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Primary cultures of neocortical neurons exhibit spontaneous Ca(2+) oscillations under zero or low extracellular [Mg(2+)] conditions. We find that mature murine neocortical neurons cultured for 9 days also produce spontaneous Ca(2+) oscillations in the presence of physiological [Mg(2+)]. These Ca(2+) oscillations were action potential mediated inasmuch as tetrodotoxin eliminated their occurrence. AMPA receptors were found to regulate the frequency of Ca(2+) oscillations. In contrast, Ca(2+) oscillations were independent of activation of L-type Ca(2+) channels, and NMDA receptors provided only a minor contribution. Release of intracellular Ca(2+) stores was involved in the oscillatory activity since thapsigargin reduced the amplitude and frequency of the oscillations. S-4-carboxyphenylglycine (S)-4CPG), an antagonist of group I metabotropic glutamate receptor (mGluR), also reduced the amplitude of oscillations. In addition, 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), a group I mGluR agonist, increased the oscillation frequency, suggesting a critical role for mGluR in the generation of Ca(2+) oscillations. The mGluR-mediated release of intracellular Ca(2+) stores appeared to be mediated by phospholipase C (PLC) since the PLC inhibitor U73122 eliminated the Ca(2+) oscillations. These results indicate that Ca(2+) oscillations in neocortical cultures in the presence of physiologic [Mg(2+)] are primarily initiated by excitatory input from AMPA receptors and involve mobilization of intracellular Ca(2+) stores following activation of mGluR.

Recent studies have shown that time windowed extraction of nonlinear parameters like an effective correlation dimension from intracranially recorded EEG of epileptic patients often allows to detect and identify an unequivocal "pre-ictal phase" preceding an epileptic seizure. In another study, however, such an anticipation could not be made. These conflicting findings may indicate that observed changes in nonlinear parameters probably depend on the type of elementary mechanisms underlying epileptic processes and/or the spatial distribution of neurons primarily involved in generation of epileptiform discharges. To test the existence of such dependencies, the transition from normal to epileptiform activity (EA) of CA3-neurons in hippocampal slices was analyzed in four epilepsy models, using a time windowed computation of an effective correlation dimension. Indeed, in xanthine and penicillin models, signal complexity in intracellular recordings was reduced before manifestation of paroxysmal depolarization shifts (PDS), whereas a preceding loss of complexity was missing in low-magnesium and veratridine models. These findings indicate that interictal-like EA is predictable only in some epilepsy models., (Copyright 1999 Elsevier Science B.V.)

Spontaneous activity is a hallmark of the thalamocortical system in vivo. Up until now, in vitro preparations of this system have been shown to be spontaneously active only when inhibition was reduced or N-methyl-D-aspartate (NMDA) receptor-mediated currents were facilitated via low extracellular magnesium levels. This study investigated the dependence of spontaneous thalamocortical activity patterns on NMDA receptor function via variation of extracellular magnesium levels (0-1 mM) and by the application of the specific NMDA receptor-antagonist D-2-amino-5-phosphonovalerate (AP5) in the absence of magnesium. We used cocultures of rat neocortical and thalamic slices which have been shown to develop reciprocal synaptic connections similar to those in vivo. Multi-site extracellular recordings revealed that the cultures were spontaneously active at all concentrations of magnesium and AP5, albeit with a high variability among cultures. Activity consisted of burst-like events which were largely synchronized within as well as among the neural tissues, and thalamic background activity during periods of neocortical quiescence. Each tissue was capable of triggering activity in the other, indicating that both thalamocortical and corticothalamic synaptic connections were functional. With increasing magnesium concentration, activity rates declined in both tissues and the site of origin of the synchronous, burst-like events shifted from neocortex to thalamus. AP5 in magnesium-free perfusion solution had qualitatively similar effects. We conclude that thalamic activity is not as dependent on the facilitation of NMDA receptor-mediated currents as neocortical activity and consequently, that the thalamus is the pacemaker of thalamocortical synchronized activity in physiological in vitro conditions., (Copyright 1999 Elsevier Science B.V.)

Regions of the thoraco-lumbar spinal cord containing sympathetic preganglionic neurones are rich in dopamine terminals. To determine the influence of this innervation intracellular recordings were made from antidromically identified sympathetic preganglionic neurones in (400 micrometers) transverse neonatal rat spinal cord slices. Dopamine applied by superfusion caused a slow monophasic hyperpolarisation in 46% of sympathetic preganglionic neurones, a slow monophasic depolarisation in 28% of sympathetic preganglionic neurones and a biphasic effect consisting of a slow depolarisation followed by a slow hyperpolarisation or vice-versa in 23% of sympathetic preganglionic neurones. Three percent of sympathetic preganglionic neurones did not respond to the application of dopamine. Low Ca2+/high Mg2+ Krebs solution or TTX did not change the resting membrane potential but abolished the slow depolarisation elicited by dopamine, indicating this was synaptic and did not prevent the dopamine induced hyperpolarisation. The dopamine induced slow hyperpolarisation was mimicked by the selective D1 agonists SKF 38393 or SKF 81297-C and blocked by superfusion with the D1 antagonist SCH 23390. It was not prevented by superfusion of the slices with alpha1 or alpha2 or beta-adrenoceptor antagonists, whereas the inhibitory or excitatory actions of adrenaline were prevented by alpha1 or alpha2 antagonists, respectively. The dopamine induced slow depolarisation occurring in a sub-population of sympathetic preganglionic neurones was mimicked by quinpirole, a D2 agonist, and blocked by haloperidol, a D2 antagonist. Haloperidol did not block the dopamine induced hyperpolarisations. Dopamine also induced fast synaptic activity which was mimicked by a D2 agonist and blocked by haloperidol. D1 agonists did not elicit fast synaptic activity., (Copyright 1999 Elsevier Science B.V.)

