Axonal release of glutamate analog, d-2,3-3H-Aspartic acid and l-14C-proline from segments of sciatic nerve following electrical and magnetic stimulation.

Segments of the mouse sciatic nerve were preloaded with either d-2,3-(3)H-Aspartic acid [nonmetabolizable analog of glutamate] or l-(14)C-proline and the release of these exogenous molecules was evaluated in the fractions of the perfusate following electrical or magnetic stimulation. The electrical stimulation (10Hz, 10Am, 20s) induced an instantaneous increase in the release of both molecules, although the release of d-2,3-(3)H-Aspartic acid was much greater. Moreover, contrary to l-(14)C-proline, the release of d-2,3-(3)H-Aspartic acid was Ca(2+)-dependent. While magnetic stimulation (15mT, 0.16Hz, 30min) also induced the release of d-2,3-(3)H-Aspartic acid in a Ca(2+)-dependent way, the release of l-(14)C-proline was negligible. These results indicate that axons can release glutamate in a specific, calcium-dependent way. This release may contribute to interaxonal interactions.

Melatonin regulates neuronal plasticity in the hippocampus.

The influence of melatonin on hippocampal evoked potentials initiated by low- and high-frequency electrical stimulations and by two pulses applied in rapid succession was investigated. In confirmation of our previous studies, melatonin attenuated the population spike triggered by low-frequency stimulation (0.03 Hz). High-frequency stimulation (HFS; 100 Hz for 1 sec, three times every 10 sec), which in control slices permanently facilitated neuronal excitability (347% +/- 32%), was also able to amplify the melatonin-depressed potential (467.8% +/- 59.6%). Because melatonin is a hydrophobic molecule, it was dissolved and applied in ethanol. Ethanol (0.4%) by itself reduced the magnitude of HFS-induced potentiation (233.5% +/- 16.8%). The slices stimulated with two pulses separated with a delay longer than 15 msec demonstrated a facilitation of the response to the second stimuli (paired-pulse facilitation; PPF). The influence of melatonin (100 microM) on PPF was biphasic: Shortly after addition of melatonin, PPF was briefly (5-10 min) reversed to paired-pulse inhibition (PPI), which gradually returned to a stable PPF. Ethanol (0.4%) applied without melatonin exerted only a marginal, facilitatory effect on PPF. The delay between two successively applied pulses, shorter than 13 msec, resulted in attenuation of the response to the second stimuli (PPI). Melatonin (100 microM) reversed the attenuation of the second potential within 15-20 min following its application. Ethanol applied by itself at the concentration of 0.4% temporarily (5-10 min), but significantly, depressed the second potential. These results demonstrate the ability of melatonin to modulate specific forms of plasticity in hippocampal pyramidal neurons.

ATP modulates Na+ channel gating and induces a non-selective cation current in a neuronal hippocampal cell line.

Extracellular ATP evoked two excitatory responses in hippocampal neuroblastoma cells (HN2). The first, an opening of a receptor-operated non-selective cation channel and the second was a leftward shift in Na+ channel activation. Both ATP (5-1000 microM) and 2',3'-(4-benzoyl)-benzoyl-ATP (Bb-ATP, 50 microM) activated a non-selective cation current reversing near 0 mV and shifted the Na+ activation and inactivation curves to the left. Based on a comparison of a series of agonists and antagonists, the inward current appeared to be partially mediated by activation of a P2X7 receptor, although hybrid channels cannot be ruled out. The shift in Na+ channel gating could be separated from the opening of the cation channel, as application of the P2Y antagonist Reactive Blue-2 and GTP shifted the Na+ current activation to the left but failed to elicit the inward cation current. Both responses to ATP and Bb-ATP were insensitive to block by the P2X antagonist suramin (300 microM) but were prevented by incubation in oxidized ATP (200 microM); a putative P2X7 receptor antagonist. Prior screening of the surface negative charge of the membrane with a high concentration of divalent cations prevented both responses. We suggest that ATP4- activates a P2X receptor and becomes trapped on a site, on or near the Na+ channel. Activation of the P2X receptor leads to the opening of a non-specific cation channel, while the binding of ATP4- leads to a modified charge sensed by the Na+ channel, similar to what occurs in the presence of charged amphiphiles as well as a number of beta-scorpion toxins.

