Valproic acid and other histone deacetylase inhibitors induce microglial apoptosis and attenuate lipopolysaccharide-induced dopaminergic neurotoxicity.

Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, has been shown to be an inhibitor of histone deacetylase (HDAC). Our previous study has demonstrated that VPA pretreatment reduces lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity through the inhibition of microglia over-activation. The aim of this study was to determine the mechanism underlying VPA-induced attenuation of microglia over-activation using rodent primary neuron/glia or enriched glia cultures. Other histone deacetylase inhibitors (HDACIs) were compared with VPA for their effects on microglial activity. We found that VPA induced apoptosis of microglia cells in a time- and concentration-dependent manner. VPA-treated microglial cells showed typical apoptotic hallmarks including phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Further studies revealed that trichostatin A (TSA) and sodium butyrate (SB), two structurally dissimilar HDACIs, also induced microglial apoptosis. The apoptosis of microglia was accompanied by the disruption of mitochondrial membrane potential and the enhancement of acetylation levels of the histone H3 protein. Moreover, pretreatment with SB or TSA caused a robust decrease in LPS-induced pro-inflammatory responses and protected DA neurons from damage in mesencephalic neuron-glia cultures. Taken together, our results shed light on a novel mechanism whereby HDACIs induce neuroprotection and underscore the potential utility of HDACIs in preventing inflammation-related neurodegenerative disorders such as Parkinson's disease.

Valproate protects dopaminergic neurons in midbrain neuron/glia cultures by stimulating the release of neurotrophic factors from astrocytes.

Valproate (VPA), one of the mood stabilizers and antiepileptic drugs, was recently found to inhibit histone deacetylases (HDAC). Increasing reports demonstrate that VPA has neurotrophic effects in diverse cell types including midbrain dopaminergic (DA) neurons. However, the origin and nature of the mediator of the neurotrophic effects are unclear. We have previously demonstrated that VPA prolongs the survival of midbrain DA neurons in lipopolysaccharide (LPS)-treated neuron-glia cultures through the inhibition of the release of pro-inflammatory factors from microglia. In this study, we report that VPA upregulates the expression of neurotrophic factors, including glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) from astrocytes and these effects may play a major role in mediating VPA-induced neurotrophic effects on DA neurons. Moreover, VPA pretreatment protects midbrain DA neurons from LPS or 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity. Our study identifies astrocyte as a novel target for VPA to induce neurotrophic and neuroprotective actions in rat midbrain and shows a potential new role of cellular interactions between DA neurons and astrocytes. The neurotrophic and neuroprotective effects of VPA also suggest a utility of this drug for treating neurodegenerative disorders including Parkinson's disease. Moreover, the neurotrophic effects of VPA may contribute to the therapeutic action of this drug in treating bipolar mood disorder that involves a loss of neurons and glia in discrete brain areas.

A role for the PI-3 kinase signaling pathway in fear conditioning and synaptic plasticity in the amygdala.

Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Modulation of voltage-dependent calcium currents by serotonin in acutely isolated rat amygdala neurons.

The modulation of voltage-dependent calcium currents (I(Ca)) by serotonin (5-HT) was studied in rat acutely dissociated amygdala neurons using whole-cell patch-clamp recording techniques. 5-HT inhibited I(Ca) in a concentration-dependent manner with a ED50 of approximately 1 microM and a maximal inhibition of approximately 50%. The inhibition was mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) and was reduced by the 5-HT1A antagonist NAN-190, indicating its mediation by 5-HT1A receptors. Pretreatment of neurons with the alkylating agent N-ethylmaleimide (NEM) or pertussis toxin (PTX) markedly reduced the action of 5-HT. The modulation was partially reversed by strong depolarization and was not seen in cell-attached patches when the agonist was applied outside the recorded patch, suggesting a membrane-delimited, G-protein-mediated signaling pathway. Nimodipine (1 microM) reduced the I(Ca) by approximately 30% without reducing inhibition of current by 5-HT significantly, ruling out L-type channels as the target of modulation. 5-HT-mediated inhibition after exposure to omega-conotoxin-GVIA (omega-CgTX, 1 microM) or omega-agatoxin-IV (omega-AgTX, 200 nM), which blocked 26% and 21% of the total I(Ca), respectively, was significantly decreased, suggesting involvement of the N- and P/Q-type channels. In the combined presence of omega-CgTX and omega-AgTX, 5-HT still caused a small but significant reduction of I(Ca), suggesting a possible involvement of R-type channels. Stimulation of beta-adrenergic receptor with isoproterenol (Iso) or activation of adenylyl cyclase with forskolin resulted in an enhancement of I(Ca). 5-HT caused the same degree of inhibition with or without Iso or forskolin pretreatment. On the other hand, application of 8-OH-DPAT inhibited I(Ca) and blocked Iso- and Sp-cAMPS-induced enhancement. These results provide the first evidence showing a dominant effect of 5-HT-mediated inhibition over Iso-mediated enhancement of I(Ca).