Perfusion of 100 microM melatonin had no effect on low frequency synaptic transmission, but prevented the induction of tetanically induced long-term potentiation (LTP) when recorded in the dendritic region of the CA1 in rat hippocampal slices. Perfusion of 100 microM melatonin in this preparation had no effect on the multiple population spikes recorded in Mg2+-free medium, and, in grease-gap recordings from the CA1-subiculum slice, 100 microM melatonin had no effect on depolarisations evoked by N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This suggests that melatonin has the ability to prevent the formation of LTP, and that this effect is not mediated by blockade of NMDA receptors.

The action of N-methyl-D-aspartic acid (NMDA) on suprachiasmatic neurones was studied using whole-cell recordings in coronal hypothalamic slices of the rat. The location of the recorded neurones within the suprachiasmatic nucleus was ascertained by intracellular labelling with biocytin, followed by histological processing of the slice. Suprachiasmatic neurones had an input resistance of 780 +/- 20 M omega (mean +/- S.E.M.; n = 106). They were voltage-clamped at or near their resting membrane potential and their responsiveness to NMDA was tested by adding this compound to the perfusion solution. NMDA generated an inward current in about 85% of the neurones. At 50 microM, the average induced peak current was 30 +/- 10 pA (n = 32); at 100 microM, it was 50 +/- 10 pA (n = 12). The NMDA-induced current was reduced by D-2-amino-5-phosphopentanoic acid (D-AP5), and NMDA receptor antagonist, and was suppressed by MK-801, and NMDA channel blocker. Reducing the extracellular magnesium concentration from 1 to 0.01 mM caused a 2- to 3-fold increase in the amplitude of this current. Thus, suprachiasmatic neurones are endowed with functional NMDA receptor-channels, which may play a role in glutaminergic transmission in this nucleus. Decreasing the extracellular calcium concentration from 2 to 0.01 mM caused a 1.3- to 4.5-fold enhancement in the whole-cell NMDA current. This effect was probably not mediated by a change in the intracellular free calcium concentration. Indeed, loading suprachiasmatic neurones with 11 or 20 mM of the calcium chelator, 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetracetic acid (BAPTA) suppressed a calcium-dependent slowly decaying outward aftercurrent but did not affect the low-calcium-induced facilitation of the NMDA response. NMDA current-voltage relations were established in normal and low-calcium perfusion solutions. In the normal solution, the net current generated by NMDA contained a region of negative slope conductance and reversed in polarity at 7 +/- 2 mV. In the low-calcium solution, this current increased in amplitude in the region of negative slope conductance, whereas at more depolarized potentials it was not altered. The NMDA-induced current was fitted using the Boltzmann equation. The effect of a low-calcium solution could be modelled by shifting the activation of the NMDA-sensitive conductance in the negative direction, by about 17 mV. We conjecture that lowering external calcium can unmask negative surface charges located on or near the NMDA channel and that this, in turn, weakens the voltage-dependent block of the channel by magnesium. A voltage-dependent blockade of the NMDA channel by calcium, however, may be also contribute to this effect. This low-calcium-induced facilitation of the NMDA response could play a regulatory role by enhancing calcium influx through the NMDA channel in case of calcium depletion in its vicinity.

Properties of aluminium fluoride (AIF) complex-activated nonselective cation (NS) channels in guinea-pig chromaffin cells were investigated using the patch clamp technique. As the membrane potential was hyperpolarized from the holding potential of -55 mV, the AIF-induced nonselective cation current (INS) diminished progressively. With hyperpolarizations to -100 mV or more negative potentials, the AIF.INS almost instantaneously disappeared. The apparent unit conductance of AIF INS was estimated to be 3 pS by fluctuation analysis. The open state probability of AIF-activated NS channels became large with a decrease in concentration of free Mg2+ ions inside the cell and was less than 0.5 at 12 microM Mg2+. It is concluded that NS channels in the chromaffin cell apparently differ from those in smooth muscle cells.

We tested the hypothesis that the release of glutamate following activation of N-methyl-D-aspartate (NMDA) receptors is mediated by nitric oxide (NO) production, using slices of the guinea pig hippocampus. The NMDA-induced glutamate release from slices of dentate gyrus or CA1, which was both concentration-dependent and Ca(2+)-dependent, was also Mg(2+)-sensitive and abolished by MK-801, a selective non-competitive NMDA receptor antagonist. In dentate gyrus, the NMDA-induced glutamate release was inhibited non-significantly by tetrodotoxin, whereas the NO synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) blocked the NMDA-induced release of glutamate in a concentration-dependent manner, but not a high K(+)-evoked release of glutamate. In addition, the L-NNA blockade of NMDA-induced release of glutamate was recovered by pretreatment with L-arginine, the normal substrate for NOS. These results suggest that activation of NMDA receptors in dentate gyrus, as well as subsequent Ca2+ fluxes, is required for the neuronal glutamate release mediated by NO production. On the other hand, the NMDA-evoked glutamate release from CA1 region was tetrodotoxin-sensitive and was not inhibited by L-NNA, thereby suggesting that activation of NMDA receptors in CA1 results in increased glutamate release in an NO-independent manner. Taken together, the NMDA receptor-mediated neuronal release of glutamate from the guinea pig dentate gyrus likely involves the recruitment of NOS activity.