The influence of NO-containing ruthenium complexes on mouse hippocampal evoked potentials in vitro.

The influence of different, nitric oxide-containing ruthenium complexes on the evoked potentials recorded from the CA1 region of the mouse hippocampus in vitro has been investigated. Of the compounds tested, only trans-[(NO)(P(OEt)3)(NH3)4Ru](PF6)3 (1-2.5 mM) exerted a strong facilitatory action on the population spike, the EPSP, and the spontaneous activity. Its activity probably depends upon its ability to release NO following reduction. The phosphito ligand is important both in terms of adjusting the reduction potential of the complex to be biologically accessible and in labilizing the coordinated NO. The effects of this compound could not be reversed by perfusion. Scavenging NO in slices preincubated with oxyhemoglobin prior to the addition of this compound eliminated its neurophysiological effects. The control molecules trans-[(P(OEt)3)2(NH3)4Ru](PF6)2, trans-[(H2O)(P(OEt)3) (NH3)4Ru](PF6)3, and [(NO)(NH3)5Ru]Cl3, which are structurally similar, but unable to generate NO, were ineffective. NaNO2 suppressed neuronal firing. Attempts to induce Long-Term Potentiation (LTP) at the time of maximal effect of trans-[(NO)(P(OEt)3)(NH3)4Ru](PF6)3 were unsuccessful, suggesting that the mechanism of amplification induced by trans-[(NO)(P(OEt)3)(NH3)4Ru](PF6)3 and LTP may share common pathways.

The modulation of neuronal activity by melatonin: in vitro studies on mouse hippocampal slices.

The influence of melatonin on evoked potentials recorded from the CAI field of mouse hippocampal slices was investigated. Melatonin (0.1-2.0 mM) and its analog, 6-chloromelatonin (0.1-0.5 mM) depressed evoked potentials (EPSP and the population spike) in a concentration-dependent manner. The melatonin-induced depression was followed by a slow recovery phase. Since the fiber potential was not affected, it was concluded that melatonin influenced synaptic efficiency and/or cell excitability. Luzindole, an antagonist of MT2 melatonin receptors, although slightly depressing evoked potentials when applied by itself (100 microM), blocked any further inhibition by melatonin when added afterwards. We concluded that melatonin reduced synaptic efficiency and/or excitability of hippocampal neurons most likely through interaction with MT2 melatonin receptors, but other possible mechanisms of melatonin action are also considered.

Dantrolene modulates the influence of steady magnetic fields on hippocampal evoked potentials in vitro.

Direct current-generated magnetic fields (2-3 mT, 20-min exposure) exerted biphasic effects on the population spike recorded from hippocampal slices. The initial decrease in the potential, observed during exposure of the slices to magnetic fields was followed by a recovery/amplification phase, which began after terminating the magnetic field action. During that phase the population spike exceeded the amplitude observed before application of the magnetic fields. The pattern of magnetic fields influence was not affected either by (+)-5-methyl-10,11-dihydro-5H-dibenzo (a,d) cyclohepten-5, 10-imine maleate (MK801), or by D,L,-2amino-5phosphonovalerate (APV), a noncompetitive and competitive NMDA receptor antagonist, respectively. The rising phase of the potential, however, was eliminated by dantrolene, an inhibitor of intracellular Ca(2 +) channels. This suggests that intracellular calcium channels participate in the mechanism of the influence of the direct current magnetic fields on the function of the hippocampal tissue.

A controlled NO-releasing compound: synthesis, molecular structure, spectroscopy, electrochemistry, and chemical reactivity of R,R,S,S-trans-[RuCl(NO)(cyclam)]2+(1,4,8,11-tetraazacyclotetradecane).