Lamotrigine inhibition of glutamate release from isolated cerebrocortical nerve terminals (synaptosomes) by suppression of voltage-activated calcium channel activity.

Lamotrigine (LAG) is an antiepileptic drug which is believed to suppress seizures by inhibiting the release of excitatory neurotransmitters. The present study was aimed at investigating the effect of LAG on the 4-aminopyridine (4AP)-evoked glutamate release in cerebrocortical nerve terminals (synaptosomes). LAG inhibited the release of glutamate evoked by 4AP in a concentration-dependent manner. This inhibitory effect was associated with a reduction in the depolarization-evoked increase in the cytoplasmic free Ca2+ concentration ([Ca2+]C). In addition, LAG did not alter the resting synaptosomal membrane potential or 4AP-evoked depolarization. Furthermore, ionomycin-evoked glutamate release was not affected by LAG. Based on these results, we suggest that presynaptic calcium influx blockade and inhibition of glutamate release may underlie the mechanism of action of LAG. These action may also contribute to their neuroprotective properties in excitotoxic injury.

Dissociation between synaptic depression and block of long-term depression induced by raising the temperature in rat hippocampal slices.

The influence of raising the bath temperature (39 degrees C) on synaptic transmission and neuronal plasticity was studied in the CA1 region of the rat hippocampus using an extracellular recording technique. Increasing the bath temperature from 32 to 39 degrees C resulted in a depression of field excitatory postsynaptic potential (fEPSP). Application of the selective A(1) receptor agonist, 2-chloro-adenosine (2-CADO, 1 microM) reduced the fEPSP and subsequently occluded the raised temperature-induced synaptic depression. On the other hand, the selective adenosine A(1) receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) blocked depression of fEPSP produced by raising the temperature. These results suggest that raising temperature-induced synaptic depression is due to an alteration of extracellular adenosine concentration. Long-term depression (LTD) could be reliably induced by the standard low-frequency stimulation (LFS, 1 Hz for 15 min) protocol at 32 degrees C but not at 39 degrees C. The raised temperature-induced block of LTD was mimicked by 2-CADO. Unexpectedly, despite the presence of DPCPX, LFS still could not elicit LTD. NMDA receptor-mediated synaptic component (fEPSP(NMDA)) was decreased when increasing the temperature to 39 degrees C and DPCPX failed to reverse such a depression. The increase in the NMDA response in 0.1 mM Mg(++) compared with 1 mM Mg(++) was significantly greater at 32 degrees C than at 39 degrees C. These results suggest that, by increasing the sensitivity of Mg(++) block, an increase in temperature modulates NMDA responses and thereby inhibits the induction of LTD.

Docosahexaenoic acid inhibits synaptic transmission and epileptiform activity in the rat hippocampus.

Docosahexaenoic acid (DHA) has been suggested to be required for neuronal development and synaptic plasticity. However, in view of the fact that DHA facilitates NMDA responses and blocks K(+) channels, it might predispose the neurons to epileptiform bursting. By using extracellular recording of population spikes in the CA1 region of rat hippocampal slices, we tested this possibility by examining the effect of DHA on the epileptiform activity induced by bicuculline or in Mg(2+)-free medium. When stimuli were delivered to the Schaffer collateral/commissural pathway every 20 or 30 sec, DHA had no significant effect on the epileptiform activity. However, when the frequency of stimulation was increased to 0.2 Hz, DHA attenuated the amplitude of the bursting activity induced by bicuculline to 57.5+/- 10.8% and those induced by Mg(2+)-free ACSF to 65.8+/-13.9% of control. DHA reduced the slope of field excitatory postsynaptic potential (fEPSP) to 77.1+/-7.4% of baseline, without significant effect on the ratio of paired-pulse facilitation (PPF). By intracellular recording of neurons in the stratum pyramidale of rat hippocampal slices, we found that DHA markedly inhibited the repetitive firing of action potentials elicited by depolarizing current pulses but did not affect the initial action potential. Thus, DHA may attenuate epileptic activity mainly through the frequency-dependent blockade of Na(+) channels.

