Rat Hippocampal CA1 Pyramidal Neurons Research Articles

The action of BDNF on GABAA currents changes from potentiating to suppressing during maturation of rat hippocampal CA1 pyramidal neurons

The action of BDNF on GABAA currents changes from potentiating to suppressing during maturation of rat hippocampal CA1 pyramidal neurons

The action of BDNF on GABA(A) currents changes from potentiating to suppressing during maturation of rat hippocampal CA1 pyramidal neurons.

During the development of the hippocampus, the action of GABA shifts from depolarizing to hyperpolarizing, and brain-derived neurotrophic factor (BDNF) has important roles in GABAergic transmission. We demonstrate that BDNF (20 ng ml-1) rapidly and reversibly potentiates postsynaptic GABAA receptor-mediated currents (by 80.5 +/- 14.3 %, n = 10) in hippocampal CA1 pyramidal neurons isolated from postnatal day (P)6 rats, using nystatin-perforated patch-clamp recordings. This potentiation is caused by an elevation of intracellular Ca2+ that occurs in response to the activation of Trk B receptor tyrosine kinase and phospholipase C-gamma. The modulation of the GABAA responses by BDNF in hippocampal CA1 pyramidal neurons isolated from P10 rats was more diverse (from potentiating to inhibitory), and at P14, BDNF induced a long-lasting inhibition. In addition, Ca2+/calmodulin-dependent protein kinase 2 plays important roles in the potentiating, but not in the inhibitory effect, of BDNF on the GABAA responses. These results suggest that changes in the intracellular signalling pathway could contribute to the developmental shift of the actions of BDNF on inhibitory systems.

Presynaptic K+ channel modulation is a crucial ionic basis of neuronal damage induced by ischemia in rat hippocampal CA1 pyramidal neurons.

The ischemia-induced synaptic potentiation (ISP) during and/or after brain ischemia has been suggested to be one of the crucial factors responsible for irreversible neuronal damage of hippocampal CA1 pyramidal neurons. However, the presynaptic modulation mechanism that leads to neuronal damage during and/or after ischemia was still unknown. By combining electrophysiological methods and infra-red differential interference contrast (IR-DIC) imaging procedures, we showed for the first time that ISP is the result of extraordinary presynaptic depolarization in association with the suppression of 4-aminopyridine (4-AP) sensitive K(+) channels at the presynaptic sites. Furthermore, we also showed that the 4-AP sensitive presynaptic K(+) channels played a crucial role in inducing neuronal damage at a very acute phase of ischemia-induced neuronal damage and would be a therapeutic target against the neuronal damage after brain ischemia.

The presynaptic modulation of glutamate release and the membrane dysfunction induced by in vitro ischemia in rat hippocampal CA1 neurons

Superfusion with an oxygen and glucose deprived medium (in vitro ischemia) of rat hippocampal CA1 pyramidal neurons in tissue slices produced a rapid depolarization within 5 min and thereafter showed no functional recovery (irreversible membrane dysfunction), even if oxygen and glucose were reintroduced. We previously suggested that such a rapid depolarization is triggered by the accumulation of extracellular glutamate (Glu). As a result, we examined the effects of either the activation or inhibition of presynaptic receptors, which modulate Glu release from the nerve terminal, on the potential change produced by in vitro ischemia. The adenosine A 1 receptor antagonist, 8-cyclopenthyl theophylline, A 2a receptor antagonist, ZM241385, and A 2b receptor antagonist, alloxazine, did not significantly alter either the latency or the maximal slope of the rapid depolarization. In addition, the GABA B receptor antagonist, 2-hydroxysaclofen, or the metabotropic Glu receptor type 4 antagonist, alpha-methylserine-O-phosphate, did not change either the latency or the maximal slope. The adenosine A 1 receptor agonist, 2-chloro-N 6-cyclopentyladenosine, A 2a receptor agonist, CGS2168, or A 2b receptor agonist, 5′-(N-ethylcarboxamido)-adenosine, did not affect these parameters either. None of these drugs restored the membrane potential to the pre-exposure level after the reintroduction of oxygen and glucose. Simultaneous intracellular recordings from CA1 and CA3 pyramidal neurons in the same slices revealed the membrane of the CA3 neurons to be hyperpolarized when a rapid depolarization occurred in the CA1 neurons. These results suggest that presynaptic Glu release does not accelerate during the generation of the rapid depolarization induced by in vitro ischemia.

Involvement of actin cytoskeleton in modulation of Ca 2+-activated K + channels from rat hippocampal CA1 pyramidal neurons

Using inside-out configuration of patch clamp techniques, actin cytoskeleton modulation of large conductance calcium-activated potassium (BK Ca) channels was studied in hippocampal CA1 pyramidal neurons from adult rat. Disruption of actin filament after brief treatment with cytochalasin D (CD) markedly decreased activity of BK Ca channels and this inhibitory action persisted even after CD washout. This CD-produced decrease in BK Ca channel activity was characterized by a shortened open lifetime and reduced opening frequency. Disruption of actin filament with another toxin cytochalasin B also exerted similar effects as CD. Phalloidin, the actin filament stabilizer, had no significant effect on BK Ca channel activity, while pre-treatment with phalloidin could prevent the CD-induced inhibitory effect on channel activity. The present study demonstrates that disruption of actin cytoskeleton can inhibit BK Ca channel activity, suggesting that BK Ca channels in rat hippocampal CA1 pyramidal neurons may be directly modulated by dynamic assembly and disassembly of submembranous actin cytoskeleton.