The synthesis of trans-[RuCl(NO)(cyclam)]2+ (cyclam = 1,4,8,11-tetraazacyclotetradecane) can be accomplished by either the addition of cyclam to K2[RuCl5NO] or by the addition of NO to trans-[RuCl(CF3SO3)(cyclam)](CF3-SO3). Crystals of trans-[RuCl(NO)(cyclam)](ClO4)2 form in the monoclinic space group P2(1)/c, with unit cell parameters of a = 7.66500(2) A, b = 24.7244(1) A, c = 16.2871(2) A, beta = 95.2550(10) degrees, and Z = 4. One of the two independent molecules in the unit cell lies disordered on a center of symmetry. For the ion in the general position, the Ru-N and N-O bond distances and the [Ru-N-O]3+ bond angle are 1.747(4) A, 1.128(5) A, 178.0(4) degrees, respectively. In both ions, cyclam adopts the (R,R,S,S) configuration, which is also consistent with 2D COSY 1H NMR studies in aqueous solution. Reduction (E degree = -0.1 V) results in the rapid loss of Cl- by first-order kinetics with k = 1.5 s-1 and the slower loss of NO (k = 6.10 x 10(-4) s-1, delta H++ = 15.3 kcal mol-1, delta S++ = -21.8 cal mol-1 K-1). The slow release of NO following reduction causes trans-[RuCl(NO)(cyclam)]2+ to be a promising controlled-release NO prodrug for vasodilation and other purposes. Unlike the related complex trans-[Ru(NO)(NH3)4(P(OEt)3)](PF6)2, trans-[RuCl(NO)(cyclam)]Cl2 is inactive in modulating evoked potentials recorded from mice hippocampal slices probably because of the slower dissociation of NO following reduction.

The effect of glutamate uptake inhibitors on hippocampal evoked potentials in vitro.

The influence of four inhibitors of the high-affinity glutamate uptake system (DL-aspartic acid beta-hydroxymate, DL-AHM; L-aspartic acid beta-hydroxymate, L-AHM; threo-beta-methylaspartate, DLM; L-transpyrrolidine-2, 4-dicarboxylate, PDC) on potentials recorded from hippocampal slices was investigated. At low concentrations of DL-AHM, L-AHM and DLM (50-150 microM) the population spike was permanently amplified. NMDA receptor antagonists blocked this facilitatory effect of L-AHM, DL-AHM and DLM. At higher concentrations (400-700 microM) DL-AHM and DLM abolished the population spike, while L-AHM did not eliminate the population spike at any concentration tested. None of these uptake inhibitors influenced an antidromic potential recorded in Ca(2+)- free Ringer solution. PDC at lower concentrations (75 microM) did not affect the population spike and at higher concentrations (150 microM-500 microM) induced only a transient elevation in population spike. Our data demonstrate that modification of glutamate uptake may be an important factor in the regulation of synaptic efficiency of glutamergic pathways.

Platelet activating factor (PAF) in memory formation: role as a retrograde messenger in long-term potentiation.

Long-term potentiation (LTP) is a neurophysiological process that has been implicated in memory formation. The elevation of intracellular Ca2+ levels in postsynaptic neurons, an essential step in the induction of LTP in the hippocampus, can lead to activation of the enzyme acetyl-CoA:lyso-PAF acetyltransferase that is required for PAF synthesis in neurons. Thus, during the induction of LTP, stimulation of Ca2+ influx by glutamate receptors would lead to a postsynaptic increase in PAF biosynthesis. A main target for PAF action in neurons is the stimulation of neurotransmitter release via Ca(2+)-dependent vesicular exocytosis, a process that occurs presynaptically. In this article we describe the evidence obtained to-date for the pre- and postsynaptic events outlined, above, and demonstrate for the first time that during the induction of LTP by high-frequency stimulation (HFS) a 9-fold increase in PAF release to the extracellular environment occurs within 60 min following HFS. This finding provides the evidence that PAF can diffuse from postsynaptic sites of synthesis to presynaptic sites of action, and thus function as a retrograde messenger in the induction of LTP. Based on these data, we present a scheme in which postsynaptic glutamate receptors cooperate with presynaptic PAF receptors in a reverberating cycle that can amplify the transmission in a Hebbian synapse.

Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.

Comparative, in vitro, studies of hippocampal tissue from homing and non-homing pigeon.