Activation of group II metabotropic glutamate receptors induces long-term depression of synaptic transmission in the rat amygdala.

An animal model most sensitive for measuring anticipatory anxiety is fear conditioning, which is expressed by an enduring increase in synaptic strength in the amygdala. A converse view predicts that agents that induce long-term depression (LTD) of synaptic efficacy in the amygdala may be useful in the amelioration of stress disorders. In the present study, we show that activation of group II metabotropic glutamate receptor (mGluR II) by (2S,3S, 4S)-2-(carboxycyclopropyl) glycine (l-ccg) induces an LTD in the basolateral amygdala neurons. The effect was concentration-dependent with a maximal inhibition of approximately 30%. The induction of l-CCG LTD required concurrent synaptic activity, required presynaptic but not postsynaptic Ca(2+) increases, and was independent of NMDA receptors. l-CCG LTD was associated with an increase in the ratio of paired-pulse facilitation and was not occluded by low-frequency stimulation-induced LTD, suggesting that these two forms of LTD did not share a common underlying mechanism. After eliciting LTD with l-CCG, application of isoproterenol increased the synaptic responses back to its original baseline, demonstrating that chemically depressed synapses could be potentiated by another chemical. A selective PKA inhibitor, KT 5720, by its own caused a depression of synaptic transmission and blocked l-CCG LTD, presumably by mimicking and thereby occluding any further depression. Together, these results suggest that l-CCG LTD is induced by presynaptically mGluR II-mediated inhibition of Ca(2+)-sensitive adenylyl cyclase, resulting in a decrease in cAMP formation and PKA activation, which leads to a long-lasting decrease in transmitter release.

Long-term depression of excitatory synaptic transmission in the rat amygdala.

In view of the fact that both kindling and fear-potentiated startle are expressed by long-term enhancement of synaptic transmission in the amygdala, synaptic plasticity in this area of the brain is of particular importance. Here, we show for the first time that low-frequency stimulation of the lateral nucleus at 1 Hz for 15 min elicited a long-term depression (LTD) in the basolateral amygdala (BLA) neurons. LTD is expressed specifically at the lateral-BLA synapses but not at ventral endopyriform nucleus-BLA synapses. The induction of LTD requires activation of both NMDA and metabotropic glutamate receptors. Loading cells with a Ca(2+) chelator BAPTA or extracellular superfusion with protein phosphatase inhibitors prevents LTD, suggesting that LTD may result from dephosphorylation of AMPA receptors. The same stimulating protocol could not elicit LTD in neurons from kindled animals, whereas neurons from sham-operated or age-matched control rats were able to exhibit LTD. Together, this study characterizes the properties of LTD in the naïve amygdala slices for the first time and demonstrates that epileptogenesis in vivo induces disruption of LTD in the in vitro preparation.

Involvement of mitogen-activated protein kinase in hippocampal long-term potentiation.

Mitogen-activated protein kinase (MAPK) cascade classically is thought to be involved in cellular transformation, including proliferation and differentiation. Recent behavioral studies suggest that MAPK may also have a role in learning and memory. Long-term potentiation (LTP), a candidate mechanism for learning and memory, has at least two distinct temporal phases: an early phase (E-LTP) which lasts for 1-2 h and a late phase (L-LTP) which can persist >/=3 h. Here, we report that PD 098059, a selective inhibitor of MAPK cascade, attenuates L-LTP induced by bath application of forskolin without affecting basal synaptic transmission. This effect was mimicked by direct injection of animals with MAPK antisense oligonucleotide into the hippocampal CA1 region. MAPK activity measured by using a synthetic peptide corresponding to the sequence surrounding the major site of phosphorylation of the myelin-basic protein by MAPK was enhanced by forskolin. The same antisense treatment also completely inhibited the increased MAPK activity. These results demonstrate an involvement of MAPK in the induction of L-LTP in the hippocampal CA1 neurons.

Masking of forskolin-induced long-term potentiation by adenosine accumulation in area CA1 of the rat hippocampus.

