Indeno[1,2-b]pyrazin-2,3-diones: a new class of antagonists at the glycine site of the NMDA receptor with potent in vivo activity.

Indeno¿1,2-bpyrazin-2,3-diones have been identified as a novel series of potent ligands on the glycine site of the NMDA receptor. To improve their in vivo activities, an acetic acid-type side chain was introduced to the 5-position, giving water-soluble compounds when formulated as the sodium salt (>10 mg/mL). Introduction of a chlorine atom in the 8-position led to a dramatic improvement of anticonvulsant activity and this was surprising since this change did not improve binding affinity. A plausible explanation is a reduced recognition by a Na(+),K(+)-ATPase active transport system responsible for the excretion of these compounds from the brain and kidney. This promising new chemical series led to the optically active isomer (-)-10i (RPR 118723), a glycine/NMDA antagonist with nanomolar binding affinity and in vivo activity in animal model of convulsions and electrophysiology at doses in the range of 2-3 mg/kg following iv administration.

Serial reconstruction of Lucifer yellow-labeled supraoptic nucleus neurons in perfused rat hypothalamic explants.

Intracellular recordings from supraoptic nucleus neurons in perfused explants of rat hypothalamus were followed by intracellular injections of the fluorescent dye, Lucifer yellow. Following fixation, 40 microns sections were processed for whole cell light-microscopic reconstruction in the horizontal or coronal plane. The somata of most supraoptic neurons were elongated (mean 25 X 13 microns) with 1-3 sparsely branched dendrites (length 30-725 microns) that displayed numerous spines. Most (95%) dendrites turned in the ventral direction to end in the glial lamina along the base of the nucleus. Each neuron had one axon: in 60% of cells, the axon arose from a dendritic profile and immediately assumed a varicose appearance; in the other 40% of cells, the axon appeared to arise directly from the soma and demonstrated in its initial 80-200 microns numerous spines and few varicosities, i.e. the morphological features of a dendrite. All axons coursed in a dorsomedial direction over the optic tract. At this point, most axons revealed smaller secondary processes 2-15 microns in length. Axons then turned ventrally towards the basal hypothalamus; some could be followed for up to 2100 microns from the cell somata. This approach to the light microscope morphology of supraoptic neurons provides a surprising array of detail on soma, dendrite and axon characteristics, while retaining the overall relationship between individual neurons and neighboring structures, including the boundaries of the nucleus itself.

Alkali cation permeability and caesium blockade of cromakalim-activated current in guinea-pig ventricular myocytes.

1. The sensitivity of cromakalim-activated current (Icrom) to manipulations of extracellular cationic composition was examined in whole-cell voltage clamp recordings from freshly-dispersed, adult guinea-pig ventricular myocytes. In bathing media with different concentrations of K+ (1, 2.5, 5.4 and 12 mM) the Icrom reversal potential (Erev) varied in strict correspondance with the K+ equilibrium potential and inward Icrom amplitude was proportional to the external K+ concentration. 2. Replacement of 12mM K+ with 12mM Rb+ induced a slight positive shift of Erev indicating that PRb+/PK+ = 1.06. K+ replacement with 12mM Cs+ reduced or abolished inward Icrom and produced a negative shift of Erev by at least 50 mV; an upper limit of PCs+/PK+ was fixed at 0.18. 3. Addition of Rb+ (1-30 mM) to 2.5 mM K(+)-containing medium produced a concentration-dependent increase in inward Icrom and positive shift of Erev suggesting that K+ and Rb+ have similar permeabilities and conductivities and do not interfere with each other in the channel. 4. CS+ (0.01-30 mM), added to medium containing 12 mM Rb+, induced a potent, voltage-dependent inhibition of inwardly rectifying current (IK1; IC50 = 0.2-3 mM). Voltage-dependent inhibition of inward Icrom was observed only at considerably higher CS+ concentrations (IC50 = 4-30 mM). Extracellular Rb+ and CS+ did not substantially alter the amplitude of outward Icrom. 5. The results support the contention that the ATP-sensitive K+ channel is the primary target of cromakalim action in ventricular myocytes.