Exposure of hippocampal slices to Mg2+ free media (0 Mg) has been shown to trigger full production of stimulus-induced seizure activity after restoration of physiological conditions [1]. In the present study employing hippocampal entorhinal cortical slices (HEC), spontaneous epileptiform discharges (SEDs) were induced using 0 Mg treatment following the return of the slices to physiological conditions. To evaluate the effect of sustained epileptiform activity on gene expression in this HEC slice preparation, changes in mRNA levels of the GABAA alpha 1 and alpha 2 and beta CaM Kinase II subunits were measured using in situ hybridization. HEC slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) in the presence or absence of Mg2+ for 3 h, then placed in oxygenated ACSF containing Mg2+ for up to 3 h. Control slices were maintained in Mg2+ containing ACSF for up to 6 h. Recurrent SEDs were observed in 0 Mg pre-treated slices while no epileptiform discharges were seen in control slices. Following induction of SEDs by 0 Mg pre-treatment, a significant decrease in mRNA encoding GABAA alpha 2 was found in the CA1, CA2, CA3 and dentate gyrus (DG) regions of the hippocampus for up to 3 h after treatment. Levels of mRNA for GABAA alpha 1 and beta CaM Kinase II were not affected. The results document a decrease in GABAA alpha 2 gene expression following the induction of SEDs in the HEC slice preparation and suggest that rapid changes in neuronal gene expression may contribute to long lasting excitability changes associated with the induction of epilepsy.

The NMDA regulation of the dendritic release of [3H]dopamine ([3H]DA) synthesized from [3H]tyrosine was investigated in vitro using a microsuperfusion procedure in the pars compacta (SNC) and the pars reticulata (SNR) of the cat substantia nigra. The spontaneous release of [3H]DA was threefold higher in the SNC than in the SNR and amphetamine (1 microM) enhanced similarly [3H]DA release in both nigral areas. In the absence of magnesium, NMDA (50 microM) stimulated markedly the release of [3H]DA in the SNC and SNR, these effects being completely prevented by MK 801 (1 microM), the NMDA receptor antagonist. The DA uptake inhibitor, nomifensine (5 microM), increased the amount of [3H]DA recovered in SNC (x2) and SNR (x3) superfusates but did not significantly modify the NMDA-evoked responses. The effects of NMDA seen in the absence or presence of nomifensine persisted when the two nigral areas were continuously superfused with tetrodotoxin (1 microM). These results are in favor of the presence of NMDA receptors on dopaminergic dendritic arborizations and indicate that the stimulation of these receptors facilitates in a similar way the release of DA from proximal and distal dendrites.

Intracellular recordings of layer V neurons from rat neocortical slices were obtained to examine the effects of reducing extracellular magnesium on inhibition. Magnesium-free solutions induced interictal and ictal-like events in cortical neurons. Changes in synaptic events underlying epileptogenesis were studied when extracellular calcium was raised (from 2 to 3-7 mM) since this delayed seizure activity. With increasing time of exposure of cells to magnesium-free solutions, there was a significant increase in the size and duration of both the depolarizing and slow synaptic hyperpolarizing responses, but the fast synaptic hyperpolarization significantly declined in amplitude. When cells were recorded with cesium acetate-filled microelectrodes slow hyperpolarizing responses were blocked, but depolarization of cells to 0 mV allowed an isolated fast hyperpolarizing response to be recorded following synaptic stimulation. The amplitude of this response was unchanged after exposure to magnesium-free solutions. Synaptic responses of cells initially bathed in an N-methyl-D-aspartate (NMDA) antagonist (CPP) were unchanged by subsequent exposure to magnesium-free solutions. CPP exposure by itself caused a decrease in depolarization duration, increase in fast hyperpolarizing amplitude, and decrease in slow hyperpolarization amplitude and duration. When the fast hyperpolarization was viewed in isolation (cesium recording electrodes) at 0 mV, the amplitude of this event was unchanged by exposure to CPP. Given these results stimulus-response characteristics of neocortical neurons were reassessed under control conditions. With higher intensity stimuli larger depolarizing and slow hyperpolarizing responses were evoked, but the fast hyperpolarization showed a decremental response. These effects were reversed when CPP was added. When NMDA activity was enhanced by exposure to magnesium-free solutions or electrical stimulation, the amplitude of excitatory events and slow hyperpolarizations increased, but fast inhibitory responses showed limited capacity for incremental recruitment. This suggests fast inhibition is saturated (maximal) at submaximal levels of excitation, and can be overcome by increasing levels of excitation. Such a process is active under physiological conditions, altering the efficacy of inhibition.

Percent recovery of CA1 field EPSP amplitude following various anoxic aglycemic (AA) periods was examined in rat hippocampal slices superfused with MK-801 (0.1 microM, 1 microM, 10 microM) or Mg(2+)-free artificial cerebrospinal fluid. Slices treated with 0.1 microM MK-801 showed greater percent recuperation of EPSP amplitude following 3 min 30 s of AA (36 +/- 12% vs 6 +/- 4% in controls). Higher concentrations of MK-801 resulted in a greater recovery of EPSP amplitudes in more than one time period of AA, with 10 microM MK-801 providing protection in up to 4 min 30 s AA. Percent recuperation of EPSP amplitude was smaller in Mg(2+)-free slices following 2 min (34 +/- 15% vs 81 +/- 11% in controls) and 2 min 30 (25 +/- 14% vs 77 +/- 10% in controls) of AA. These results suggest that the activation of the N-methyl-D-aspartate (NMDA) receptor channel may contribute to irreversible AA induced synaptic failure in CA1.