At hippocampal Schaffer collateral-CA1 synapses, activation of beta-adrenergic receptors and adenylyl cyclase increases transmitter release. However, this effect is transient, which is in contrast to that seen at mossy fiber-CA3 synapses, where activation of cyclic-AMP-dependent protein kinase results in long-lasting facilitation of transmitter release, a phenomenon known as a presynaptic form of long-term potentiation. The present study was aimed at investigating whether forskolin, an adenylyl cyclase activator, could produce long-term effects at the Schaffer collateral-CA1 synapses using extracellular recording techniques. As has been reported previously, forskolin persistently increased the amplitude of evoked population spikes without having a long-term effect on the field excitatory postsynaptic potentials. However, under the conditions where adenosine A1 receptors are inhibited, cyclic-AMP metabolism is disrupted or the transport of cyclic-AMP is blocked, forskolin induces long-term potentiation. Forskolin-induced potentiation is associated with a decrease in paired-pulse facilitation and is blocked by the cyclic-AMP-dependent protein kinase inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate. Activation of N-methyl-D-aspartate receptors is not required for forskolin-induced long-term potentiation, because pretreatment of slices with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate did not prevent forskolin-induced potentiation. These results suggest that blockade of adenosine A1 receptors unmasks forskolin-induced long-term potentiation, and activation of cyclic-AMP-dependent protein kinase induces a form of long-term potentiation which is different from that induced by tetanic stimulation.

Cross-modulation of synaptic plasticity by beta-adrenergic and 5-HT1A receptors in the rat basolateral amygdala.

Neurotransmitter receptors are often colocalized in a neuron with other receptors, and activation of one receptor can either amplify or antagonize the response to a colocalized receptor. The aim of this study was to investigate the cross-regulation of synaptic transmission by beta-adrenergic and serotonin 1A (5-HT1A) receptors and to elucidate their underlying mechanisms. Stimulation of presynaptic beta-adrenergic receptors with isoproterenol (Iso) in the basolateral amygdala resulted in a long-lasting increase in synaptic transmission. This effect was mimicked by forskolin, an activator for adenylyl cyclase and a cAMP analog. In addition, the effect of forskolin was blocked by catalytic and regulatory site antagonists for cAMP-dependent protein kinase (PKA), indicating a PKA-mediated mechanism. Application of 5-HT depressed the synaptic transmission and blocked Iso- and forskolin-induced potentiation. The effect of 5-HT was mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin and was blocked by the selective 5-HT1A antagonist 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl]piperazine, indicating its mediation by 5-HT1A receptors. To determine the locus of interaction, Sp-cAMPS, a membrane-permeable activator of PKA, was applied, and the potentiation produced by Sp-cAMPS was completely blocked in slices pretreated with 5-HT. These results suggest that the interaction between the intracellular signaling pathways activated by 5-HT1A and beta-adrenergic receptors occurs at a step downstream from cAMP production.

Promotion of forskolin-induced long-term potentiation of synaptic transmission by caffeine in area CA1 of the rat hippocampus.

Caffeine which is present in soft drinks has been shown to increase alertness and allays drowsiness and fatigue. The aim of this study is to investigate whether caffeine could produce a long-term effect on the synaptic transmission using extracellular recording technique in the hippocampal slices. Bath application of caffeine (100 microM) reversibly increased the slope of field excitatory postsynaptic potential (fEPSP). Forskolin (25 microM) by its own did not affect the fEPSP significantly. However, in the presence of caffeine, forskolin induced a long-term potentiation (LTP) of fEPSP. Enprofylline which has been shown to exhibit some actions like caffeine but with a low adenosine antagonistic potency did not affect the normal synaptic transmission or the effect of forskolin at a lower concentration (10 microM). However, when the concentrations were increased to 20 and 50 microM, enprofylline significantly enhanced the fEPSP slope and promoted forskolin-induced LTP. The parallel increase of fEPSP and promotion of LTP observed with enprofylline suggests that adenosine A1 antagonism is the primary mechanism behind caffeine's effect. This hypothesis was further strengthened by the finding that promotion of forskolin-induced LTP was mimicked by the non-xanthine adenosine antagonist 9-chloro-2-(furyl)[1,2,4]triazolo [1,5-c]quinazolin-5-amine (CGS 15943). The promotion of forskolin-induced LTP provides a cellular basis behind caffeine's increase in capacity for sustained intellectual performance.

Frequency-dependent inhibition of neuronal activity by topiramate in rat hippocampal slices.