ICE/Caspase-1 inhibitors as novel anti-inflammatory drugs.

In recent years, several strategies that selectively inhibit pro-inflammatory cytokines, have yielded effective protein-based therapies for inflammatory disorders, validating the therapeutic hypothesis that intervention in cytokine signalling can provide clinical benefit. However, these protein-based products must be administered by injection, a constraint associated with inconvenience, adverse effects and expense for patients, caregivers and insurers. Besides interfering with the effects of cytokines such as TNF-alpha or IL-1beta that have already been produced, inhibition of pro-inflammatory cytokine production or signalling with low-molecular weight orally-active drugs would combine the convenience of conventional pharmaceuticals with the focused efficacy of the protein therapies. Reducing IL-1beta and IL-18 production by inhibition of IL-1beta converting enzyme (ICE, caspase-1) is one promising strategy because of the key roles of these cytokines in many inflammatory diseases. Pralnacasan, the first orally available, potent and selective ICE inhibitor to enter clinical trials, is currently under investigation in rheumatoid arthritis.

Opposing alpha- and beta-adrenergic mechanisms mediate dose-dependent actions of noradrenaline on supraoptic vasopressin neurones in vivo.

The effects of pressure-applied noradrenaline (NA) on the activity of neurosecretory cells of the supraoptic nucleus (SON) were examined in anaesthetized male rats. Spontaneously active, antidromically identified neurosecretory cells were classified as vasopressin (VP)-secreting on the basis of activity patterns and responsiveness to baroreceptor activation. The probability of encountering VP units was enhanced by confining electrode penetrations to the caudal aspect of the SON. Application of low concentrations of NA (50-150 microM) excited 75% of putative VP neurones tested (n = 45), while very high concentrations (1-100 mM) were inhibitory (79%, n tested = 14). The excitatory effects of NA were blocked by the alpha 1 antagonist prazosin (0.1-5 microM, n = 9) and mimicked by application of the alpha 1 agonist methoxamine (300 microM-1 mM, n = 29). The alpha 2 agonist clonidine (800 microM-1 mM) also frequently elicited mild excitations (92%, n tested = 13); however, this was commonly followed by an extended period of quiescence. Neither the alpha 2 antagonist yohimbine (5 microM, n = 4) nor the beta-adrenoreceptor antagonist timolol (5-20 microM, n = 6) blocked NA-induced excitations. The inhibitory effects of high concentrations of NA, however, were blocked by the application of timolol (5-20 microM, n = 5). It is suggested that the excitatory effect of low concentrations of NA on VP neurones reflects the actions of this substance when endogenously secreted at normal sites of release within the SON.

Alpha-adrenergic activation of rat hypothalamic supraoptic neurons maintained in vitro.

Extracellular recordings from 141 rat supraoptic nucleus neurons maintained in vitro in a perfused hypothalamic explant indicated that the excitability of 85% of cells was enhanced by the addition of 10-200 microM norepinephrine (NE) and the alpha-agonist methoxamine (MOXY), but not by isoproterenol, to the perfusion medium or by pressure application. Similar responses were observed from 7 cells maintained in synaptic isolation in medium containing 15 mM Mg2+. At the lowest effective concentration (10 microM), NE and MOXY induced bursting activity without an overall increase in firing frequency; at higher concentrations an initial increase in firing frequency was followed by the appearance of phasic activity patterns. The actions of NE and MOXY were reversibly blocked by administration of the alpha-adrenergic antagonists phenoxybenzamine (1-10 microM), yohimbine (greater than 10 microM) and irreversibly blocked by prazosin (10 nM-1 microM). These observations suggest that NE has a predominantly facilitatory role to enhance the excitability and promote bursting activity in supraoptic neurosecretory neurons through an alpha-1 adrenoreceptor mechanism.

Allosteric potentiation by diazoxide of AMPA receptor currents and synaptic potentials.

