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A variety of polyamine spider and wasp toxins are known to block N-methyl-D-aspartate receptor channels and recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the edited glutamate receptor (GluR)2 subunit. Recently, inward rectification of GluR2-lacking AMPA receptors was shown to be caused by voltage-dependent block by intracellular spermine. Here we demonstrate that, when applied extracellularly, the endogenous polyamines spermine and spermidine, as well as monoacylated spermine analogs and the polyamine toxins ageltoxin-489 and philanthotoxin-433, exerted a use-dependent and weakly voltage-dependent block of AMPA receptors that lack the edited GluR2 subunit, when the recombinant receptors were expressed in Xenopus oocytes. External spermine and polyamine toxins were also effective blockers of AMPA receptor mutants that did not not show inwardly rectifying kainate responses but had high calcium permeability. The polyamines and polyamine toxins also markedly reduced inwardly rectifying currents of native AMPA receptors expressed by a class of hippocampal interneurons in rat CA3 stratum radiatum that appear not to express the GluR2 subunit. In contrast, polyamines had little or no effect on the linear or outwardly rectifying kainate responses of other interneurons or CA3 pyramidal cells in which GluR2 mRNA was routinely detected. Together with previous reports, these data suggest that endogenous polyamines may bind to GluR2-lacking AMPA receptors at two or more distinct sites, one near the cytoplasmic side of the pore and the other nearer the outer side of the pore.

1. Short iontophoretic pulses of acetylcholine (ACh) inhibited almost every spontaneously active cell encountered in the nucleus reticularis thalami of cats anaesthetized with a mixture of halothane, nitrous oxide and oxygen. On 200 cells the mean current needed to eject an effective inhibitory dose of ACh was 67 +/‐ 2 nA. When the ACh‐evoked inhibition was mimicked by gamma‐aminobutyric acid (GABA) or glycine on the same cell, the current required to release ACh was found to be approximately twice as great as that required to release an equally effective dose of GABA or glycine. 2. ACh inhibitions developed with a latency which was very much shorter than that for ACh excitation in cells of the ventrobasal complex. The latency of the ACh‐evoked inhibition was as rapid as the onset and offset of the excitation of the same cells glutamate and their inhibition by GABA or glycine. 3. The firing pattern of ACh‐inhibited neurones in the nucleus reticularis was characterized by periods of prolonged, high frequency bursts, and their mean firing frequency was 22 Hz. Raster dot displays and interspike interval histograms showed that whereas ACh suppressed the spikes that occurred between bursts much more readily than those that occurred during bursts, all spikes were equally sensitive to the depressant action of GABA and glycine. Large doses of ACh provoked or exaggerated burst activity. 4. ACh‐evoked inhibition was extremely sensitive to blockade by short iontophoretic applications of atropine, which had no effect on the inhibitions evoked on the same cell equipotent doses of GABA or glycine. The ACh‐evoked inhibitions were also antagonized by dihydro‐beta‐erythroidine released with slightly larger currents. When tested on the same cell, small iontophoretic applications of picrotoxin and bicuculline methoiodide blocked the inhibition evoked by GABA but had no effect on that evoked by ACh. Iontophoretic strychnine only rarely affected the inhibition evoked by ACh, while readily blocking the inhibition evoked on the same cell by an equipotent dose of glycine. In two cats, intravenous strychnine (1‐2 mg/kg) had no effect on the ACh‐evoked inhibition, while greatly reducing the sensitivity of the cell under study to glycine. 5. Only four out of forty‐eight ACh‐inhibted cells tested were inhibited by iontophoretic applications of either guanosine or adenosine 3':5'‐phosphate. 6. Cells of the nucleus reticularis have been shown to have an inhibitory action on the thalamic relay cells, which are excited by ACh. It is suggested that the presence of both ACh excited and inhibited cells in different nuclei of the thalamus could be of considerable functional significance in gating sensory transmission through the thalamus.

