injection showed that at the mean resting membrane potential (RMP; -67.2 mV) the input resistance (RN) and time constant for membrane charging (tau) were near maximal, and that both values were reduced with membrane hyperpolarization, suggesting an intrinsic regulation of synaptic efficacy. 3. Responses to stimulation of the ST or LA consisted of an EPSP followed by either a fast inhibitory postsynaptic potential (f-IPSP) only, or by a fast- and subsequent slow-IPSP (s-IPSP). The EPSP was graded in nature, increasing in amplitude with increased stimulus intensity, and with membrane hyperpolarization after DC current injection. Spontaneous EPSPs were also observed either as discrete events or as EPSP/IPSP waveforms. 4. In physiological Mg2+ concentrations (1.2 mM), at the mean RMP, the EPSP consisted of dual, fast and slow, glutamatergic components. The fast-EPSP (f-EPSP) possessed characteristics of kainate/quisqualate receptor activation, namely, the EPSP increased in amplitude with membrane hyperpolarization, was insensitive to the NMDA receptor antagonist, APV (50 microM), and was blocked by the non-NMDA receptor antagonist, CNQX (10 microM). In contrast, the slow-EPSP (s-EPSP) decreased in amplitude with membrane hyperpolarization, was insensitive to CNQX (10 microM), and was blocked by APV (50 microM), indicating mediation by NMDA receptor activation. 5. In the presence of CNQX (10 microM), ST stimulation evoked an APV-sensitive s-EPSP. In contrast, LA stimulation evoked a f-IPSP, which when blocked by subsequent addition of bicuculline methiodide (BMI; 30 microM) revealed a temporally overlapping APV-sensitive s-EPSP. These data suggest that EPSP amplitude and duration are determined, in part, by the shunting of membrane conductance caused by a concomitant IPSP. 6. Superfusion of either CNQX or APV in BLA neurons caused membrane hyperpolarization and blockade of spontaneous EPSPs and IPSPs, suggesting that these compounds may act to block tonic excitatory amino acid (EAA) release within the nucleus, and that a degree of feed-forward inhibition occurs within the nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices.

1. Conventional intracellular and extracellular recording techniques were used to investigate the physiology and pharmacology of epileptiform bursts induced by 4-aminopyridine (4-AP, 50 microM) in the CA3 area of rat hippocampal slices maintained in vitro. 2. 4-AP-induced epileptiform bursts, consisting of a 25-to 80-ms depolarizing shift of the neuronal membrane associated with three to six fast action potentials, occurred at the frequency of 0.61 +/- 0.29 (SD)/s. The bursts were generated synchronously by CA3 neurons and were triggered by giant excitatory postsynaptic potentials (EPSPs). A second type of spontaneous activity consisting of a slow depolarization also occurred but at a lower rate (0.04 +/- 0.2/s). 3. The effects of 4-AP on EPSPs and inhibitory postsynaptic potentials (IPSPs) evoked by mossy fiber stimulation were studied on neurons impaled with a mixture of K acetate and 2(triethyl-amino)-N-(2,6-dimethylphenyl) acetamide (QX-314)-filled microelectrodes. After the addition of 4-AP, the EPSP became potentiated and was followed by the appearance of a giant EPSP. This giant EPSP completely obscured the early IPSP recorded under control conditions and inverted at -32 +/- 3.9 mV (n = 4), suggesting that both inhibitory and excitatory conductances were involved in its generation. IPSPs evoked by Schaffer collateral stimulation increased in amplitude and duration after 4-AP application. 4. The spontaneous field bursts and the stimulus-induced giant EPSP induced by 4-AP were not affected by N-methyl-D-aspartate (NMDA) receptor antagonists 3-3 (2-carboxy piperazine-4-yl) propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV) but were blocked by quisqualate/kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). CNQX also abolished the presence of small spontaneously occurring EPSPs, thereby disclosing the presence of bicuculline-sensitive (BMI, 20 microM) IPSPs. 5. Small, nonsynchronous EPSPs played an important role in the generation of 4-AP-induced epileptiform activity. 1) After the addition of 4-AP, small EPSPs appeared randomly on the baseline and then became clustered to produce a depolarizing envelope of irregular shape that progressively formed an epileptiform burst, 2) These small EPSPs were more numerous in the 100 ms period that preceded burst onset. 3) The frequency of occurrence of small EPSPs was positively correlated with the frequency of occurrence of synchronous bursts. 4) Small EPSPs and bursts were similarly decreased after the addition of different concentrations of CNQX (IC50 in both cases of approximately 1.2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of synaptic transmission in hippocampal CA1 neurons by a novel neurotoxin (beta-pompilidotoxin) derived from wasp venom.