1. Topiramate is a structurally novel anticonvulsant which was recently approved for adjunctive therapy in partial and secondarily generalized seizures. The present study was aimed at elucidating the mechanisms underlying the anticonvulsant efficacy of topiramate using intra- and extracellular recording techniques in the in vitro hippocampal slices. 2. When stimuli were delivered every 20 s, topiramate had no measurable effect on both field excitatory postsynaptic potentials (fEPSPs) and population spikes (PSs). However, increasing the stimulation frequency from 0.05-0.2 Hz, topiramate significantly decreased the slope of fEPSP and the amplitude of PS in a concentration-dependent manner. The amplitude of presynaptic fiber volley was also reduced. 3. Topiramate did not affect the magnitude of paired-pulse inhibition and monosynaptically evoked inhibitory postsynaptic potentials (IPSPs). 4. Sustained repetitive firing was elicited by injection of long duration (500 ms) depolarizing current pulses (500-800 pA). Superfusion with topiramate significantly reduced the number of action potentials evoked by a given current pulse. 5. After blockade of GABA receptors by bicuculline, burst firing which consisted of a train of several spikes riding on a large depolarizing wave termed paroxysmal depolarizing shift (PDS) was recorded. Application of topiramate reduced the duration of PDS and later spikes with less effect on the initial action potential. 6. These results suggest that frequency-dependent inhibition of neuronal activity due to blockade of Na+ channels may account largely for the anticonvulsant efficacy of topiramate.

Potentiation of N-methyl-D-aspartate currents by isoproterenol in the acutely dissociated rat amygdalar neurons.

Whole-cell patch-clamp recordings in the acutely dissociated amygdalar neurons were performed to investigate the effect of isoproterenol (Iso) on NMDA currents. Bath application of Iso selectively enhanced the NMDA currents without affecting the AMPA currents. Iso potentiated the NMDA currents to the same extent at various holding potentials and the reversal potential for the NMDA response was not altered. These results suggest that adrenergic innvervations in this area may play an important role in synaptic plasticity.

Serotonin depresses excitatory synaptic transmission and depolarization-evoked Ca2+ influx in rat basolateral amygdala via 5-HT1A receptors.

The actions of serotonin on rat basolateral amygdala neurons were studied with conventional intracellular recording techniques and fura-2 fluorimetric recordings. Bath application of 5-hydroxytryptamine (5-HT or serotonin) reversibly suppressed the excitatory postsynaptic potential in a concentration-dependent manner without affecting the resting membrane potential and neuronal input resistance. Extracellular Ba2+ or pertussis toxin pretreatment did not affect the depressing effect of 5-HT suggesting that it is not mediated through activation of Gi/o protein-coupled K+ conductance. The sensitivity of postsynaptic neurons to glutamate receptor agonist was unaltered by the 5-HT pretreatment. In addition, the magnitude of paired-pulse facilitation was increased in the presence of 5-HT indicating a presynaptic mode of action. The effect of 5-HT was mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) and was blocked by the selective 5-HT1A antagonist 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl]piperazine oxadiazol-3-yl]methyl]phenyl]-methanesulphonamide. In contrast, the selective 5-HT2 receptor antagonist ketanserin failed to affect the action of 5-HT. The effects of 5-HT and 8-OH-DPAT on the high K+-induced increase in [Ca2+]i were studied in acutely dissociated basolateral amygdala neurons. High K+-induced increase in [Ca2+]i was blocked by Ca2+-free solution and Cd2+ suggesting that Ca2+ entry responsible for the depolarization-evoked increase in [Ca2+]i occurred through voltage-dependent Ca2+ channels. Application of 5-HT and 8-OH-DPAT reduced the K+-induced Ca2+ influx in a concentration-dependent manner. The effect of 5-HT was completely abolished in slices pretreated with Rp-cyclic adenosine 3',5'-monophosphothioate (Rp-cAMP), a regulatory site antagonist of protein kinase A, suggesting that 5-HT may act through a cAMP-dependent mechanism. Taken together, these results suggest that functional 5-HT1A receptors are present in the excitatory terminals and mediate the 5-HT inhibition of synaptic transmission in the amygdala.

Endogenous serotonin inhibits epileptiform activity in rat hippocampal CA1 neurons via 5-hydroxytryptamine1A receptor activation.