To evaluate actions of glutamate on excitatory synaptic transmission in the central nervous system, we examined glutamate-induced changes in the paired pulse facilitation of monosynaptic excitatory post-synaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Glutamate (1 mM) depolarized motoneurons both in the presence and absence of Mg2+. In most cells perfused with Mg(2+)-free or high Ca(2+)-Mg2+ solutions, the glutamate potential was accompanied by a reduction in peak amplitude of EPSPs, although the degree of change varied with the cells. Glutamate enhanced the EPSP amplitude in a few cells with Mg(2+)-free and high Ca(2+)-Mg2+ solutions, and in most cells with high Mg2+ medium. In 3/5 cells tested, the paired pulse facilitation of EPSPs was reduced by glutamate when the EPSP amplitude either increased or decreased. NMDA (50 microM), kainate (50-100 microM), quisqualate (5-50 microM) and L-2-amino-4-phosphonobutyrate (L-AP4, 1 mM) also decreased the facilitation in about half of the cells tested. The glutamate-induced decrease in the facilitation was observed in both the presence and absence of Mg2+ and was not affected by the concomitant application of glutamate and antagonists for non-NMDA or NMDA receptors, such as 6-cyano-7-nitro-quinoxalinediones (CNQX, 60 microM) or 2-amino-5-phosphonovalerate (APV, 250 microM). Glutamate reduced the facilitation of excitatory post-synaptic currents (EPSCs) recorded at a constant membrane potential under voltage clamp, when the EPSC amplitude either increased or decreased and when the input conductance either increased or decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

This study was designed to assess some of the contributions of excitatory amino acids to locally evoked responses in neurons in slices from frontal motor cortex in Sprague-Dawley rats. Intracellular recordings were obtained from 54 cortical neurons. Paroxysmal depolarization shifts (PDS) were evoked by local single pulse stimulation in cortex or in a small number of cases (n = 2) occurred spontaneously. These potentials could be abolished by application of kynurenic acid, a broad spectrum excitatory amino acid receptor antagonist. They were enhanced in Mg(2+)-free medium and could then be antagonized by application of D,L-2-amino-5-phosphonovalerate (AP5), a selective blocker of the N-methyl-D-aspartate (NMDA) receptors.

The effects of phospholipase blockers on tetanus-induced long-term potentiation (LTP) and of diacylglycerol (DG) and arachidonic acid (AA) on synaptic transmission were studied in CA1 neurons of guinea pig hippocampal slices to evaluate the role of protein kinase C (PKC) and AA on the maintenance of LTP. Tetanus-induced LTP was suppressed by perfusion with neomycin (1 mM) or 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC, 0.1 mM), blockers of phospholipase. 1-Oleoyl-2-acetyl-glycerol (OAG, 100 micrograms/ml) and AA (100 microM) produced a temporal increase in both the amplitude of the population spike (PS) and the slope of the field excitatory postsynaptic potentials (EPSPs) but failed to produce LTP. Application of OAG or AA in low-Mg2+ (0.1 mM) solution induced LTP. OAG- and AA-induced LTP was blocked by DL-2-amino-phosphopentanoic acid (AP5; 50 microM). The administration of a potent activator of PKC, phorbol-12,13-dibutyrate (PDBu), in low-Mg2+ (0.1 mM) solution enhanced the PS and EPSPs for 2 or 3 h but this enhancement did not persist. These results suggest that PKC activation is not as important as AA for the maintenance of LTP and that OAG and AA play important roles in the maintenance of LTP in synergy with the influx of Ca2+ through NMDA receptor-coupled channels.

Intracellular recordings were made from antidromically identified motoneurons in neonate (12-22 days) rat transverse spinal cord slices and the transmitters and receptors probably involved in initiating the excitatory (EPSP) and inhibitory (IPSP) postsynaptic potentials were investigated. Stimulation of dorsal roots elicited in motoneurons an EPSP, an IPSP, or an EPSP followed by an IPSP. EPSPs in 70% of motoneurons had a short latency (less than or equal to 1 ms) and in the remaining cells a latency longer than 1 ms. The IPSPs had a long latency (greater than or equal to 1 ms). Short- and long-latency EPSPs were enhanced by the acidic amino acid uptake inhibitor L-aspartic acid-beta-hydroxamate (AAH) and depressed by the non-selective glutamate receptor antagonists gamma-D-glutamylglycine (DGG) and kynurenic acid. Short-latency EPSPs were suppressed by the quisqualate/kainate (QA/KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) but not by the N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino-5-phosphonovaleric acid (APV) and ketamine. Long-latency EPSPs were reduced by DNQX as well as by APV and ketamine. Superfusion of the slices with a Mg-free solution increased the EPSPs and unmasked a late, APV-sensitive component. The IPSP was reduced by the glycine antagonist strychnine as well as by APV and ketamine but resistant to DNQX. The results indicate that stimulation of dorsal roots elicited in motoneurons a monosynaptic EPSP mediated by glutamate/aspartate acting predominantly on the QA/KA subtype of glutamate receptors; an NMDA component can be unveiled in Mg-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Power spectra were used to analyse spontaneous field potentials (SFPs) recorded in the CA3 distal apical dendritic region of guinea pig hippocampal slices perfused with either penicillin or reduced Mg2+. High concentrations of penicillin (2000 IU/ml) progressively converted the low amplitude, irregular oscillations observed in control medium to higher amplitude, low frequency, rhythmic oscillations at approximately 2-3 Hz just prior to the onset of spontaneous, synchronized bursting. Low concentrations (50-300 IU/ml) increased the power of frequencies below 10 Hz and suppressed higher frequencies in a dose-dependent fashion. Although Mg2(+)-free medium also increased the magnitude of the SFPs prior to the onset of synchronous bursting, the changes were smaller than with penicillin and the frequency distribution was completely different. Low concentrations of Mg2+ (0.0-0.5 mM) increased the power across all frequencies, however, the maximal effect was on frequencies between 5 and 25 Hz. The transition from normal to epileptiform activity may proceed through at least 2 distinct intermediate states. When recurrent inhibition is blocked (penicillin), synchronous synaptic activity precedes the onset of bursting, whereas non-specific increases in excitability and activation of NMDA receptors (reduced Mg2+) produce an asynchronous transition state.