We examined the effects of beta-pompilidotoxin (beta-PMTX), a neurotoxin derived from wasp venom, on synaptic transmission in the mammalian central nervous system (CNS). Using hippocampal slice preparations of rodents, we made both extracellular and intracellular recordings from the CA1 pyramidal neurons in response to stimulation of the Schaffer collateral/commissural fibers. Application of 5-10 microM beta-PMTX enhanced excitatory postsynaptic potentials (EPSPs) but suppressed the fast component of the inhibitory postsynaptic potentials (IPSPs). In the presence of 10 microM bicuculline, beta-PMTX potentiated EPSPs that were composed of both non-NMDA and NMDA receptor-mediated potentials. Potentiation of EPSPs was originated by repetitive firings of the presynaptic axons, causing summation of EPSPs. In the presence of 10 microM CNQX and 50 microM APV, beta-PMTX suppressed GABA(A) receptor-mediated fast IPSPs but retained GABA(B) receptor-mediated slow IPSPs. Our results suggest that beta-PMTX facilitates excitatory synaptic transmission by a presynaptic mechanism and that it causes overexcitation followed by block of the activity of some population of interneurons which regulate the activity of GABA(A) receptors.     

Synaptic mechanisms of inspiratory off-switching evoked by pontine pneumotaxic stimulation in cats

To elucidate synaptic mechanisms and the involvement of N-methyl-D-aspartate (NMDA) receptors in inspiratory off-switching (IOS) evoked by the stimulation of the nucleus parabrachialis medialis (NPBM), excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) were recorded from bulbar augmenting inspiratory (aug-I) and postinspiratory (PI) neurons in vagotomized cats. Stimulation of NPBM produced either transient inhibition or premature termination of inspiration (reversible or irreversible IOS), depending on the stimulus intensity. Each neuron displayed four-phasic postsynaptic responses during the reversible IOS, i.e. Phase 1 EPSPs, Phase 2 IPSPs, Phase 3 EPSPs and Phase 4 IPSPs in aug-I neurons, and Phase 1 plus 2 EPSPs, Phase 3 IPSPs and Phase 4 EPSPs in PI neurons. During the irreversible IOS, Phase 4 responses were replaced by sustained hyperpolarization in aug-I neurons and decrementing depolarization in PI neurons. Blockade of NMDA receptors by dizocilpine (0.3 mg kg(-1) i.v.) selectively increased Phase 4 potentials in both types of neurons and decreased the thresholds for evoking the irreversible IOS. The NPBM-induced responses had a pattern and time-course similar to those induced by vagal stimulation. The present results suggest that pneumotaxic and vagal inputs converge on the common IOS circuit, and the effectiveness of both inputs is modulated by NMDA receptors.     

Excitatory amino acid antagonists inhibit synaptic responses in the guinea pig hypothalamic paraventricular nucleus.

1. The effects of specific excitatory amino acid (EAA) antagonists on evoked excitatory synaptic responses were studied in the hypothalamic paraventricular nucleus (PVN) of the guinea pig, by the use of the in vitro slice preparation. Intracellular recordings were obtained from paraventricular neurons, and excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were induced by perifornical electrical stimulation. To reduce the influence of a potential gamma-aminobutyric acidA (GABAA) inhibitory component on the synaptic responses, all experiments were performed in the presence of 50 microM picrotoxin. 2. Of 20 cells tested, 13 had electrophysiological characteristics similar to magnocellular neuropeptidergic cells (MNCs) and 7 displayed low-threshold Ca2+ spikes (LTSs). No difference was detected in the effect of the antagonists on the synaptic responses of cells with or without LTS potentials. 3. The broad-spectrum EAA antagonist kynurenic acid decreased the amplitude of the EPSPs and EPSCs in a dose-dependent manner: the mean decrease was 5% for 100 microM, 43% for 300 microM, and 70% for 1 mM. 4. The quisqualate/kainate-receptor-selective antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) induced a dose-dependent decrease of the EPSPs and EPSCs: 1 microM had no detectable effect, 3 and 10 microM caused 30 and 70% decreases, respectively, and 30 microM blocked the response almost completely. This effect was not accompanied by a change in resting membrane potential or input resistance and was slowly reversible. 5. The N-methyl-D-aspartate (NMDA)-receptor-selective antagonist DL-2-amino-5-phosphonopentanoic acid (AP5), applied at 30 and 300 microM, reduced slightly the amplitude of the decay phase of the EPSP but did not significantly affect the peak amplitude. In some cells, the current-voltage relationship of the decay phase of the EPSC revealed a region of negative slope conductance between -70 and -40 mV. 6. These results suggest that 1) glutamate or a related EAA is responsible for the fast excitatory input to magnocellular and parvocellular neurons in the PVN and probably also for cells around PVN, 2) a quisqualate/kainate receptor type is responsible for the rising phase and peak amplitude of the synaptic current, and 3) an NMDA receptor contributes to the late part of the synaptic response.     