The modulatory effects of endogenous serotonin on the synaptic transmission and epileptiform activity were studied in the rat hippocampus with the use of extracellular and intracellular recording techniques. Field excitatory postsynaptic potential was reversibly depressed by serotonin in a concentration-dependent manner. Intracellular recordings revealed that serotonin-mediated synaptic depression was unaffected by extracellular Ba2+ or intracellular application of Cs+ while the postsynaptic hyperpolarizing effect was completely blocked. Epileptiform activity induced by picrotoxin (50 microM), a GABA(A) receptor antagonist, was also dose-dependently suppressed by serotonin. The antiepileptic effect was mimicked by 5-hydroxytryptamine1A agonist and was blocked by 5-hydroxytryptamine1A antagonists. 5-Hydroxytryptamine2 antagonist had no effect on the modulation. Similarly, fluoxetine, a selective serotonin re-uptake blocker, potently inhibited the epileptiform activity and this effect was blocked by 5-hydroxytryptamine1A receptor antagonist. Depletion of endogenous serotonin by pretreating the slices with p-chloroamphetamine completely prevented the antiepileptic action of fluoxetine, without modifying the action of serotonin in the same cells. These results suggest that the antiepileptic action of fluoxetine is due to an enhancement of endogenous serotonin which in turn is mediated by 5-hydroxytryptamine1A receptor. Endogenous serotonin transmission in the hippocampus is therefore capable of limiting the development and propagation of seizure activity.

Lamotrigine inhibits depolarization-evoked Ca++ influx in dissociated amygdala neurons.

Spectrophotometry with the Ca(++)-sensitive dye fura-2 was used to study the effect of lamotrigine (LAG) on the depolarization-evoked Ca++ influx in the acutely isolated basolateral amygdala neurons. Depolarization of the neurons with high K+ resulted in the elevation of intracellular Ca++ concentration [Ca++]i in a concentration-dependent manner. The K(+)-induced Ca++ influx was completely blocked in the Ca(++)-free solution or by Cd++, indicating that depolarization-induced increases in [Ca++]i were triggered largely, if not at all, by Ca++ entry from extracellular space and Ca++ entry occurred through voltage-dependent Ca++ channels. Application of LAG reduced the depolarization-evoked Ca++ influx in a concentration-dependent manner. The effect of LAG was markedly reduced in the presence of N-type Ca++ channel blocker omega-conotoxin-GVIA (omega-CgTX). These results suggest that the action of LAG is mediated, at least in part, by the modulation of N-type Ca++ channels.

Cancellation of low-frequency stimulation-induced long-term depression by docosahexaenoic acid in the rat hippocampus.

The effects of docosahexaenoic acid (DHA) on low-frequency stimulation (LFS)-induced long-term depression (LTD) were investigated in the CA 1 subfield of rat hippocampal slices. LTD was routinely produced by LFS of 900 pulses at 1 Hz. The field excitatory postsynaptic potential (fEPSP) 40 min after LFS was 59 +/- 4% (n = 18) of baseline response. However, in experiments from 18 neurons pretreated with DHA (50 microM), fEPSP returned to baseline levels within 20 min after LFS in eight cells and was slightly potentiated in three cells. Only in seven cells was LTD induced. The effect of DHA on LTD was concentration dependent. The slopes of fEPSP 40 min after LFS were 67 +/- 4% (n = 6), 72 +/- 7% (n = 7) and 80 +/- 5% (n = 18) of baseline response, with pretreatment of 1, 10 and 50 microM DHA, respectively. The blockade of LTD induction suggests that DHA may play a role in learning and memory.

Selective enhancement of P-type calcium currents by isoproterenol in the rat amygdala.

We investigated activation of beta-adrenergic receptor-adenylyl cyclase-cAMP cascade on the whole-cell voltage-dependent Ca2+ currents (ICa) in acutely isolated rat basolateral amygdala neurons. Application of beta-receptor agonist isoproterenol (Iso) caused a long-term enhancement of ICa. The effect of Iso was blocked by concurrent application of beta-receptor antagonist propranolol. However, delayed application of propranolol after the ICa enhancement did not affect Iso-induced potentiation, suggesting that the sustained effect was not caused by a slow washout of Iso. Nimodipine and omega-conotoxin-GVIA reduced the ICa by approximately 35 and approximately 29%, respectively, without reducing enhancement of ICa by Iso significantly. The modulation appeared to involve P-type current, because the enhancement was abolished after pretreatment with omega-agatoxin-IVA. Forskolin, an adenylyl cyclase activator, mimicked the action of Iso in enhancing ICa, and this effect was blocked by an inhibitor of cAMP cascade, indicating a cAMP-dependent mechanism. Iso also induced a long-term potentiation (LTP) of synaptic transmission, which could be prevented by P-type Ca2+ channel blockers. These results suggest that P-type Ca2+ channels were selectively upregulated in the basolateral amygdala neurons, and enhancement of P-type currents could contribute to presynaptic form of LTP.