Spatial segregation and interaction of calcium signalling mechanisms in rat hippocampal CA1 pyramidal neurons.

Postsynaptic [Ca2+]i increases result from Ca2+ entry through ligand-gated channels, entry through voltage-gated channels, or release from intracellular stores. We found that these sources have distinct spatial distributions in hippocampal CA1 pyramidal neurons. Large amplitude regenerative release of Ca2+ from IP3-sensitive stores in the form of Ca2+ waves were found almost exclusively on the thick apical shaft. Smaller release events did not extend more than 15 microm into the oblique dendrites. These synaptically activated regenerative waves initiated at points where the stimulated oblique dendrites branch from the apical shaft. In contrast, NMDA receptor-mediated increases were observed predominantly in oblique dendrites where spines are found at high density. These [Ca2+]i increases were typically more than eight times larger than [Ca2+]i from this source on the main aspiny apical shaft. Ca2+ entry through voltage-gated channels, activated by backpropagating action potentials, was detected at all dendritic locations. These mechanisms were not independent. Ca2+ entry through NMDA receptor channels or voltage-gated channels (as previously demonstrated) synergistically enhanced Ca2+ release generated by mGluR mobilization of IP3.

Calcium channel subtypes on single GABAergic presynaptic terminal projecting to rat hippocampal neurons

High voltage-activated Ca 2+ channel subtypes triggering GABA release from nerve terminals (boutons) projecting to rat hippocampal CA1 pyramidal neurons were studied. Evoked GABAergic inhibitory postsynaptic currents (eIPSCs) were recorded in response to focal stimulation of single boutons in mechanically dissociated neurons and in response to stimulation of nerve bundle in slice preparations. Nilvadipine (3 μM), an L-type Ca 2+ channel blocker, completely inhibited eIPSCs evoked by stimulation of single boutons, but had no effect on eIPSCs evoked by stimulation of nerve bundle at low frequencies. Nilvadipine (3 μM) did, however, prevent the potentiation of eIPSC amplitude following high-frequency stimulation of nerve bundles in the slice preparation. ω-Conotoxin-GVIA (3 μM), an N-type Ca 2+ channel blocker, and ω-agatoxin-IVA (0.3 μM), a P/Q-type Ca 2+ channel blocker, completely inhibited single bouton evoked eIPSCs in 33.3 and 83.3% of recordings, respectively. In response to low-frequency nerve bundle stimulation in the slice preparation, ω-conotoxin-GVIA (3 μM), ω-agatoxin-IVA (0.1 μM) both partially reduced eIPSC amplitude, with the residual component being abolished by Cd 2+ (0.1 mM). From these results, the following hypotheses could be drawn. (1) The distribution of P/Q- and N-type channels at a single bouton is nonuniform. (2) When a focal stimulation is applied to a single bouton, L-type channels play a significant role in a generation of an action potential which subsequently activates P/Q- and N-type channels at GABA release sites. (3) Action potentials conducted through axons in the slice preparation are sufficient to depolarize the bouton membrane, even when L-type channels are suppressed.

Beta-agkistrodotoxin inhibits large-conductance calcium-activated potassium channels in rat hippocampal CA1 pyramidal neurons

Effect of β-agkistrodotoxin (β-AgTx), a presynaptic neurotoxin purified from snake venom, on large-conductance calcium-activated potassium channels (BK Ca) was studied in rat hippocampal CA1 pyramidal neurons using inside-out configuration of patch-clamp technique. The results showed that in equimolar K + (150 mM) and 1 μM intracellular Ca 2+ conditions, internal application of β-AgTx inhibited the activity of BK Ca by reducing open probability ( P o) of the channels in a concentration-dependent manner. High concentration (74 nM) of β-AgTx completely eliminated opening of the channels. However, 37 nM β-AgTx (at −40 mV) decreased P o from 0.49±0.07 to 0.03±0.03, switched two open time constants (0.51±0.32 and 8.77±1.63 ms) to be a single time constant of 0.46±0.40 ms. The results indicate that inhibition of BK Ca by β-AgTx may account for the facilitatory phase of the toxin on acetylcholine release from nerve terminals.

Inhibition of slow Ca(2+)-activated K(+) current by 4-aminopyridine in rat hippocampal CA1 pyramidal neurones.