The purpose of this research was to characterize morphologically and electrophysiologically tissue slices obtained from the hippocampus of homing and non-homing pigeons. When hippocampal slices from the brain of homing and non-homing pigeons are observed under the dissecting microscope, diffuse fiber paths can be seen. These fiber pathways appeared to be identical with the medial fiber tract (VM) previously described histologically in the hippocampus of homing pigeon. Visualization of these tracts in living slices allowed placement of stimulating and recording electrodes in corresponding locations in these slices in both homing and non-homing pigeons. Extracellular potentials recorded from VM regions of the brains of both homing and non-homing pigeons were sensitive to CNQX indicating that glutamate may be a neurotransmitter in this area of pigeon hippocampus. These potentials could undergo long-term potentiation (LTP) following high frequency stimulation. This LTP was blocked by NMDA receptor antagonist APV in the hippocampus of homing pigeon, but was APV-resistant in the hippocampus of non-homing pigeon. Extracellular potentials from the hippocampus of homing pigeons were increased in amplitude when slices were perfused with Mg(2+)-free Ringer, while potential recorded from hippocampal slices from non-homing pigeons wre unaffected by Mg(2+)-free solutions. Intracellular recordings from the hippocampal slices of homing pigeons revealed that about half the cells demonstrated excitatory synaptic potentials evoked by extracellular stimulation. The EPSP was sometimes large enough to trigger an action potential. Neurons filled with the fluorescent dye, Lucifer Yellow, in the hippocampus of homing pigeons showed multipolar structure. The response of these cells to extracellular stimulation provides the activity responsible for the extracellular potentials which can undergo LTP.

The influence of steady magnetic fields on the mouse hippocampal evoked potentials in vitro.

Direct current-generated EM fields modulated evoked potentials recorded from hippocampal slices. Both the synaptic efficiency represented by the slope of EPSP and the number of activated pyramidal cells represented by the population spike were modified by EM fields. While the weak EM fields (2-3 mT) exerted a biphasic effect observed as a transient small depression followed by a long-lasting amplification of the potentials, stronger EM fields (8-10 mT) depressed these potentials. This depression could be partially reversed by increasing the strength of the stimulation. It is suggested that EM-induced fluctuations in the intracellular Ca2+ concentration are responsible for the observed effect.

Extracellular ATP as a neurotransmitter: its role in synaptic plasticity in the hippocampus.

Growing evidence indicates that ATP may play a very important role in Long-Term Potentiation (LTP), a neurophysiological process that has been implicated in memory formation. LTP is an enhancement of synaptic strength induced by a specific pattern of high frequency stimulation, or by application of exogenous ATP. In the hippocampus LTP-inducing stimulation is accompanied by a massive, Ca(2+)-dependent release of ATP from presynaptic terminals. Released extracellular ATP may either interact with numerous types of ATP receptors present on the neuronal surface, or serve as a substrate for ecto-protein phosphorylation. The results of combined electrophysiological and biochemical experiments indicate that participation of extracellular ATP in the ecto-protein phosphorylation process is most likely involved in the permanent amplification of the synaptic response in the hippocampus.

Facilitation of hippocampal potentials by suramin.

The influence of suramin, a suggested purino-receptor antagonist, on the evoked synaptic potentials recorded from hippocampal slices was evaluated. The suramin induced a nondecremental, concentration-dependent amplification of the slope of excitatory postsynaptic potential and magnitude of the population spike (long-term potentiation, LTP). The effect of suramin was completely abolished by adenylimidodiphosphate, a non-hydrolyzable analogue of ATP, and markedly reduced by NMDA-receptor antagonists DL-2-amino-5-phosphonovaleric acid and MK-801. These results indicate that, in addition to acting as an antagonist of P2 receptors, suramin is also able to facilitate hippocampal potentials in a way that involves mechanisms participating in induction of LTP.

Metaphit amplifies long-term potentiation (LTP) in the mouse hippocampus.

The influence of metaphit, a phencyclidine derivative, on the amplitude of Long-Term Potentiation (LTP) in the mouse hippocampus was investigated. Mice (C57BL/6) of both sexes were injected with metaphit (80 mg/kg) and hippocampal slices were prepared at 3, 24, 48 hrs and 6 days following injection. The extracellular evoked potentials were recorded from the pyramidal cell layer following Schaffer collateral stimulation. The threshold value, defined as the minimum strength of the stimuli to evoke a 0.1 mV potential, was about 5 fold greater in metaphit slices than in control slices 3 hr following injection, and then declined to the control value within 6 days. The magnitude of LTP was also amplified by metaphit in a time-dependent fashion. The effect was visible three hours after injection, reached its maximum at 48 hr and then declined to a level slightly higher than control at 6 days following injection. These results demonstrate that metaphit, a compound known to induce audiogenic seizures, is able to modify synaptic plasticity in the hippocampus. Presented results are also in agreement with our previous data which demonstrated an interaction between the mechanisms of LTP and audiogenic seizure.