Diazoxide (100-560 microM) reversibly increased the amplitude and duration of excitatory post-synaptic field potentials recorded in the dentate gyrus of hippocampal slices following stimulation of the perforant pathway. In rat cortex mRNA-injected Xenopus oocytes diazoxide (1-1000 microM) alone had little effect on membrane current, but rapidly and reversibly increased (up to 5-fold) current responses to (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA, 30 microM), L-glutamate (100 microM), quisqualate (3 microM), kainate (100 microM) and domoate (3 microM), an effect that was neither mimicked by other activators of ATP-sensitive potassium channels nor blocked by glibenclamide. Diazoxide increased current amplitudes for all concentrations of the 'inactivating' ligands, AMPA, L-glutamate and quisqualate but had little effect on their EC50 values. In contrast, diazoxide increased the apparent potency of the 'non-inactivating' ligands, kainate and domoate, but increased the efficacy of saturating concentrations by only 10-20%. Diazoxide did not modify the competitive inhibition of AMPA and kainate currents by 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and thus does not compete for the agonist site as do AMPA and kainate. Similarly, diazoxide neither inhibited the binding of [3H]AMPA or [3H]kainate to rat cortical membranes in competition experiments nor consistently modified the apparent [3H]AMPA affinity (Kd) or receptor density (Bmax) in saturation experiments. These data suggest that diazoxide acts at an allosteric site on the AMPA receptor/channel to potentiate activation in a manner dependent upon the properties of the excitatory agonist.

Competitive inhibition by NBQX of kainate/AMPA receptor currents and excitatory synaptic potentials: importance of 6-nitro substitution.

We evaluated the inhibitory potencies at excitatory amino acid receptors of 2,3-dihydroxy-7-sulfamoyl-benzo[f]quinoxaline (BQX) and its 6-nitro derivative, NBQX. Currents activated by kainate or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in two-electrode voltage-clamp recordings of Xenopus oocytes injected with rat cortex mRNA were inhibited by BQX and NBQX: the apparent Ki values versus kainate were 14 microM and 78 nM, respectively, and versus AMPA were 23 microM and 63 nM, respectively. Thus, to a degree even more marked than with other quinoxalinedione derivatives, 6-nitro substitution of BQX to yield NBQX increases potency (200-fold) at the non-NMDA ionotropic receptor, but does not confer selectivity for kainate or AMPA. Schild analysis of the NBQX inhibition of the kainate and AMPA currents yielded pA2 values of 7.17 +/- 0.05 and 7.05 +/- 0.10, respectively, and slopes near unity confirming the competitive nature of the inhibition. Neither BQX nor NBQX significantly inhibited the current activated by glycine plus NMDA. The selectivity ratio of NBQX (greater than 5000-fold) is by far the greatest of any quinoxalinedione derivative antagonist of the kainate/AMPA receptor. BQX and NBQX also inhibited the excitatory postsynaptic field potentials mediated by kainate/AMPA receptors in the CA1 region of hippocampal slices after stimulation of the Schaffer collateral-commissural pathways with IC50 values of 130 and 0.90 microM, respectively. The 10-fold differences between the IC50 values in hippocampal slices and the Ki values in Xenopus oocytes correlate closely with data for other quinoxalinedione derivative antagonists.

Ca2+ channel inhibition by a new dihydropyridine derivative, S11568, and its enantiomers S12967 and S12968.

Biochemical and electrophysiological techniques were used to describe the Ca2+ channel blocking properties of a new dihydropyridine derivative, S11568 (+/-)- ([(amino-2-ethoxy)-2-ethoxy]methyl)-2-(dichloro-2',3'-phenyl)-4- ethoxy-carbonyl-3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyridine and its enantiomers S12967 ((+)-S11568) and S12968 ((-)-S11568). In binding studies, S11568 and S12968 displaced specifically bound [3H]PN 200-110 from cardiac and vascular smooth muscle preparations with potencies of 5.6-51 nM, respectively. S12967 was 6- to 18-fold less potent than S12968. A good correlation was found between the IC50 value for the inhibition of 45Ca2+ uptake by A7r5 aortic smooth muscle cells and binding data. Whole-cell patch clamp studies in both guinea-pig ventricular myocytes and A7r5 cells yielded similar results. At holding potential (VH) -50 mV, S12968 inhibited L-type Ca2+ current with an IC50 value near 70 nM, 2- to 3-fold more potently than S11568 and 30-fold more potently than S12967. With VH -100 mV, all three compounds were less potent, with IC50 values ranging from 500 nM to 3 microM. These results demonstrate conclusively that S12968 is the more active enantiomer. Furthermore, the pronounced voltage dependence of its actions in vitro suggests that in vivo it could exhibit good selectivity for vascular smooth muscle over cardiac muscle.