Role of EPSPs in initiation of spontaneous synchronized burst firing in rat hippocampal neurons bathed in high potassium

1. Spontaneous discharges that resemble interictal spikes arise in area CA3 b/c of rat hippocampal slices bathed in 8.5 mM [K+]o. Excitatory postsynaptic potentials (EPSPs) also appear at irregular intervals in these cells. The role of local synaptic excitation in burst initiation was examined with intracellular and extracellular recordings from CA3 pyramidal neurons. 2. Most (70%) EPSPs were small (less than 2 mV in amplitude), suggesting that they were the product of quantal release or were evoked by a single presynaptic action potential in another cell. It is unlikely that most EPSPs were evoked by a presynaptic burst of action potentials. Indeed, intrinsic burst firing was not prominent in CA3 b/c pyramidal cells perfused in 8.5 mM [K+]o. 3. The likelihood of occurrence and the amplitude of EPSPs were higher in the 50-ms interval just before the onset of each burst than during a similar interval 250 ms before the burst. This likely reflects increased firing probability of CA3 neurons as they emerge from the afterhyperpolarization (AHP) and conductance shunt associated with the previous burst. 4. Perfusion with 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a potent quisqualate receptor antagonist, decreased the frequency of EPSPs in CA3 b/c neurons from 3.6 +/- 0.9 to 0.9 +/- 0.3 (SE) Hz. Likewise, CNQX reversibly reduced the amplitude of evoked EPSPs in CA3 b/c cells. 5. Spontaneous burst firing in 8.5 mM [K+]o was abolished in 11 of 31 slices perfused with 2 microM CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kindling-induced long-lasting changes in synaptic transmission in the basolateral amygdala.

1. Intracellular current-clamp recordings were obtained from neurons of the basolateral amygdala (BLA) in an in vitro slice preparation from control and kindled animals. Postsynaptic potentials, elicited by stimulation of the stria terminalis (ST) or lateral amygdaloid nucleus (LA), were used to investigate the role of excitatory and inhibitory amino acid transmission in kindling-induced epileptiform activity. The contributions of glutamatergic and GABAergic receptor subtypes were analyzed by use of the non-N-methyl-D-aspartate (non-NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the NMDA antagonist DL-2-amino-5-phosphonovaleric acid (APV), and the GABAA antagonist bicuculline methiodide (BMI). 2. The synaptic waveform evoked in control neurons consisted of an excitatory postsynaptic potential (EPSP), a fast inhibitory postsynaptic potential (f-IPSP), and a slow inhibitory postsynaptic potential (s-IPSP). Stimulation of the ST or LA pathways evoked a burst-firing response in BLA neurons contralateral from the site of stimulation of kindled animals. 3. APV (50 microM) reduced, but CNQX (10 microM) completely blocked, the burst-firing response in BLA neurons from kindled animals and bicuculline-induced bursting in control neurons. 4. Kindling significantly increased the amplitude of both the slow NMDA- and the fast non-NMDA-receptor-mediated components of synaptic transmission (s- and f-EPSPs, respectively). Furthermore, the stimulus intensities required to evoke EPSPs just subthreshold for action potential generation were significantly lower in slices from kindled animals. 5. In kindled neurons no significant change was observed in the membrane input resistance and resting membrane potential or in the number of action potentials elicited in response to depolarizating current injection. 6. Kindling resulted in a pathway-specific loss of ST- and LA-evoked feedforward GABAergic synaptic transmission and of spontaneous IPSPs. In the same BLA neurons, direct GABAergic inhibition via stimulation of the LA was not affected by kindling. 7. The enhanced glutamatergic transmission was not due to disinhibition, because, in the presence of BMI (and CNQX to prevent BMI-induced bursting), the s-EPSP amplitude was still greater in kindled than in control neurons. 8. These results provide evidence that the epileptiform activity observed in BLA neurons after kindling results from an increase in excitatory NMDA- and non-NMDA-receptor-mediated glutamatergic transmission and a decrease in inhibitory gamma-aminobutyric acid (GABA)-receptor-mediated transmission; the enhanced excitatory transmission cannot be accounted for by reduced inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of long-term potentiation without participation of N-methyl-D-aspartate receptors in kitten visual cortex.