All-or-none electrographic seizures (EGSs) were studied in hippocampal slices from young (21- to 38-day-old) rats in medium containing low (0 mM) or physiological (0.9 mM) levels of magnesium, with and without the GABAB agonist baclofen. Extracellular recording and stimulation were performed in stratum pyramidale and stratum radiatum of CA3, respectively. EGS activity was induced by exposure to low-Mg medium or by delivering repetitive stimulus trains in physiological Mg medium. After EGS activity had stabilized, the EGSs were tested for all-or-none behavior by varying the number of pulses in a train. An EGS was considered all-or-none if subthreshold stimulation produced no afterdischarge bursts, and if the EGS duration was largely independent of the number of suprathreshold stimulus pulses. According to this measure, EGSs in Mg-free + baclofen medium were all-or-none. EGSs evoked in physiological Mg medium were also all-or-none, although the threshold was higher, and the EGS duration lower, than in Mg-free medium. This all-or-none characteristic was observed whether the EGSs were induced by prior exposure to Mg-free medium or by repetitive stimulation, and in the presence and absence of baclofen. The all-or-none characteristic suggests that while the triggering mechanism for EGSs is strongly dependent on stimulus intensity, regenerative mechanisms--independent of stimulus intensity--are responsible for the maintenance of EGSs. EGSs are also terminated by mechanisms not dependent on stimulus intensity.

It has previously been found that the sensitivity of hippocampal slices to adenosine depends on magnesium ions. The addition of 2-amino-5-phosphonopentanoic acid (2AP5) in magnesium-free medium, thus blocking receptors for N-methyl-D-aspartate (NMDA) did not modify this dependence. Here it is shown that if 2AP5 is present before the removal of magnesium ions the sensitivity to adenosine is maintained. The channel blocker MK-801 also restores adenosine responsiveness. Superfusing slices with NMDA itself directly suppresses adenosine responses and can reverse adenosine inhibition of synaptic potentials. Increasing neuronal excitability non-selectively with 8.5 mM potassium in the presence of 2AP5 has little effect on adenosine sensitivity, while removing the 2AP5 causes a loss of sensitivity. The results indicate that activation of NMDA receptor-coupled channels causes a reduction of adenosine sensitivity. This sequence of events may be viewed as a positive feedback and may be of fundamental importance in phenomena such as long-term potentiation, seizure generation and kindling.

The components of an HPLC system can be easily reassembled into an instrument for transient-state enzyme kinetic experiments based on the continuous-flow technique. The scheme of reassembly, operation and data evaluation is described in detail for the Pharmacia FPLC system. The working quality of the suggested scheme was tested using a well-studied process of bacterial beta-galactosidase activation by Mg2+ ions. The found parameters were in a good agreement with the literature data. The pulse-flow mode was developed for the delivery of working solutions to reduce the required quantities of enzyme and/or other reaction components. The work presents new approach to HPLC modules as building blocks for sophisticated liquid flow reactors, such as automatic solid phase synthesizers, sequencers and so on.

Extracellular recordings were obtained from spontaneously active, presumed dopaminergic neurons of the ventral tegmental area (VTA) of the rat in a slice preparation. Bath-applied N-methyl-D-aspartate (NMDA) (1-20 microM) activated all neurons tested (n = 36). This effect was clearly concentration-dependent (n = 14), quickly reversible and reproducible. No bursting type of discharge was observed during NMDA infusion. The NMDA receptor blocker DL-2-amino-5-phosphonovaleric acid (50 microM) reversibly antagonized the increase in cell firing produced with 10 microM NMDA by 83.5 +/- 3% (mean +/- S.E.M.) (n = 8, P less than 0.05). Lowering the Mg2+ concentration of the perfusion fluid to one-third of its normal value significantly enhanced the excitatory effect of 5 microM NMDA (n = 7, P less than 0.05), but not of 500 nM carbachol (n = 6). Finally, NMDA did not modify the sensitivity of dopaminergic autoreceptors of VTA neurons (n = 8), when compared to controls (n = 10). These observations strongly support the presence of specific NMDA receptors in the VTA.