Alpha 1-adrenergic receptor activation releases vasopressin and oxytocin from perfused rat hypothalamic explants.

Norepinephrine and the alpha-agonist phenylephrine in concentrations of 10(-5) to 10(-3) M prompted the release of radioimmunoassayable vasopressin (up to 150 pg/min) and oxytocin (up to 20 pg/min) from intraarterially perfused explants of rat basal forebrain. Drug effects were markedly reduced or abolished in the presence of the non-specific alpha-antagonists phentolamine and phenoxybenzamine, and the specific alpha 1-antagonist prazosin. In concert with recent in vivo and in vitro electrophysiological observations, these data imply that endogenous noradrenergic pathways to magnocellular neurosecretory cells are excitatory, mediated through activation of their alpha 1-receptors, thereby enhancing the release of both vasopressin and oxytocin in the neurohypophysis.

Cardiovascular input to hypothalamic neurosecretory neurons.

In vivo extracellular recordings from rat supraoptic and paraventricular magnocellular neurosecretory cells (MNCs) indicate that putative vasopressin-secreting MNCs may be identified by an abrupt and brief cessation in firing consequent to a transient drug-induced rise in arterial pressure sufficient to activate arterial baroreceptors. In the diagonal band of Broca (DBB), a population of neurons projecting towards the supraoptic nucleus are activated during this drug-induced hypertension. Electrical stimulation in DBB selectively depresses supraoptic vasopressin-secreting MNCs. Intracellular recordings in perfused hypothalamic explants confirm a DBB-evoked bicuculline-sensitive and chloride-dependent postsynaptic inhibition, similar to that associated with the application of gamma-aminobutyric acid (GABA) in approximately half of supraoptic MNCs. Since bicuculline also selectively blocks baroreceptor-induced inhibition in supraoptic MNCs, it is proposed that the depressant baroreflex input to vasopressin-secreting MNCs involves a population of DBB neurons and GABAergic interneurons located close to MNCs. An excitatory and selective input to vasopressin-secreting MNCs follows chemoreceptor activation, possibly mediated by the A1 noradrenergic cell group in the ventrolateral medulla. Another excitatory input to both vasopressin- and oxytocin-secreting MNCs is triggered by circulating angiotensin II and appears to be relayed centrally through an angiotensinergic projection from the subfornical organ.

Tetrabutylammonium induces a voltage-dependent block of kainate current in Xenopus oocytes injected with rat brain mRNA.

Following injection of rat striatal and cerebrellar mRNA, Xenopus oocytes were voltage clamped and current responses to the excitatory amino acid receptor agonist, kainate, were recorded. This nonspecific cationic current is carried principally by Na+ and K+ and reverses polarity at a membrane potential of approximately -5 mV. When the membrane potential was voltage clamped to -60 mV, bath-applied tetrabutylammonium (0.1-30 mM) produced a rapid, concentration dependent and reversible block of kainate-induced inward current with an IC50 of 1.3 mM. Tetraalkylammonium derivatives having shorter chains (methyl, ethyl, and propyl) were relatively ineffective blockers. Longer alkyl chain derivatives (pentyl, hexyl, and heptyl) were more potent than tetrabutylammonium but limited in their usefulness by their toxicity. The antagonism of kainate-induced current by tetrabutylammonium displayed apparently uncompetitive kinetics, in contrast with the competitive antagonism by gamma-D-glutamylaminomethylsulfonate. The block by tetrabutylammonium was strongly voltage dependent; an e-fold change in IC50 was observed for a 27 mV change in holding potential. Replacement of the Na+ in the medium with a more permeant cation (NH4+), a less permeant cation (tetramethylammonium), or an uncharged solute (mannitol) had little effect on the block of kainate-induced current by tetrabutylammonium. The rates of association and dissociation of tetrabutylammonium with the kainate receptor-channel are clearly rapid. These observations suggest that tetrabutylammonium enters and blocks the kainate receptor-associated cation selective channel. Tetrabutylammonium appears to traverse 80-90% of the membrane electrical field to reach a relatively low-affinity binding site that may simply be a narrowing of the channel.