1. Intracellular recording was made from layer II-III cells in slice preparations of kitten (30-40 days old) visual cortex. Low-frequency (0.1 Hz) stimulation of white matter (WM) usually evoked an excitatory postsynaptic potential (EPSP) followed by an inhibitory postsynaptic potential (IPSP). The postsynaptic potentials (PSPs) showed strong dependence on stimulus frequency. Early component of EPSP and IPSP evoked by weak stimulation both decreased monotonically at frequencies greater than 0.5-1 Hz. Strong stimulation similarly depressed the early EPSP at higher frequencies (greater than 2 Hz) and replaced the IPSP with a late EPSP, which had a maximum amplitude in the stimulus frequency range of 2-5 Hz. 2. Very weak WM stimulation sometimes evoked EPSPs in isolation from IPSPs. The falling phase of the EPSP revealed voltage dependence characteristic to the responses mediated by N-methyl-D-aspartate (NMDA) receptors and was depressed by application of an NMDA antagonist DL-2-amino-5-phosphonovalerate (APV), whereas the rising phase of the EPSP was insensitive to APV. 3. The early EPSPs followed by IPSPs were insensitive to APV but were replaced with a slow depolarizing potential by application of a non-NMDA antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX), indicating that the early EPSP is mediated by non-NMDA receptors. The slow depolarization was mediated by NMDA receptors because it was depressed by membrane hyperpolarization or addition of APV. 4. The late EPSP evoked by higher-frequency stimulation was abolished by APV, indicating that it is mediated by NMDA receptors, which are located either on the recorded cell or on presynaptic cells to the recorded cells. 5. Long-term potentiation (LTP) of EPSPs was examined in cells perfused with solutions containing 1 microM bicuculline methiodide (BIM), a gamma-aminobutyric acid (GABA) antagonist. WM was stimulated at 2 Hz for 15 min as a conditioning stimulus to induce LTP, and the resultant changes were tested by low-frequency (0.1 Hz) stimulation of WM. 6. LTP of early EPSPs occurred in more than one-half of the cells (8/13) after strong conditioning stimulation. The rising slope of the EPSP was increased 1.6 times on average. 7. To test involvement of NMDA receptors in the induction of LTP in the early EPSP, the effect of conditioning stimulation was studied in a solution containing 100 microM APV, which was sufficient to block completely synaptic transmission mediated by NMDA receptors. LTP occurred in the same frequency and magnitude as in control solution.     

Role of GABAB receptor-mediated inhibition in reciprocal interareal pathways of rat visual cortex