Extra- and intracellular recording techniques were used to study the epileptiform activity generated by rat hippocampal slices perfused with Mg2(+)-free artificial cerebrospinal fluid (ACSF). This procedure induced in both CA1 and CA3 subfields the appearance of synchronous, spontaneously occurring epileptiform discharges which consisted of extracellularly recorded 100-800 ms long, positive shifts with superimposed negative going population spikes. Simultaneous, extracellular recordings from CA1 and CA3 subfields revealed that the epileptiform discharges in CA3 preceded those occurring in CA1 by 5-25 ms. Surgical separation of the two areas led to the disappearance of spontaneous events in the CA1 but not in the CA3 subfield. In this type of experiment CA1 pyramidal cells still generated epileptiform discharges following orthodromic stimuli. The intracellular counterpart of both spontaneous and stimulus-induced epileptiform discharges in CA1 and CA3 pyramidal cells was a large amplitude depolarization with high frequency discharge of action potentials which closely resembled the paroxysmal depolarizing shift recorded in the experimental epileptogenic focus. A hyperpolarizing potential triggered by alvear stimuli was recorded in CA1 cells perfused with Mg2(+)-free ACSF. This hyperpolarization was blocked by bicuculline methiodide (BMI) indicating that it represented a GABAergic inhibitory postsynaptic potential (IPSP). BMI also caused a prolongation of both spontaneous and stimulus-induced Mg(+)-free epileptiform discharges. Perfusion of the slices with the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphono-valerate (APV) reduced and eventually abolished the Mg(+)-free epileptiform discharges. These effects were more pronounced in the CA1 than in the CA3 subfield. APV also reduced the amplitude and the duration of the alveus-induced IPSP. These data demonstrate that Mg(+)-free epileptiform activity is present in the hippocampal slice at a time when inhibitory GABAergic potentials are operant as well as that in the CA1 subfield this type of epileptiform activity is dependent upon NMDA-activated conductances. Our experiments also indicate that NMDA receptors might be involved in the neuronal circuit responsible for the hyperpolarizing IPSP generated by CA1 pyramidal neurons.

At frog neuromuscular junctions immersed in solutions containing 0.5 mM Mn2+, verapamil (40 microM) reduced the increase in miniature end-plate potential (MEPP) frequency produced by tetanic stimulation (50 Hz, 2 min) of the motor nerve to 5% of that in the absence of verapamil. In solutions containing 5 mM Mg2+, verapamil reduced the tetanic increase in MEPP frequency to 8% of that in the absence of verapamil. Verapamil added to solutions containing 0.15 mM Ca2+ decreased the tetanic rise in MEPP frequency to 6% of the control value. In low Ca2+ (nominally Ca2(+)-free) solutions, verapamil decreased the tetanic rise to 70% of the control value. The present results suggest that Mn2+ and Mg2+, as well as Ca2+, enter the nerve terminal through Ca2+ channels during nerve stimulation and promote transmitter release. In addition to its effect on the Ca2+ channel, verapamil at higher concentrations appears to have inhibitory effects on the acetylcholine-gated end-plate channel and on the Na+ channel as suggested by its depressive effects on the amplitudes of MEPPs, end-plate potentials and nerve terminal action potentials.

Brief exposure to Mg2(+)-free medium (MFM) enhanced the population response of CA1 neurons to stratum radiatum stimulation in hippocampal slices from normal (+/?) and epileptic tottering (tg/tg) mice. The enhancement was maintained in both groups for at least 2 h following reperfusion with normal medium (NM). Excitability curves obtained from the extracellular records suggest that, while both synaptic activation and postsynaptic excitability are enhanced during MFM perfusion, only the latter enhancement is maintained at significant levels after reperfusion with NM. The long-term increase in postsynaptic excitability was comparable in strength to that produced by long-term potentiation (LTP) inducing tetanic stimuli, was accompanied by an increase in the slope of the population spike/field excitatory postsynaptic potential (PS/fEPSP) curve and did not appear to depend on the induction of epileptiform activity by MFM. Both the short- and the long-term effects of MFM on synaptic activation and postsynaptic excitability were qualitatively similar in normal and epileptic mice and any quantitative differences were not statistically significant. Thus, epileptogenesis in the tottering mutant may not involve a change in the NMDA receptor-mediated control of excitability, at least in the CA1 area of hippocampus.

The effect of superfusion of the internal surface of the skin of Xenopus laevis with saline containing Co2+, Ca2+, Mg2+, or Ba2+, on the frequency of spontaneous action potentials of the lateral line nerve, was studied to investigate the role of extracellular Ca2+ in spontaneous neural activity. Addition of divalent cations to frog saline, either singly or as a mixture of two different ions, produced concentration-dependent suppression of spontaneous rate. The rank order of potency for suppression by each ion, perfused alone, was Co2+ greater than Ca2+ greater than Mg2+ greater than Ba2+. Suppression by combinations of Mg2+ and Co2+, or of Ca2+ and Co2+, was approximated by the sum of the suppressive effects of each cation. Ca2+ was more suppressive than Mg2+ when each of these ions was paired with the same amount of Co2+, while Ca2+ was approximately as suppressive as Co2+ when similarly paired with Mg2+. One interpretation of the suppression by Ca2+ invokes the hypothesis that divalent cations suppress spontaneous activity by charge screening of voltage-sensitive Na+ channels on afferent dendrites and that release of neurotransmitter by the influx of extracellular Ca2+ through voltage-sensitive Ca2+ channels of hair cells may not be the sole mechanism for generation of spontaneous activity in the lateral line. These results quantify the relative suppressive potency of common divalent cations in the lateral line, and serve as a caveat to investigators who interpret a blockade of action potentials by high concentrations of Co2+ or Mg2+ as sufficient evidence for dependence of neurotransmission upon extracellular Ca2+, particularly in acousticolateralis systems.

An enzyme from Leishmania donovani that decatenates kinetoplast DNA networks into covalently closed monomeric circles has been isolated and characterized. The enzyme also relaxes supercoiled plasmid pBR322. The decatenation and relaxation reactions both require ATP and Mg2+. In both reactions the formation of topological isomers of unique linking numbers suggests that the enzyme is a type II DNA topoisomerase. Both the relaxation and decatenating activities are inhibited by novobiocin at a very high concentration.