Intracellular recordings were made from CA1 pyramidal cells in the rat hippocampal slice to study the processes that influence the time course of inhibitory post‐synaptic potentials (i.p.s.p.s) mediated by gamma‐aminobutyric acid (GABA), and conductance changes evoked by ionophoretically applied GABA. The GABA‐uptake inhibitors, nipecotic acid and cis‐4‐OH‐nipecotic acid (1 mM), greatly prolonged conductance increases associated with both hyperpolarizing and depolarizing responses to ionophoretically applied GABA. In contrast to their effects on GABA‐evoked conductances, uptake inhibitors only slightly prolonged antidromically evoked i.p.s.p.s. Their primary effect occurred after the i.p.s.p. had decayed to 5‐30% of its peak. 4‐OH‐isonipecotic acid, a nipecotic acid analogue that does not inhibit GABA uptake, did not prolong i.p.s.p.s or ionophoretically evoked conductance changes. Sodium pentobarbitone (100 microM), a drug that prolongs the open time of GABA‐activated chloride channels, potentiated both i.p.s.p.s and responses to ionophoretically applied GABA. Whereas pentobarbitone also prolonged i.p.s.p.s, it did not prolong responses to ionophoretically applied GABA. The prolongation of i.p.s.p.s by pentobarbitone occurred equally in both the early and late phases of the i.p.s.p., in contrast to the effects of GABA‐uptake inhibitors. I.p.s.p.s did not usually decay exponentially. The observation that uptake inhibitors prolonged the late but not the early decay phase of the i.p.s.p., together with the previous finding that the conductance change persists for the duration of the i.p.s.p., indicate that GABA is present in the synapse throughout much of the i.p.s.p. These data suggest that diffusion of GABA out of the synapse, a non‐exponential process, is an important determinant of the i.p.s.p. decay time course. Increasing the extracellular potassium concentration from 3.5 to 8.5 mM resulted in spontaneously occurring, synchronous burst firing of pyramidal cells. Cis‐4‐OH‐nipecotic acid significantly reduced the number and amplitude of extracellularly recorded population spikes within each burst. We conclude that diffusion, channel open time and GABA uptake all influence the time course of GABA‐mediated i.p.s.p.s. The time course of a single, brief i.p.s.p. is determined predominantly by post‐synaptic channel kinetics and diffusion of GABA out of the synapse, whereas the inhibition produced by prolonged synaptic bursts or relatively long application of exogenous GABA can be markedly influenced by GABA uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

To determine if electrophysiological properties of hippocampal pathways are altered in kindled rats, extracellular recordings were made from hippocampal slices of rats kindled in the lateral entorhinal cortex and compared with those from implanted but unstimulated controls. Studies were made either 24 h or 28 days after the last kindled seizure and done in normal (3.5 mM) or elevated (7 mM) K+. The preparation of slices, data accumulation, and data analyses were done blind. One day or 28 days after the last kindled seizure, the proportion of slices with spontaneous epileptiform bursts recorded from the CA2/3 region in elevated K+ was significantly (P less than 0.001) increased in the kindled animals. The frequency of spontaneous burst firing was also increased and reached significance (P less than 0.02) at 28 days following the last kindling stimulus. One day after the last kindling stimulus, paired-pulse (GABAergic) inhibition in the CA1 region was decreased (P less than 0.001). Several measures suggested an increased synaptic inhibition in the dentate gyrus of slices from the kindled groups 1 day after kindling. Paired-pulse inhibition was increased (P less than 0.01), the current required to evoke a near-threshold population spike was increased (P less than 0.05), and the population spike amplitude was reduced for a given field excitatory postsynaptic potential (EPSP) (P less than 0.01). Twenty-eight days after the last kindling stimulus, however, paired-pulse inhibition in the dentate was slightly less in slices from kindled rats (P less than 0.005). In other respects the CA1 and dentate regions did not differ between kindled and control groups within 24 h of the last stage V seizure. Thus the maximum amplitudes of presynaptic fiber volley, population spike, and field-excitatory postsynaptic potential (EPSP) slope, and the number of population spikes evoked by a near-maximally effective afferent stimulus, were unchanged. In the CA1 region the input-output curve of field EPSP versus population spike, and the current intensity required to evoke a near-threshold population spike were also unchanged. In addition, no spontaneous bursts were recorded from CA1 in 3.5 mM K+. We conclude that either synapses or neurons intrinsic to the hippocampus are altered by kindling stimuli applied outside this brain area. The transient increase in inhibition in the dentate gyrus suggests that it may reflect a compensatory reaction to kindled seizures. In contrast, the long-lasting (at least 28 days) increase in burst firing in CA2/3 may represent a mechanism for the initiation or propagation of kindled seizures.