In neocortex, synaptic inhibition is mediated by gamma-aminobutyric acid-A (GABAA) and GABAB receptors. By using intracellular and patch-clamp recordings in slices of rat visual cortex we studied the balance of excitation and inhibition in different intracortical pathways. The study was focused on the strength of fast GABAA- and slow GABAB-mediated inhibition in interareal forward and feedback connections between area 17 and the secondary, latero-medial visual area (LM). Our results demonstrate that in most layer 2/3 neurons forward inputs elicited excitatory postsynaptic potentials (EPSPs) that were followed by fast GABAA- and slow GABAB-mediated hyperpolarizing inhibitory postsynaptic potentials (IPSPs). These responses resembled those elicited by horizontal connections within area 17 and those evoked by stimulation of the layer 6/white matter border. In contrast, in the feedback pathway hyperpolarizing fast and slow IPSPs were rare. However weak fast and slow IPSPs were unmasked by bath application of GABAB receptor antagonists. Because in the feedback pathway disynaptic fast and slow IPSPs were rare, polysynaptic EPSPs were more frequent than in forward, horizontal, and interlaminar circuits and were activated over a broader stimulus range. In addition, in the feedback pathway large-amplitude polysynaptic EPSPs were longer lasting and showed a late component whose onset coincided with that of slow IPSPs. In the forward pathway these late EPSPs were only seen with stimulus intensities that were below the activation threshold of slow IPSPs. Unlike strong forward inputs, feedback stimuli of a wide range of intensities increased the rate of ongoing neuronal firing. Thus, when forward and feedback inputs are simultaneously active, feedback inputs may provide late polysynaptic excitation that can offset slow IPSPs evoked by forward inputs and in turn may promote recurrent excitation through local intracolumnar circuits. This may provide a mechanism by which feedback inputs from higher cortical areas can amplify afferent signals in lower areas.     

Hyperpolarizing and depolarizing GABAA receptor-mediated dendritic inhibition in area CA1 of the rat hippocampus.

1. gamma-Aminobutyric acidA (GABAA) receptor-mediated inhibition of pyramidal neuron dendrites was studied in area CA1 of the rat hippocampal slice preparation with the use of intracellular and extracellular recording and one-dimensional current source-density (CSD) analysis. 2. Electrical stimulation of Schaffer collateral/commissural fibers evoked monosynaptic excitatory postsynaptic potentials (EPSPs) and population EPSPs, which were followed by biphasic inhibitory postsynaptic potentials (IPSPs). In the presence of the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-2-amino-5-phosphonovalerate (APV), stimulation in stratum radiatum evoked monosynaptic fast, GABAA and late, GABAB receptor-mediated IPSPs and fast and late positive field potentials recorded in s. radiatum. 3. Fast monosynaptic IPSPs and fast positive field potentials evoked in the presence of DNQX and APV were reversibly abolished by the GABAA receptor antagonist bicuculline methiodide (BMI; 30 microM) and were not changed by the GABAB receptor antagonist P-[3-aminopropyl]-P-diethoxymethylphosphinic acid (CGP 35,348; 0.1-1.0 mM). CGP 35,348 (0.1 mM) reversibly blocked late monosynaptic IPSPs and late positive field potentials. These results suggest that fast field potentials are GABAA receptor-mediated population IPSPs (GABAA, fast pIPSPs) and that late field potentials are GABAB receptor-mediated population IPSPs (GABAB, late pIPSPs). 4. Fast pIPSPs were reversibly abolished when the extracellular Cl- concentration [( Cl-]o) was reduced from 132 to 26 mM in parallel with a depolarizing shift in the reversal potential of fast IPSPs. Paired or repetitive stimulation in s. radiatum reversibly depressed fast pIPSPs and fast IPSPs. Paired-pulse depression of fast pIPSPs was reversibly antagonized by CGP 35,348 (0.4-0.8 mM). 5. Laminar analysis of s. radiatum-evoked fast pIPSPs and one-dimensional CSD analysis revealed active current sources in s. radiatum and passive current sinks in s. oriens and s. lacunosum moleculare. S. radiatum sources were abolished by pressure application of BMI in s. radiatum but not in s. oriens. Stimulation in s. oriens, s. pyramidale, or s. lacunosum moleculare evoked GABAA current sources horizontal to the stimulation site. Changes in the dendritic location of inhibitory current with changes in stimulus location paralleled changes in the distribution of excitatory current. 6. In the presence of 4-aminopyridine (50-100 microM), DNQX and APV long-lasting depolarizing GABAA receptor-mediated responses (LLDs) occurred spontaneously or could be evoked. Current sinks associated with s. radiatum-evoked LLDs were located in the same dendritic area as sources associated with hyperpolarizing fast IPSPs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Responses of spinal dorsal horn neurones evoked by myelinated primary afferent stimulation are blocked by excitatory amino acid antagonists acting at kainate/quisqualate receptors

The effects of excitatory amino acid antagonists on responses, evoked by myelinated primary afferent stimulation, in neurones of the spinal cord dorsal horn has been investigated in an isolated hemisected spinal cord preparation from the neonatal rat. The first evoked action potential of most responses was reversibly blocked by either 5 mM kynurenic acid (80%) or 10 microM CNQX (91%). The small latency variability and the ability of some of these responses to follow repetitive stimulation suggested that they were evoked monosynaptically. The selective NMDA receptor antagonist, CPP, blocked some later components of responses consisting of bursts of action potentials but not the first evoked action potential. The results support the suggestion that myelinated primary afferents release excitatory amino acids which act at kainate/quisqualate receptors.     