Actions of gamma-aminobutyric acid on rat supraoptic nucleus neurosecretory neurones in vitro.

1. Intracellular recordings were obtained from thirty-eight rat supraoptic nucleus (s.o.n.) neurosecretory neurones in perfused hypothalamic explants. Changes in membrane potential and conductance were monitored following application of gamma-aminobutyric acid (GABA), and related agonists and antagonists. 2. GABA depressed action potential discharge of all of thirty-five s.o.n. neurones tested and induced either membrane hyperpolarization or depolarization. Neurones that displayed membrane hyperpolarization in response to lower GABA concentrations (30-300 microM) demonstrated a biphasic membrane voltage change with a later depolarizing phase as a response to higher concentrations (up to 3000 microM). 3. GABA (10-3000 microM) induced a prominent concentration-dependent increase in membrane conductance in all neurones. The critical slope for the log-log plot of [GABA] vs. GABA-induced membrane conductance was 1.7, indicating co-operativity in the GABA receptor-induced conductance change. 4. Muscimol (0.3-30 microM) potently mimicked all the effects of GABA. Bicuculline (1-100 microM) antagonized the effects of GABA and muscimol in a competitive manner. 5. Glycine and taurine (1-10 mM) had weak effects, although comparatively similar to those of GABA. These actions were blocked both by bicuculline (100 microM) and by strychnine (1 microM). At higher concentrations (greater than 10 microM), strychnine also antagonized the actions of GABA. 6. In recordings with potassium-acetate-filled micropipettes, the reversal potential of hyperpolarizing membrane voltage responses to GABA was -72.5 +/- 1.5 mV in close agreement (+/- 5 mV) with the reversal potential of inhibitory post-synaptic potentials (i.p.s.p.s) recorded in the same neurones. Depolarizing responses to GABA reversed polarity at -50 +/- 1.6 mV. In recordings with KCl-filled micropipettes, voltage responses to GABA were always depolarizing and reversed near -40.0 +/- 4.3 mV. Similarly, reduction of the concentration of chloride ions in the perfusion medium from 134 to 10.4 mM induced a positive shift of the GABA reversal potential by 40-50 mV. 7. From measurements of input resistance (Rin) and cell time constant (tau O), input capacitance (Cin; representing total membrane capacitance) was calculated as 78.9 +/- 2.1 pF. During responses to GABA or muscimol, decreased Rin was accompanied by a linearly related decrease in tau o indicating that these substances had no effect on the membrane capacitance of s.o.n. neurones.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of L-type but not T-type calcium channel current by a new dihydropyridine derivative, S11568.

S11568, (+-)[((amino-2-ethoxy)-2-ethoxy]-methyl)-2-(dichloro-2', 3'-phenyl)-4-ethoxycarbonyl-3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyr idine HCl, is a new dihydropyridine derivative that is water soluble and relatively insensitive to light. The Ca2+ channel inhibitory activity of S11568 was tested in whole-cell patch clamp recordings from cultured embryonic chick cardiomyocytes in 40 mM Ba2(+)-containing medium that revealed T-type and L-type components of inward current through calcium channels. S11568 inhibited L-type Ca2+ current with an IC50 value near 1 microM but was without effect on the T-type barium current.

Characterization of spontaneous and evoked inhibitory postsynaptic potentials in rat supraoptic neurosecretory neurons in vitro.