As measured by extracellular single-cell recording, the responses to adenosine triphosphate (ATP) by ATP-sensitive chemoreceptors (ATP cells) on the olfactory organ of the spiny lobster are markedly suppressed by adenosine diphosphate (ADP), adenosine monophosphate (AMP) and to a lesser extent, adenosine, when each is presented in binary mixture with ATP. In the presence of ADP, the dose-response function for ATP exhibits an apparent parallel displacement to the right suggesting that this antagonism may occur via competition at the ATP receptor. Structure-activity relationships reveal that the structural requirements for antagonism by diphosphate analogs of ADP bear little relationship to the requirements for the agonistic activity of corresponding triphosphate analogs. Under Mg2+-free conditions, the desensitization of ATP cells tends to be delayed resulting in enhanced responses to ATP. Desensitization does not appear to be related to the generation of the antagonist, ADP, from ATP via ecto-ATPase activity. The results of this study suggest that the responses of ATP cells to the ATP contained in natural stimulus (odor) mixtures can be tempered by the suppressive interactions of other nucleotides in the mixtures. Furthermore, these interactions may be mitigated and/or intensified by the actions of sensillar ectonucleotidases.

After 10-60 min of normothermic complete ischemia, hippocampal slices were prepared and allowed to recover for 60 min. The presence or absence of an evoked transsynaptic response was measured in CA1, CA3, and dentate gyrus. A selective vulnerability of the field excitatory postsynaptic potential to ischemia was found (CA1 greater than CA3 greater than dentate gyrus). Recovery of synaptic transmission in CA1 and CA3 was significantly improved by decreasing extracellular Ca2+ and increasing Mg2+ after ischemia. Addition of an N-methyl-D-aspartate antagonist further improved functional recovery. Postischemic reduction in extracellular Cl- increased recovery in CA1 and CA3, whilst reduction in Na+ was deleterious.

The effect of magnesium-free medium on the electrical activity in CA3 of the rat hippocampal slice was examined. Magnesium removal resulted in the development of spontaneous and triggered interictal-like bursting, followed by spontaneous ictal-like events and finally periodic clustered bursts. The ictal-like events consisted of a tonic firing phase and a phase of clustered burst discharges resembling the tonic and clonic phases of seizures. The return to normal medium resulted in spontaneous and triggered interictal-like bursts.

Short-term potentiation of extracellular field excitatory postsynaptic potentials (EPSP's) was investigated in the stratum radiatum of CA1 of the rat hippocampal slice in vitro. A presynaptic tetanus consisting of a single train of stimuli at 250 Hz evoked short-term potentiation (STP), but not long-term potentiation (LTP) of small amplitude, just suprathreshold, EPSP's in a medium containing 2 mM Ca and 2 mM Mg. Increasing the number of stimuli in the tetanus enhanced the initial amplitude of the STP to a maximum of 40-70% with 30-40 stimuli, and enhanced the decay time constant of STP to a maximum of 60-80 s. Increasing the Ca influx by raising external Ca, lowering Mg or adding the agent 4-aminopyridine caused a large increase in the initial amplitude of STP but only a very slight increase in the decay time of STP. LTP was frequently evoked in high Ca/Mg ratios of 2.0 or 4.0, but not in 1.0 or 0.5.

The in vitro effects of ethanol and acetaldehyde on polymerization of calf brain microtubular proteins (MTP) were examined. While ethanol up to 100 mM had no effect on the polymerization of MTP, acetaldehyde above 0.5 mM had an inhibitory effect. This effect was not dependent on the presence of microtubule-associated proteins (MAPs), since acetaldehyde had a similar effect on the polymerization of highly purified tubulin. Electron microscopy revealed that the number and the length of microtubules at equilibrium was reduced by the presence of acetaldehyde. Acetaldehyde raised the critical concentration for tubulin assembly and caused greater inhibition at lower tubulin concentrations. Acetaldehyde augmented the depolymerizing effects of Ca2+ on preassembled microtubules. In addition, acetaldehyde itself caused depolymerization of microtubules but only in the absence of MAPs. Long-term (19.5 h) incubation of MTP with acetaldehyde led to significant loss of polymerization ability which could not be reversed by removal of acetaldehyde. This loss of activity was apparently independent of the observed formation of reducible adducts between acetaldehyde and MTP.

Spontaneous discharges were observed in recordings from dorsal and ventral roots (DRs and VRs) of hemisected mouse spinal cords in vitro when bath [Ca2+] was raised from the control level of 1.2 to 1.8 or 2.4 mmol/l, and when bath [Mg2+] was lowered resembling drug-induced 'interictal' discharges described earlier. Maximum discharge frequency was reached at 2.4 or 3.6 mmol/l [Ca2+] while at higher concentrations mean frequency diminished but mean amplitude still increased somewhat. The frequency distribution of wave amplitudes suggests recruitment of neurons by groups or assemblies. The pacemaker is located in the dorsal spinal quadrant. The GABAA receptor antagonists picrotoxin and bicuculline in low concentration blocked the spontaneous activity, indicating an obligatory GABAergic link in the pacemaker circuit. The NMDA antagonists D.L-APV and D-APV in high concentration variably depressed but did not abolish spontaneous activity. The propensity of spinal neuron assemblies for self-paced discharge may contribute to the pathologic irritability of injured spinal cords.