Glutamate as a putative neurotransmitter in the buccal central pattern generator of Helisoma trivolvis.

1. The effects of L-glutamate superfusion over identified neurons within the buccal ganglia of Helisoma trivolvis were examined. Glutamate mirrored the effect of activity of subunit 2 (S2) of the tripartite feeding central pattern generator (CPG) on S2 postsynaptic neurons. Neurons that are excited by S2 are depolarized by glutamate, whereas neurons that are inhibited by S2 are hyperpolarized by glutamate. Glutamate also stimulated rhythmic S2 activity. 2. Different glutamate agonists could mimic specific components of the effects of glutamate on buccal neurons. Kainate produced depolarizations in neurons that receive S2 excitatory postsynaptic potentials (EPSPs) and activated rhythmic S2 activity. Quisqualate produced hyperpolarizations in neurons that receive S2 inhibitory postsynaptic potentials (IPSPs). 3. The non-N-methyl-D-aspartate glutamate receptor antagonist cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked the effects of S2 EPSPs and depolarizations produced by application of glutamate and kainate, but was ineffective in blocking S2 IPSPs or hyperpolarizations produced by application of glutamate and quisqualate. 4. These data support the hypothesis that glutamate is the transmitter of S2 of the feeding CPG in Helisoma, acting at CNQX-sensitive kainate-like receptors at excitatory synapses and CNQX-insensitive quisqualate-like receptors at inhibitory synapses.     

Long-term enhancement of excitatory synaptic inputs to layer V parahippocampal neurons by low frequency stimulation in rat brain slices

Excitatory inputs to layer V neurons of the parasubiculum and medial entorhinal cortex were examined in rat brain slices with intracellular and field potential recordings. Single extracellular stimuli to layer V evoked subthreshold excitatory postsynaptic potentials (EPSPs) or a long duration (>100 ms) depolarization that sustained high frequency firing. Repetitive stimulation at low frequencies (from 1/10 s to 1/min) induced stable long-lasting decreases in the threshold for firing in individual cells or population events, and also induced stable long-lasting increases in evoked intracellular or field response amplitudes. More stimuli were required to produce the equivalent changes in threshold and amplitude in the presence of MCPG (200 microM). Smaller changes in amplitude, but equivalent changes in threshold were elicited in the presence of CPP (10 microM), or CPPG (20 microM). No changes in threshold or amplitude were detected in the presence of CNQX (10 microM), even when used in combination with picrotoxin (100 microM). EPSP facilitation was enhanced greatly by firing in postsynaptic cells. It is suggested that stable changes in excitatory inputs to layer V parahippocampal neurons involve the activation of NMDA and metabotropic glutamate receptors, but requires AMPA receptor activation and postsynaptic cell firing.     

The kainate/quisqualate receptor antagonist, CNQX, blocks the fast component of spontaneous epileptiform activity in organotypic cultures of rat hippocampus

Intracellular recordings were made from CA1 pyramidal neurones of hippocampus maintained in organotypic culture. Both spontaneous interictal and ictal epileptiform activity was observed. CNQX, an antagonist at kainate/quisqualate but not at N-methyl-D-aspartate (NMDA)-sensitive excitatory amino acid receptors depressed but did not abolish spontaneous epileptiform activity. Addition of the specific NMDA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) abolished the remaining activity. Similar effects were observed on electrically evoked excitatory post synaptic potentials (EPSPs). This suggests a role for endogenous excitatory amino acids acting at both kainate/quisqualate and NMDA sensitive excitatory amino acid receptors in the generation and maintainance of epileptiform activity within these organotypic cultures.     

Synaptic responses of guinea pig cingulate cortical neurons in vitro.