Intracellular recordings from 52 supraoptic nucleus neurosecretory neurons in perfused explants of rat hypothalamus revealed abundant spontaneous inhibitory postsynaptic potentials (sIPSPs) and a compound evoked inhibitory postsynaptic potential (eIPSP) following electrical stimulation in the diagonal band of Broca (DBB). These IPSPs were characterized in terms of the magnitude and ionic specificity of the underlying current and in terms of the transmitter responsible for their activation. sIPSPs rose rapidly to peak within 3-5 ms and decayed exponentially with a mean time constant of 20.2 +/- 1.9 ms (mean +/- SE), a value 1.6-fold greater than the mean cell time constant of 13.8 +/- 1.0 ms. The eIPSPs rose rapidly to peak within 3-10 ms and decayed exponentially over 60-100 ms with a mean time constant of 37.0 +/- 2.8 ms, which is 2.6-fold greater than the mean cell time constant. These features imply a brief persistence of the conductance underlying the IPSPs. In recordings with KAcetate-filled micropipettes, sIPSPs were hyperpolarizing at membrane potentials in the range of -50 to -70 mV and reversed polarity when the membrane was hyperpolarized beyond -80 mV. The mean reversal potential (EsIPSP) was -72.4 +/- 1.1 mV. eIPSPs were hyperpolarizing at resting membrane potential and could be reversed by membrane hyperpolarization beyond a mean reversal potential (EIPSP) of -67.4 +/- 1.4 mV. In recordings with KCl-filled micropipettes, sIPSPs were depolarizing at all membrane potentials more negative than -50 mV. Under these conditions, EsIPSP was estimated at -44 mV. sIPSPs were absent when chloride ions were removed from the perfusion medium. eIPSPs were depolarizing at all membrane potentials and often evoked action potentials; mean EeIPSP was 43.2 mV. Reversal potentials of spontaneous and evoked IPSPs were similar. At a given membrane potential, sIPSP amplitudes varied widely between 1 and 20 mV. The conductance increase underlying individual sIPSPs was estimated to vary between 0.17 and 3.0 nS (avg 0.6 nS) against a mean resting input conductance of 3.78 +/- 0.41 nS. Estimates of the conductance underlying eIPSPs varied widely between cells, from 0.8 to 22.0 nS (mean 72 nS). Accordingly, the ratio of evoked to spontaneous IPSP conductance varied from 1.6 to 43.7 (mean 13.1). The reversal potential of evoked IPSPs shifted with the extracellular concentration of Cl- ions ([Cl-]0) with a mean slope of 41 mV/log [Cl-]0.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha 1-adrenergic receptor activation depolarizes rat supraoptic neurosecretory neurons in vitro.

Intracellular data were obtained from 35 supraoptic nucleus neurosecretory neurons maintained in vitro in intra-arterially perfused explants of rat hypothalamus. Addition of norepinephrine, phenylephrine, or methoxamine, but not isoproterenol (30-200 microM), consistently induced membrane depolarization, bursting activity, and an associated prolongation in action potential duration, effects that were reversibly antagonized by the alpha 1-antagonist prazosin. Norepinephrine-evoked depolarizations demonstrated no consistent change in membrane resistance and were reduced both by membrane hyperpolarization and by raising extracellular K+. Norepinephrine shortened the time course of spike hyperpolarizing afterpotentials and increased the magnitude of late depolarizing afterpotentials. It is proposed that one of norepinephrine's actions on supraoptic neurons involves K+ channels, perhaps by modulation of a transient K+ current known as A current.

Non-synaptic depolarizing potentials in rat supraoptic neurones recorded in vitro.