Dual effect of sodium nitroprusside on potentials recorded from mouse hippocampal slices.

The purpose of this research was to estimate the usefulness of sodium nitroprusside (SNP) as a donor of nitric oxide (NO) in the study of synaptic plasticity. The influence of SNP, K3Fe(CN)6 and K4Fe(CN)6 on the evoked potential recorded from mouse hippocampal slices was evaluated. When slices were exposed to 1 mM SNP for no longer than 15 min, a facilitation of the population spike was observed following perfusion. Longer, 60 min exposure of the slices to 1 mM SNP prevented this perfusion-induced amplification of the potential and blocked the induction of LTP by subsequently applied high frequency stimulation (HFS). SNP at lower concentrations (0.2 mM) had no effect. SNP (1 mM) suppressed the NMDA component of the population spike. Light inactivated, 1.0 mM SNP, when applied for 15 min, reduced the potential and subsequently induced LTP. However, longer exposure of the slices to the light inactivated 1 mM SNP made the potential unstable and blocked induction of LTP. K3Fe(CN)6 and K4Fe(CN)6, molecules structurally related to SNP but unable to generate NO, transiently amplified the population spike and its NMDA component. We suggest that SNP, which is often used as nitric oxide donor, exerts a dual inhibitory and facilitatory action on hippocampal evoked potentials. The possible mechanisms of these actions are discussed.

On the role of extracellular ATP in the induction of long-term potentiation in the hippocampus.

The involvement of a purinergic system in the mechanisms of ATP- and electrically induced long-term potentiation (LTP) has been investigated in mouse hippocampal slices. Extracellular ATP (500 nM) and its slowly hydrolyzable analogue adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S; 2.5 microM) amplified permanently the magnitude of the population spike. This effect was antagonized by adenylimidodiphosphate (AMPPNP), a non-hydrolyzable analogue of ATP. AMPPNP, other ATP analogues [2-methylthioadenosine triphosphate (2-MeSATP) and alpha, beta-methyleneadenosine 5'-triphosphate (alpha, beta-methyleneATP)], or a purinergic receptor antagonist (Cibacron Blue 3G) tested in the concentration range of 3-40 microM did not exert agonistic activity similar to that of ATP or ATP-gamma-S, suggesting that ATP hydrolysis is required to exert this effect. All the tested nonhydrolyzable analogues reduced or prevented the establishment of stable, nondecremental LTP without blocking the short-lasting increase in the magnitude of the population spike immediately after electrical stimulation (short-term potentiation). These results indicate that ATP released by high-frequency stimulation contributes to the maintenance of stable LTP. The underlying mechanism operating in this process may involve a new type of ATP receptors or hydrolysis by ecto-ATPase. However, the findings that ATP-gamma-S is less potent than ATP and that other ATP analogues known to act as agonists of purinergic receptors did not induce LTP but rather inhibited its maintenance are more consistent with the possibility that ecto-protein kinase, using extracellular ATP as a cosubstrate, plays a role in mechanisms underlying synaptic plasticity.

Influence of audiogenic seizures on synaptic facilitation in mouse hippocampal slices is mediated by N-methyl-D-aspartate receptor.

The influence of audiogenic seizures (AGS) on synaptic facilitation was studied in DBA/2J (D2) and C57BL/6J (B6) mice. AGS susceptibility is inherited in D2 mice, but can be acquired in AGS-resistant B6 mice through acoustic priming. The experiments were performed on hippocampal slices obtained from D2 and B6 mice both with and without seizures. Long-term potentiation (LTP) and low-Mg(2+)-induced synaptic facilitation (LMISF) were evaluated after stimulation of Schaffer collaterals. The magnitude of LTP and LMISF was significantly greater in slices obtained from mice with seizures than from mice without seizures in both strains. Seizure-induced enhancement of LTP and LMISF was markedly reduced by the N-methyl-D-aspartate (NMDA) receptor antagonist AP-5. The noncompetitive NMDA receptor antagonist MK 801 reduced the efficiency of priming in B6 mice and abolished AGS in D2 mice and primed B6 mice. The data suggest that audiogenic seizures can enhance synaptic facilitation through activation of the NMDA receptor.