1. Intracellular recordings were made from layer V/VI neurons of the guinea pig anterior cingulate cortex to investigate postsynaptic potentials (PSPs) evoked by electrical stimulation of the subcortical white matter (forceps minor). 2. Four distinct types of PSPs were recorded (at the resting potential) under normal physiological conditions; 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive excitatory postsynaptic potentials (EPSPs) were followed by bicuculline- or picrotoxin-sensitive depolarizing or hyperpolarizing inhibitory postsynaptic potentials (IPSPs), which were further followed by phaclofen-sensitive, long-lasting hyperpolarizing postsynaptic potentials (LPSPs). The average times-to-peak for the EPSP, depolarizing and hyperpolarizing IPSPs, and LPSP were 10, 22, 28, and 146 ms, respectively. 3. In the presence of CNQX and bicuculline, high-intensity electrical stimulation elicited a longer lasting EPSP with a time-to-peak of 21 ms. The amplitude and duration of the EPSP decreased with membrane hyperpolarization and increased with membrane depolarization. The EPSP was reversibly abolished by D,L-2-amino-5-phosphonovaleric acid (D,L-APV). 4. The bicuculline- or picrotoxin-sensitive depolarizing and hyperpolarizing IPSPs and the phaclofen-sensitive LPSP were markedly suppressed by CNQX, suggesting that glutamate (Glu) and/or aspartate nerve terminals project to GABAergic interneurons, and that the GABAergic interneurons are activated mainly by non-N-methyl-D-aspartate (non-NMDA) receptors. 5. In the presence of picrotoxin, the average reversal potential for the compound EPSP was 0 mV, which was similar to that (-6 mV) for the Glu-induced depolarization. In a solution containing D,L-APV at low concentrations, the average reversal potentials for the depolarizing and hyperpolarizing IPSPs and for the early and late components of the gamma-aminobutyric acid (GABA)-induced responses were -62, -72, -70, and -61 mV, respectively. Thus the value for the depolarizing IPSP was similar to that for the late response to GABA, whereas the value for the hyperpolarizing IPSP was almost the same as that for the early response to GABA. The average reversal potential of -90 mV for the LPSP was similar to -93 mV for the baclofen-induced hyperpolarization and to -94 mV for the spike afterhyperpolarization. 6. Application of phaclofen decreased the interspike interval of the spontaneous firing and reversed the increase in the interspike interval after subcortical stimulation. This result indicates that, even in a slice preparation, the anterior cingulate neurons are under tonic inhibitory control exerted by spontaneously active GABAergic interneurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Receptor subtypes involved in callosally-induced postsynaptic potentials in rat frontal agranular cortex in vitro

Summary A slice preparation of rat frontal agranular cortex preserving commissural inputs has been used for intracellular recording from layer V pyramidal cells, in order to characterize the synaptic potentials induced by stimulation of the corpus callosum and to reveal the subtypes of amino acid receptors involved. Stimulation of the corpus callosum induced EPSPs followed by early IPSPs with a peak latency of 30 ± 2 ms and late IPSPs with a peak latency of 185 ± 18 ms. Reversal potentials for early and late IPSPs were –75 ± 5 mV (early) and –96 ± 5 mV (late). Late IPSPs were more dependent on extracellular K⁺ concentration. The early IPSPs were blocked by GABA_A antagonists, bicuculline and picrotoxin, whereas the late IPSPs were reduced by the GABA_B antagonist, phaclofen. CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), an antagonist of non-NMDA (N-methyl-D-aspartate) receptors, suppressed both EPSPs and late IPSPs at 5 µM. Early IPSPs remained at this concentration but were suppressed by 20 µM CNQX. In Mg²⁺-free solution, EPSPs were larger and more prolonged than in control solution. These enhanced EPSPs persisted after 5 to 20 µM CNQX, but were reduced in amplitude, and their onset was delayed by 3.6 ± 0.8 ms. The remaining EPSPs were suppressed by 50 µM APV (DL-2-amino-5-phosphono-valeric acid), an antagonist of NMDA receptors. In Mg²⁺-free solution containing 5 to 20 µM CNQX, the late IPSPs were not diminished. The remaining late IPSPs were suppressed by APV or by phaclofen. By contrast, the amplitude of early IPSPs was not affected by APV in Mg²⁺-free solution containing 5 µM CNQX. These results show that stimulation of the corpus callosum can induce GABA_A and GABA_B dependent IPSPs and NMDA and non-NMDA dependent excitation. It is suggested that these four types of amino acid-based transmission are conveyed by intracortical pathways with different characteristics.     