Intracellular recordings obtained from eighty-two supraoptic nucleus neurones in perfused explants of rat hypothalamus revealed a mean resting membrane potential of -66 +/- 5 mV (S.D.) and spike amplitudes of 70-106 mV. When recorded with K acetate-filled micropipettes, non-spike membrane voltage fluctuations included spontaneous depolarizing and hyperpolarizing potentials. Spontaneous hyperpolarizing potentials peaked in 3-5 ms and decayed exponentially with a mean time constant of 20.2 +/- 0.1 ms, 1.6 times the membrane time constant of 13.8 +/- 0.1 ms. These potentials were identified as spontaneous inhibitory post-synaptic potentials, and all demonstrated a common reversal potential near -80 mV, a depolarizing shift of this reversal potential during intracellular Cl- accumulation, and reversible blockade by raising [Mg2+] to 15 mM in the perfusate. Depolarizing potentials with features typical of spontaneous excitatory post-synaptic potentials i.e. brief (8-20 ms) depolarizing transients, were rarely recorded with K acetate-filled micropipettes. Instead, most neurones demonstrated what are termed non-synaptic depolarizing potentials (n.s.d.p.s) lasting 20-125 ms (mean 86.4 +/- 8.6 ms (S.E. of mean)) with a rise time 21.1 +/- 2.8 ms and a decay time of 16.3 +/- 2.8 ms (n = 28 measured). Unlike typical spontaneous post-synaptic potentials, these events could sustain a constant peak amplitude for most of their duration. These n.s.d.p.s displayed a strong voltage-dependent behaviour and were detected only at membrane potentials within 5-7 mV of the threshold for spike initiation. Spontaneous slow depolarizing membrane shifts preceding or following phasic bursts, or any manipulation (e.g. current step, sinusoid, depolarizing after-potential) causing the membrane potential to enter this range of activation, prompted their appearance. N.s.d.p.s were completely insensitive to the presence of 15 mM-Mg2+ but they were reduced in size and frequency when Ca2+ were replaced with Co2+ or Mn2+. They were detected at a more positive membrane potential when Na+-dependent action potentials were blocked with tetrodotoxin. The size, voltage-dependent and non-synaptic nature of these depolarizing potentials raises the possibility that they reflect the activity of individual (or small clusters of) ionic channels carrying inward current. Their ability to serve as prepotentials to trigger spikes is deemed to be particularly important for promoting the onset of phasic bursts in supraoptic neurosecretory neurones.

Calcium-dependent potassium conductance in rat supraoptic nucleus neurosecretory neurons.

Intracellular recordings of rat supraoptic nucleus neurons were obtained from perfused hypothalamic explants. Individual action potentials were followed by hyperpolarizing afterpotentials (HAPs) having a mean amplitude of -7.4 +/- 0.8 mV (SD). The decay of the HAP was approximated by a single exponential function having a mean time constant of 17.5 +/- 6.1 ms. This considerably exceeded the cell time constant of the same neurons (9.5 +/- 0.8 ms), thus indicating that the ionic conductance underlying the HAP persisted briefly after each spike. The HAP had a reversal potential of -85 mV and was unaffected by intracellular Cl- ionophoresis of during exposure to elevated extracellular concentrations of Mg2+. In contrast, the peak amplitude of the HAP was proportional to the extracellular Ca2+ concentration and could be reversibly eliminated by replacing Ca2+ with Co2+, Mn2+, or EGTA in the perfusion fluid. During depolarizing current pulses, evoked action potential trains demonstrated a progressive increase in interspike intervals associated with a potentiation of successive HAPs. This spike frequency adaptation was reversibly abolished by replacing Ca2+ with Co2+, Mn2+, or EGTA. Bursts of action potentials were followed by a more prolonged afterhyperpolarization (AHP) whose magnitude was proportional to the number of impulses elicited (greater than 20 Hz) during a burst. Current injection revealed that the AHP was associated with a 20-60% decrease in input resistance and showed little voltage dependence in the range of -70 to -120 mV. The reversal potential of the AHP shifted with the extracellular concentration of K+ [( K+]o) with a mean slope of -50 mV/log[K+]o.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-activity relationships in a series of 3-sulfonylamino-2-(1H)-quinolones, as new AMPA/kainate and glycine antagonists.

This paper describes the design and synthesis of a new class of molecules, the 3-sulfonylamino-2-(1H)-quinolones, which are potent and selective antagonists at both the AMPA/kainate site as well as at the NMDA-associated glycine site. The molecules were characterized by their binding affinities to rat cortical membranes and by electrophysiology on Xenopus oocytes injected with mRNA isolated from rat cerebral cortex. The most potent compound 61 has an IC50 of 0.09 microM for binding at the AMPA/kainate site, and 0.16 microM in oocyte electrophysiology.

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage.

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3-1000 microM) induced a smooth inward current that was competitively inhibited by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 microM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (Er) of -6.1 +/- 0.5 mV. In cerebellar mRNA-injected oocytes; Er was nearly identical (-5.1 +/- 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd++ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.