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Intracellular recordings were made from CA1 pyramidal cells in the hippocampal slice in vitro. The responses to orthodromic and antidromic activation and to ionophoretically applied GABA were studied. 2. The epileptogenic agent sodium benzyl penicillin reduced the recurrent i.p.s.p. evoked by subthreshold antidromic stimulation. Reversal potential studies of the i.p.s.p. and resistance measurements showed that this reduction was mainly due to a decrease in i.p.s.p. conductance. 3. Penicillin also reduced the conductance and associated membrane potential changes induced by ejecting GABA near the soma or into the apical dendritic region. 4. The mixed e.p.s.p.‐i.p.s.p. evoked by orthodromic stimulation was converted to a pure depolarizing potential as the i.p.s.p. was blocked. Concurrently the probability of discharge to a constant orthodromic stimulus was increased. Similar changes were seen in a low chloride solution. 5. The time course of the reduction of inhibition was similar to that of the enhanced orthodromic response seen after penicillin treatment. 6. We conclude that reduction of postsynaptic inhibition is partly responsible for the increased probability of orthodromic discharge caused by penicillin. The longer latency all‐or‐nothing burst seen in some cells, however, seems to require an additional mechanism, although reduced inhibition may facilitate the triggering of this burst.

1. Extra‐ and intracellular recordings were made from CA1 cells in hippocampal slices in vitro. The effects of ionophoretically applied GABA on somatic and dendritic regions were studied. 2. Ionophoresis of GABA at dendritic sites gave a reciprocal effect by inhibiting the effect of excitatory synapses close to the dendritic application, while facilitating those lying further away. For example, GABA delivered to the mid‐radiatum dendritic region reduced the population spike generated by a radiatum volley, while facilitating the population spike evoked by oriens fibre stimulation. Similarly, when single cells were recorded from, mid‐apical dendritic delivery of GABA abolished the synaptically driven discharges evoked by fibres terminating at this part of the dendritic tree, but facilitated the responses to input from fibres terminating on the basal dendrites of the same cell. 3. With intracellular recording two effects were observed. Applied near the soma, GABA induced a hyperpolarization associated with an increased membrane conductance. When applied to dendrites, GABA caused a depolarization also associated with an increased membrane conductance. Both types of GABA applications could inhibit cell discharges, although in some cases the depolarizing response could facilitate other excitatory influences or cause cell firing by itself. 4. Both the hyperpolarizing and depolarizing GABA responses persisted after blockade of synaptic transmission by applying a low calcium high magnesium solution, indicating mediation via a direct effect upon the cell membrane. 5. The reversal potential for the hyperpolarizing GABA effect was similar to the equilibrium potential for the i.p.s.p. evoked from alveus or orthodromically, and was 10‐12 mV more negative than the resting potential. The size of the depolarizing response was also dependent upon the membrane potential. By extrapolation an estimated equilibrium potential was calculated as about ‐40 mV. 6. Our results support the idea that the hyperpolarizing basket cell inhibition at the soma is mediated by the release of GABA. This hyperpolarizing response causes a general inhibition of firing. The dendritic effects of GABA, however, seem to represent another type of inhibition, which by shunting synaptic currents makes possible a selective inhibitory influence on afferents synapsing locally while facilitating more remotely placed excitatory synapses. We propose the term discriminative inhibition for this postulated new type of control of pyramidal cell discharges.