Differential blocking action of Joro spider toxin analog on parallel fiber and climbing fiber synapses in cerebellar Purkinje cells.

Synaptic potentials were recorded intracellularly from Purkinje cells in guinea pig cerebellar slices. EPSPs evoked by stimulation of parallel fibers were effectively blocked by perfusion of a slice with the synthetic analog of Joro spider toxin, 1-naphthylacetyl-spermine (NAS) at 250 microM. However, it did not influence those responses evoked by stimulation of climbing fibers. This action of NAS is in contrast to other commonly used glutamate antagonists, CNQX or APV: CNQX (5 microM) blocked both parallel fiber- and climbing fiber-induced responses, while APV (up to 1 mM) did not influence either except for a weak reduction observed in climbing fiber responses. NAS thus provides a useful tool for pharmacologically distinguishing parallel fiber and climbing fiber synapses.     

An intracellular analysis of amino acid induced excitations of deep dorsal horn neurones in the rat spinal cord slice

The rat spinal cord slice preparation has been used to investigate the sensitivity of deep dorsal horn neurones to the excitatory amino acids N-methyl-D-aspartate (NMDA), quisqualate and L-glutamate. Intracellular recordings were made from 44 neurones in laminae III-VI of 14- to 16-day rats. Superfusion of quisqualate (30 microM) excited all neurones, NMDA (50 microM) excited 72% and L-glutamate (0.5-1 mM) 63% of the neurones. Depolarizations were retained after tetrodotoxin but with a reduced amplitude. The NMDA antagonist D-aminophosphonovalerate (D-APV, 10 microM) reduced NMDA and L-glutamate depolarizations by 66% and by 40%, respectively, while the quisqualate responses were enhanced by 27%. Dorsal root stimulation elicited two main patterns of activity; short-latency single/double spikes followed by subthreshold excitatory postsynaptic potentials (EPSPs) or a burst of spikes rising from a long duration composite EPSP. D-APV reduced the long-latency components of the first type and reduced the amplitude and duration of the composite EPSP of the second. These results support a specialized role for NMDA receptors in synaptic transmission in the dorsal horn.     

Slow excitatory amino acid receptor-mediated synaptic transmission in turtle cerebellar Purkinje cells

1. The excitatory synaptic responses of turtle Purkinje cells to climbing and parallel fiber (CF and PF) stimulation have been studied by the use of intrasomatic and intradendritic recordings in intact cerebellum and brain stem-cerebellum preparations in vitro. 2. Activation of CF inputs from the cerebellar peduncle or the region of the inferior olive evoked complex spikes followed by slow excitatory postsynaptic potentials (EPSPs), both of which were evoked in an all-or-none fashion. 3. Single stimuli applied to the cerebellar molecular layer activated fast PF-mediated EPSPs; brief trains of PF stimuli (2-5 stimuli, 50-100 Hz) evoked volleys of fast EPSPs followed by a slow, long-lasting EPSP. The amplitude of the fast and slow PF-mediated EPSPs were both graded with stimulus intensity. 4. Slow EPSPs evoked both by CF and PF stimulation were associated with an increase in membrane conductance and were increased in amplitude by hyperpolarization. 5. The CF-evoked slow EPSP was profoundly attenuated by repetitive activation at interstimulus intervals of less than 15-20 s, whereas the PF-evoked slow EPSP was not reduced by repetitive activation. 6. The PF-evoked slow EPSP readily triggered dendritic pacemaker discharges when activated at or near resting membrane potential. The activation of this potential by phasic PF volleys may, therefore, provide an appropriate synaptic drive to cerebellar Purkinje cells to entrain the intrinsic pacemaker properties of these cells to cycles of motor activity. 7. Both slow synaptic potentials were blocked by the excitatory amino acid antagonists kynurenate and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by DL-2-amino-5-phosphonovalerate (DL-AP5) or L-serine-O-phosphate (L-SOP). The PF-evoked slow EPSP was selectively antagonized by L-2-amino-4-phosphonobutyrate (L-AP4; 20-100 microM). 8. It is suggested that the CF- and PF-evoked slow EPSPs observed in this study represent a novel class of excitatory amino acid receptor-mediated slow synaptic potentials activated by Purkinje cell afferents, which may play a role in synaptic integration and motor pattern generation in the cerebellum.