Seizure-like discharges were observed in slices of human epileptogenic neocortex maintained in vitro when [Mg2+]o was lowered near to zero. This type of epileptiform activity: (1) could occur spontaneously or following extracellular focal stimuli; (2) resembled the electrographic pattern associated with tonic-clonic seizures; (3) was accompanied by increases in [K+]o (maximally 6.2 mM from a baseline of 3.25 mM) and decreases in [Ca2+]o (maximally 0.23 mM from a baseline of 1.8 mM). Application of the selective antagonist of N-methyl-D-aspartate (NMDA) receptors, DL-2-amino-5-phosphonovalerate, suppressed in a reversible manner both spontaneous and stimulus-induced seizure-like discharges, suggesting that NMDA-activated conductances are important for the genesis of prolonged epileptiform discharges generated by human epileptogenic neocortical slices.

Hemisected mouse spinal cords were maintained in vitro and the concentrations of calcium [CA2+] and of magnesium [Mg2+] in the bath were varied. In control solution containing 1.2 mM of both [Ca2+] and [Mg2+] stimulation of a dorsal root (DR) evoked in an adjacent DR an initial fiber volley representing 'input'; a postsynaptic compound spike recorded by volume conduction from dorsal gray matter, the dorsal horn response (DHR); and a slow dorsal root potential (DRP). In the ventral root of the stimulated segment a monosynaptic reflex (VRR1) was evoked. The fiber volley was enhanced by lowering [Ca2+] or [Mg2+] and depressed when either ion concentration was raised. The DRP, DHR and VRR1 were enhanced in low [Mg2+] and in moderately elevated [Ca2+]. At 1.8 mM [Ca2+] and above, the 'classical' dorsal root reflex (DRR) and a GABA-dependent delayed VR reflex (VRR2) appeared. Transmission of reflexes was maximal between 2.4 and 3.6 mM [Ca2+], while DRP was maximal at about 4.8 mM. In elevated [Mg2+] and in low [Ca2+] all synaptically transmitted responses (DRR, DRP and VRR) were depressed. The influence of both [Ca2+] and [Mg2+] was stronger on DRP, DRR and VRR2 than on DHR and VRR1. The effects of simultaneous changes of [Ca2+] and [Mg2+] partially cancelled each other. We conclude that low to moderately elevated [Ca2+] mainly influences the release of transmitter from presynaptic terminals; at very high [Ca2+] the depression of neuronal excitability dominates. The effects of Ca2+ and Mg2+ on GABAergic transmission are especially marked.

The dependence of the spontaneous and sound driven activity of single cochlear nerve fibers on the calcium and magnesium content of the perilymph was studied by perfusion of the perilymphatic space. It was possible to study these effects under steady-state conditions by continuously perfusing scala tympani at low rates while simultaneously recording from units in the chinchilla auditory nerve. Preparations were stable for many hours. As previously reported [Robertson and Johnstone (1979) Pflügers Arch. 380, 7-12], perfusion with solutions containing elevated concentrations of magnesium reduces both the spontaneous and driven activity. When calcium was eliminated from the perfusate, activity was completely abolished for stimuli with sound pressure levels below 100 dB. During partial blocks, a relatively frequency-independent threshold elevation was seen for frequencies well below the characteristic frequency (CF) of the unit, with greater elevations closer to CF. When the threshold elevation at CF was 30-40 dB, the width of the 'tip' portion of the tuning curve was reduced, resembling that of naturally-occurring units with low spontaneous rates of discharge. These effects are similar to that of raising the criterion for response during threshold measurement and are probably related to a frequency-dependent nonlinearity exhibited by the motion of the basilar membrane. The dynamic range for the growth of average rate with level was increased and saturation was shifted to higher stimulus levels during elevated magnesium perfusion. Raising the calcium content of the perfusate increased both spontaneous and driven rates, even in the saturated portion of the rate-intensity plot. Under these conditions, the response of the unit may more directly correspond to the intracellular potential of the presynaptic hair cell. It is argued that the primary site of divalent cation interaction is in the control of transmitter release. Inner hair cells of the mammalian cochlea apparently do not release transmitter in the absence of a calcium influx. The size of the pool of 'readily-available' transmitter appears to be influenced by divalent cations. Even though this synapse is probably specialized for the transmission of auditory signals, the mechanism of synaptic transmission is probably not fundamentally different from that of other well-characterized synapses.

Glass microelectrode recordings were made from single fibres of the posterior ampullary nerve in the isolated labyrinth of the frog (Rana esculenta). Potentials were recorded both at rest and during rotatory stimulation of the canal. At rest, the tracings revealed an intense background of small, largely summated potentials (0.5-10 mV amplitude, 3-6 msec duration), which underlay the discharge of spikes in all the impaled units. The frequency of the subthreshold events was related to the frequency of the propagated spikes, the latter ranging from 0 to 40/sec. Stimulation modulated the frequency of both spikes and subthreshold potentials, whose summation during excitation led to a positive shift of the fibre membrane potential. The small potentials proved to be dependent on Ca2+ and Mg2+ levels in the bath. Antidromic-stimulation of the posterior ampullary nerve indicated that the observed events do not represent an artifact due to extracellular field interference related to spike activity in the neighbouring fibres. Tetrodotoxin (10(-7)-10(-6) g/ml) applied externally to the preparation or previously perfused through the frog vessels abolishes the propagated spikes but left unaffected the small potentials which, even under drug treatment, were normally modulated by the stimulus. The subthreshold potentials thus appear to be EPSPs generated at the cyto-neural junction between the hair cells and the endings of the ampullary nerve fibres.