The depolarizing actions of N‐methyl‐DL‐aspartate (NMA) and L‐glutamate on pyramidal neurones were compared in a hippocampal slice preparation. Tetrodotoxin (1 microM) was added to the perfusion solution to suppress regenerative Na conductances. Depolarization evoked by ionophoretic application of NMA triggered slow, high‐threshold regenerative spikes. These are considered to be Ca spikes since the amplitude and rate of rise could be reduced by verapamil, D‐600, Co2+ and Mn2+, and increased by Ba2+. Multiple Ca‐spike thresholds could be demonstrated in the same cell. In contrast, depolarizations evoked by L‐glutamate only rarely triggered Ca‐spikes. The minimum latency to the onset of depolarization evoked by NMA was less than 20 ms. The latency and amplitude of NMA‐evoked responses were highly dependent on the position of the ionophoretic pipette; movements of the pipette by as little as 10‐50 micron could markedly change the size of the response. Spatially separate hot spots for NMA and glutamate were not found. Depolarizations evoked by small to moderate ionophoretic currents of NMA were usually associated with an apparent rise in input resistance, as tested by the response to transmembrane current pulses. Ionophoresis of L‐glutamate, or high NMA doses, however, usually caused a fall in input resistance. Both the depolarization and the conductance change evoked by NMA were highly voltage‐dependent within the approximate range ‐50 to ‐80 mV; they could be increased by modest depolarization and reduced by hyperpolarization of the membrane. No reversal potential could be demonstrated in the hyperpolarizing direction. Rather, the NMA response approached zero asymptotically at sufficiently hyperpolarized membrane potentials. Subthreshold depolarizations and conductance changes elicited by NMA could be blocked by Co2+, Mn2+ and Cd2+, and reduced by D‐600 and verapamil. These Ca2+ antagonists had little or no effect on resting membrane potential or input resistance, or on responses to L‐glutamate. Ba2+ increased the amplitude of subthreshold NMA responses. Intracellular injection of Cs+ plus tetraethylammonium caused cells to fire large, prolonged (up to 15 s) Ca spikes, presumably because most K+ conductances were blocked. Under these conditions the effect of NMA was unchanged or enhanced. Raising [K+]o to 10.5 mM (from the normal 3.5 mM) caused a depolarization and fall in input resistance, but did not change the amplitude or voltage dependence of the NMA response. Reducing [Na+]o caused an initial increase, then usually a delayed decrease in the amplitude of the NMA response.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of the N‐methyl‐D‐aspartate (NMDA) receptor antagonist, D‐2‐amino‐5‐phosphonovaleric acid (D‐APV), and other excitatory amino acid antagonists, were studied on CA1 pyramidal neurones treated with picrotoxin or bicuculline to reduce synaptic inhibition mediated by gamma‐aminobutyric acid (GABA). Under these conditions epileptiform burst firing is readily produced by orthodromic stimulation of the pyramidal cell population. D‐APV reduced the plateau amplitude and duration of the depolarization underlying evoked and spontaneous bursts without affecting membrane potential, input resistance or the ability of the cell to fire a Ca2+ spike or a short train of Na+ spikes. A late component of the subthreshold excitatory post‐synaptic potential (e.p.s.p.) was voltage dependent, being reduced in amplitude on membrane hyperpolarization. D‐APV selectively removed this component of the e.p.s.p. in disinhibited slices. In contrast, in the absence of GABA antagonists, D‐APV had no noticeable effect on the e.p.s.p. as studied with field potential recordings. The concentration‐response relationship of the inhibitory effect of D‐APV and L‐APV on population spike bursts was studied. The action of APV was highly stereoselective; the EC50 of D‐APV was approximately 700 nM, whereas a similar inhibition required 540 microM‐L‐APV. A number of other excitatory amino acid antagonists were tested at a fixed concentration (100 microM). Among them, the quisqualate antagonist gamma‐D‐glutamylaminomethyl sulphonic acid was ineffective against epileptiform bursts. In the low nanomolar concentration range both D‐ and L‐APV potentiated bursting. These results suggest that in the absence of GABAergic inhibition, a significant component of the slow depolarization underlying burst firing is voltage dependent, synaptic in origin and mediated by NMDA receptors. We propose that, under normal (non‐epileptic) physiological conditions, the balance between synaptic inhibition mediated by GABA receptors and synaptic excitation mediated by NMDA receptors may modulate the excitability of pyramidal cell dendrites.

The effects of the opioid peptides morphiceptin and [N-MePhe3-D-Pro4]morphiceptin (PL017), both mu receptor agonists, were examined by electrophysiological techniques in the rat hippocampal slice and ligand binding techniques in hippocampal membrane preparations. The electrophysiological actions of the mu agonists were similar to those of the previously studied delta receptor agonist [D-Ala2, D-Leu5]enkephalin. Thus, for a given size field excitatory postsynaptic potential the amplitude of both population spike and intracellular excitatory postsynaptic potential was increased by morphiceptin. These effects were concentration dependent and reversed by naloxone. The EC50 for morphiceptin was 1.6 microM, which is consistent with the mu-selective binding properties of this peptide. Similar results were obtained with the more potent analog PL017. Morphiceptin and morphine had similar displacement profiles in competition experiments performed with hippocampal membranes and a variety of radioligands. In Tris buffer morphiceptin potently inhibited the binding of the mu receptor marker [125I]FK 33,824 but displayed the expected shallow displacement isotherm against binding of the delta receptor marker [125I][D-Ala2, D-Leu5]enkephalin. A significant interaction of either morphiceptin or morphine with kappa binding sites is improbable since neither agonist could fully displace binding of [3H]ethylketocyclazocine or [3H]diprenorphine. The potency of morphiceptin in displacing [3H]naloxone from mu binding sites was reduced by inclusion of 100 mM NaCl or 100 microM GTP in the assay. The dissociation constant of morphiceptin for mu binding sites in physiological saline was 0.78 microM, comparable to its EC50 determined in electrophysiological experiments. It appears, therefore, that the electrophysiological properties of opioid peptides in the hippocampal slice may be mediated by both mu and delta receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative pharmacological studies were done to determine the properties of excitatory amino acid receptors expressed in Xenopus oocytes injected with rat brain mRNA. Smooth currents with properties indicative of N-methyl-D-aspartate (NMDA) and quisqualate/kainate receptors were observed in mRNA-injected oocytes. Schild analysis of currents evoked by NMDA indicated that the EAA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) exerted a competitive block of the oocyte NMDA receptor, because the Schild regression was linear with a slope not significantly different from unity (1.03 +/- 0.025) up to 100 microM D-APV. The pA2 estimated for D-APV antagonism of NMDA currents (5.87 +/- 0.043) was nearly identical to that for D-APV as an L-aspartate antagonist (pA2 = 5.86 +/- 0.073, slope = 0.97 +/- 0.036), suggesting that these two agonists are selective for NMDA receptors in oocytes up to concentrations well above 1 mM. 6-Nitro-7-cyano-quinoxaline-2,3-dione (CNQX) reduced the maximum NMDA response significantly (70% reduction by 15 microM CNQX) but had no effect on the NMDA EC50. CNQX exerted a mixed competitive-noncompetitive block of the glycine site on NMDA receptors; 15 microM CNQX increased the glycine EC50 by 5-fold and reduced the maximum glycine response by 35%. In addition, CNQX exerted a potent and competitive antagonism of currents evoked by kainate. The Schild regression was linear up to 30 microM CNQX with a slope of 1.02 +/- 0.014 and a pA2 of 6.53 +/- 0.029. The block of kainate or NMDA currents by 2 microM CNQX was not voltage dependent. D-APV exerted a weak antagonism of kainate-evoked currents, with a pA2 of 3.39 +/- 0.044, but the slope of the Schild regression was slightly less than 1 (0.90 +/- 0.03). These data demonstrate a clear pharmacological distinction between receptors that mediate the kainate- and NMDA-induced currents and quantify the potency of CNQX and D-APV acting at NMDA/glycine and quisqualate/kainate receptors. The implications of these data for the identification of EAA receptors in oocytes and the classification of neuronal EAA receptors are discussed.

Morphine, which inhibits release of acetylcholine from neurons in the myenteric plexus, also inhibits the spontaneous electrical activity of some myenteric neurons. To determine whether morphine acts at a site presynaptic to these neurons, we investigated this morphine effect under conditions of synaptic transmission blockade. Synaptically driven action potentials evoked by point stimulation were recorded extracellularly, and it was shown that all synaptic responses were eliminated or greatly reduced in Ca-free, high-Mg Ringer's with ethylenebis [(oxyethylenenitrilo)]-tetraacetic acid (EGTA), suggesting that synaptic transmission was blocked. Under these conditions, the ability of morphine to inhibit spontaneous electrical activity was virtually unimpaired. Assuming a single locus of narcotic action in the myenteric plexus, it is unlikely, therefore, that the primary action of opiates is to stimulate release of an inhibitory transmitter, to prevent release of an excitatory transmitter or to block the postsynaptic receptor for an excitatory transmitter. Rather, opiates may raise the membrane threshold of a class of neurons. Electric field stimulation activates myenteric neurons, resulting in a morphine-sensitive release of acetylcholine and a contraction of the longitudinal muscle. The ability of field stimulation to induce contractions and of morphine to inhibit these contractions, was virtually unchanged when the only two known excitatory inputs to the cholinergic motor neuron were eliminated by receptor blockade. These observations, taken together, suggest that opiates act directly on the cholinergic motor neuron of the myenteric plexus.

1. The CA1 region of rat hippocampal slices bathed in 8.5 mM interstitial K+ ([K+]o) exhibited spontaneous 20- to 90-s electrographic seizures at regular intervals of 1-8 min. In these same slices CA3 neurons generated spontaneous interictal bursts that propagated throughout the pyramidal cell subfields. CA1 electrographic seizures contained components reminiscent of discharges recorded in vivo during tonic-clonic motor seizures. The tonic phase lasted 1-10 s, consisted of a sustained depolarization and firing of CA1 pyramidal cells, and was associated with a negative extracellular potential in the cell layer. The clonic phase lasted tens of seconds and was composed of paroxysmal bursts with afterdischarges in pyramidal cells. 2. Electrographic seizures in CA1 were focal in nature in that they did not invade the CA3 region. Moreover, in approximately 85% of all slices the frequency and amplitude of interictal bursts in CA3 did not change during a CA1 seizure. 3. Both the tonic phase and each clonic discharge of an electrographic seizure were triggered synaptically by a CA3 interictal burst. Microlesions of the Schaffer collateral input abolished CA1 seizures in high [K+]o, and electrical stimulation of these afferents, in a pattern designed to mimic interictal input, restored seizures. Likewise, similarly patterned electrical stimulation of these fibers in slices bathed in high [K+]o with the CA3 region removed reliably evoked electrographic seizures with period and duration similar to spontaneous seizures in whole slices. 4. Electrographic seizures but not CA3 interictal bursts could be reversibly abolished by lowering the temperature from 35-37 to 28-30 degrees C or by the competitive N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (5-10 microM). The inactive isomer, L-2-amino-5-phosphonovaleric acid (25 microM) did not eliminate seizures. 5. Neither the frequency nor intensity of interictal bursts recorded in the CA3 region changed in the minute preceding seizure initiation. Thus, although the presence of interictal input from the CA3 region is required for CA1 seizure generation, it appears that electrographic seizures do not result from a change in the quality or quantity of interictal input to the CA1 region. 6. During the 30- to 60-s period leading to a seizure the excitability of CA1 pyramidal cells appeared to increase gradually. Over the interseizure interval both CA1 pyramidal cells and glia gradually depolarized, the intensity of interictal bursts recorded in the CA1 region increased, and the extracellular DC potential recorded in the CA1 cell layer drifted negative.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Focal electrographic seizures arose in the CA1 region of rat hippocampal slices bathed in elevated (8.5 mM) external potassium [( K+]o). High [K+]o also induced spontaneous interictal bursts that originated in area CA3 and propagated to CA1. To examine the contribution to electrographic seizure initiation of excitatory mechanisms that are influenced by extracellular volume, we studied the effect of hyperosmotic expansion of interstitial volume on seizure occurrence, interictal bursts, and excitatory synaptic transmission. The tissue electrical resistance was also measured leading up to and during seizures. 2. Media made 5-30 mosmol/kg hyperosmotic by addition of agents restricted to the extracellular space (mannitol, sucrose, raffinose, L-glucose, dextran) rapidly and reversibly abolished [K+]o-induced spontaneous CA1 seizures in 86% of slices tested. However, similar increases in osmolality effected by agents that access the intracellular compartment (D-glucose, glycerol) did not influence electrographic seizure occurrence. Hyperosmotic changes with plasma membrane impermeable compounds, but not permeable compounds, produced significant concentration-dependent decreases (1-10%) in the electrical resistance of CA1 stratum pyramidale. Because tissue resistance is proportional to extracellular volume, these results suggest that hyperosmotic suppression of electrographic seizures is associated with expansion of the extracellular space in hippocampal slices. 3. Measurement of electrical resistance of the CA1 stratum pyramidale during spreading depression and electrographic seizure revealed an increase in tissue resistance to 122% and 108% of control, respectively. Furthermore, a slight (approximately 2%) but significant increase in electrical resistance gradually occurred over the 20 s immediately preceding seizure generation. The observed increase in tissue resistance suggests extracellular space is decreased during these events. 4. Hyperosmolality did not alter CA3 interictal burst frequency. However, burst intensity, estimated from the total length of the burst waveform, was significantly reduced in both the CA3 (83% control) and CA1 region (67% control) when osmotic changes were imposed by plasma membrane impermeant compounds. Additionally, media made hypoosmotic by removal of 7.5 mM NaCl reversibly increased burst intensity. 5. High [K+]o potentiated excitatory synaptic transmission and excitatory postsynaptic potential (EPSP) spike coupling.(ABSTRACT TRUNCATED AT 400 WORDS)

The endogenous neurotransmitter candidates L-aspartate, L-cysteine sulfinate (CSA), L-glutamate, L-homocysteate (HCA), and the endogenously occurring analogue quinolinate were compared in terms of potency, maximal activity, and selectivity for steady state activation of N-methyl-D-aspartate (NMDA) and non-NMDA [(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)] types of glutamate receptors expressed in Xenopus oocytes injected with mRNA isolated from rat brain (minus cerebellum). Selective activation of NMDA receptors was achieved by deleting Mg2+ and including 3-10 microM glycine in the perfusion medium and by applying ligands in the presence of 30 microM quisqualate, which blocks the AMPA receptor and desensitizes the oocyte's own Ca(2+)-dependent Cl- current. Oocytes were voltage clamped, and steady state inward currents were measured in response to perfusion with agonists at known concentrations. Under the NMDA receptor-preferring condition, the potency rank order was L-glutamate (EC50 = 2.2 microM, 95% confidence interval = 1.4-3.6 microM) greater than L-aspartate (13 microM) = HCA (13 microM) greater than CSA (59 microM) greater than quinolinate (greater than or equal to 7200 microM). All amino acids tested evoked similar maximal currents, which were 120-159% that of NMDA itself. The Hill coefficient was greater than 1 for all agonists except L-HCA (0.6), which might reflect heterogeneity of NMDA receptors expressed. This was supported by the finding that glycine was more potent in combination with HCA than NMDA, in activating NMDA receptors. To study the activity of agonists at AMPA receptors, glycine and quisqualate were omitted and 1 mM Mg2+ was included to block NMDA receptors. Ca(2+)-dependent Cl- currents activated by L-glutamate were prevented by inclusion of 0.4 M ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the recording electrode. All amino acids were less potent at AMPA receptors than at NMDA receptors; the potency rank order for steady state activation of AMPA receptors was L-glutamate (EC50 = 11 microM, 95% confidence interval = 7.3-18 microM) greater than HCA (430 microM) greater than CSA (3300 microM). L-Aspartate and quinolinate produced little or no inward current even up to 10 mM, i.e., were inactive at forebrain AMPA receptors. The maximal currents activated by all amino acids at steady state were 5-10% that of kainate, presumably due to severe desensitization of the AMPA receptor by the natural agonists. These results are consistent with L-glutamate acting as a mixed agonist at both AMPA and NMDA synaptic receptors and L-aspartate being involved exclusively in NMDA receptor-mediated synapses.

The GluR1 cDNA clone encodes a functional excitatory amino acid receptor (Hollmann et al., Nature 342: 643-648 (1989]. We have studied the pharmacological properties of this homomeric (single subunit) receptor expressed in Xenopus oocytes and compared these properties with those of receptors encoded by rat forebrain mRNA. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate, quisqualate, and glutamate were partial agonists at both GluR1 and forebrain non-N-methyl-D-aspartate (-NMDA) receptors. The potency of the agonists kainate, domoate, and glutamate was higher, and that of the antagonists 6-cyano-7-nitro-quinoxalinedione and 6,7-dichloro-3-hydroxy-2-quinoxaline carboxylic acid lower, for GluR1 receptors as compared with forebrain non-NMDA receptors. The GluR1 receptor differed strikingly from forebrain-derived non-NMDA receptors, however, in that it exhibited slow, calcium-dependent desensitization. Thus, most properties of the GluR1 receptor are similar but not identical to those of non-NMDA receptors expressed from forebrain mRNA. These results indicate that the ligand recognition sites on GluR1 homomeric receptors are subtly different from those of non-NMDA receptors expressed from a mixture of forebrain mRNA.

Xenopus oocytes injected with rat brain mRNA were used to identify and characterize the effects of compounds that are antagonists at both the glycine site on N-methyl-D-aspartate (NMDA) receptors and the quisqualate/kainate receptor. Oocytes were voltage-clamped at -60 mV and inward currents were measured at equilibrium following perfusion with agonists and antagonists. Application of 7-chlorokynurenic acid (7-Cl-Kyn) or 6,7-dichloro-3-hydroxy-2-quinoxaline carboxylic acid (6,7-diCl-HQC), each at 15 microM, produced a parallel shift to the right of the glycine concentration-response curve. Schild analysis indicated a KB of 300 nM for 6,7-diCl-HQC and 350 nM for 7-Cl-Kyn. The slopes of the Schild plots were 1.01-1.02 in each case, suggesting that both compounds are competitive glycine antagonists. Both compounds also blocked the receptor mediating kainate-induced inward current. Schild analysis of 6,7-diCl-HQC (KB = 3.0 microM, slope = 0.98) indicated competitive antagonism of kainate currents, but with a potency 10-fold lower than at the glycine site. 7-Cl-Kyn antagonized kainate-evoked currents (KB = 14.1 microM), but the slope of the Schild regression was less than 1 (0.72 +/- 0.11; p less than 0.05). Thus, 7-Cl-kyn was approximately 40-fold more potent at the glycine site than at the receptor mediating kainate currents but is probably not entirely competitive at the latter receptor. Omission of the Cl groups from these antagonists drastically reduced activity at both glycine and kainate sites. 6,7-Dinitro- and 6-cyano-7-nitro-quinoxalinedione were both more potent antagonists of kainate than glycine, but substitution of Cl at the 6-position and especially the 6- and 7-positions increased potency at the glycine site. These results suggest that the glycine coagonist site of the NMDA receptor and the agonist binding site of the quisqualate/kainate receptor have some structural similarity. Halogenated derivatives of quinoxalines and kynurenines should be useful in evaluating the function of the glycine site in synaptic transmission mediated by NMDA receptors. In this regard we found that 7-Cl-kyn (5 and 15 microM) selectively attenuated NMDA receptor-mediated epileptiform bursts in the CA1 region of hippocampal slices perfused with zero-Mg medium, without reducing the amplitude of the primary population spike. This block could be overcome by 300 microM D-serine, which alone did not influence bursting. These results together indicate that the glycine site plays a role in epileptiform bursting mediated by NMDA receptors in adult rat hippocampus.