1. The effect of 4-aminopyridine (4-AP) on the slow afterhyperpolarization (sAHP) seen after high frequency dendritic or somatic firing was investigated in rat hippocampal CA1 pyramidal neurones (PC). Intracellular recordings were obtained from the distal apical dendrites and somata and suprathreshold depolarizing current pulses were used to evoke a sAHP. The sAHP was blocked by low concentrations of carbacholine (Cch) but insensitive to high concentrations of apamin. 2. In the presence of extracellular 4-AP, the first dendritic sAHP evoked was reduced compared to a maximal sAHP evoked in the absence of 4-AP. The reduction was evident at submillimolar concentration and increased to about 80% with 4 mM 4-AP. 3. The stability of the 4-AP-induced block was affected by the type of anion used in the electrode solution. With K(+) acetate (KAc) or K(+) methylsulphate (KMeSO(4)) containing electrodes, the block was progressively removed during the initial 300 - 400 s of recordings. With KCl containing electrodes, the block remained stable and was 10% larger than that obtained with acetate. Detailed investigations showed that intracellular acetate promotes the removal of the 4-AP-induced block in an activity-dependent manner. 4. Intracellularly applied 4-AP also induced an acetate-sensitive block of the dendritic sAHP. 5. 4-AP also blocked the somatic sAHP and the stability of the block showed the same sensitivity towards anions as the dendritic sAHP. 6. Thus 4-AP appears to block the slow Ca(2+)-activated K(+) current underlying the sAHP in a complex manner which is sensitive to certain types of anions.

Photolysis-induced suppression of inhibition in rat hippocampal CA1 pyramidal neurons.

1. Whole cell patch clamp recording, Ca(2+) measurement with ratiometric fluorescent dyes and photolysis of caged Ca(2+) were combined to investigate the depolarization- and photolysis-induced suppression of inhibition (DSI and PSI) in rat hippocampal CA1 pyramidal cells. 2. A 5-s depolarization from -70 mV to 0 mV or a 6-s photolysis of nitrophenyl-EGTA (NPE) in cell bodies could each depress the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) and the amplitude of evoked IPSCs while elevating intracellular Ca(2+) concentration ([Ca(2+)](i)). 3. Within a cell the elevation of [Ca(2+)](i) induced by depolarization was inversely related to that induced by photolysis, suggesting that higher [NPE] is more effective in releasing caged Ca(2+) but also increases buffer capacity to reduce [Ca(2+)](i) rises caused by Ca(2+) influx through voltage-dependent Ca(2+) channels. 4. Both DSI and PSI were linearly related to [Ca(2+)](i), with a 50 % reduction in transmission occurring at about 3.6--3.9 microM. 5. [Ca(2+)](i) recovered more quickly than DSI, indicating that the duration of DSI is not set simply by the duration of [Ca(2+)](i) elevation, but rather entails other rate-limiting processes. 6. We conclude that DSI is activated by micromolar [Ca(2+)](i) acting far from sites of Ca(2+) entry through channels in the plasma membrane.

The nootropic drug vinpocetine inhibits veratridine-induced [Ca2+]i increase in rat hippocampal CA1 pyramidal cells.

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3alpha,16alpha-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca2+]i, this study investigated the effect of vinpocetine on [Ca2+]i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na+ influx, causing depolarization and increasing [Ca2+]i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca2+]i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 microM) significantly inhibited the elevation in [Ca2+]i induced by veratridine (10 microM). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca2+]i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca2+]i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.

Properties of large conductance calcium-activated potassium channels in pyramidal neurons from the hippocampal CA1 region of adult rats.

The properties of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels were studied in rat hippocampal CA1 pyramidal neurons by using the patch-clamp technique in the excised-inside-out-patch configuration. The lowest [Ca(2+)](i) in which BK(Ca) channel activities were observed was 0.01 microM with the membrane potential of +20 mV and the [Ca(2+)](i) at which P(O) of the channel is equal to 0.5 was 2 microM. The unitary conductance of the single BK(Ca) channel was 245.4 pS with symmetrical 140 mM K(+) on both sides of the excised membrane. With a fixed [Ca(2+)](i) of 2 microM, P(O) increased e-fold with a 17.0 mV positive change in the membrane potential. Two exponentials, with time constants of 2.8 ms and 19.2 ms at the membrane potential of +120 mV with 2 microM [Ca(2+)](i), were required to describe the observed open time distribution of BK(Ca) channel, suggesting the existence of two distinct open channel states with apparently normal conductance. A BK(Ca) channel occasionally entered an apparent third open channel state with the single channel current amplitude about 45% of the normal amplitude. The properties of BK(Ca) channel, which were found in this study to be more steeply dependent on voltage and more sensitive to [Ca(2+)](i) in adult hippocampal neurons than in cultured or immature hippocampal neurons, may be responsible for the shortened duration of action potential in hippocampal CA1 pyramidal neurons of adult rat.

Action of carbamazepine on epileptiform activity of the verartidine model in CA1 neurons

The veratridine epileptiform model was utilized to assess the antiepileptic effect of Carbamazepine (CBZ) in rat hippocampal CA1 pyramidal neurons using conventional intracellular recording techniques. In the veratridine model, where brain slices are treated with veratridine (0.3 μM), a single intracellular stimulus evokes epileptiform bursting. Additionally, spontaneous epileptiform activity commonly appears on prolonged exposure to veratridine in this model. In this model, therapeutic (7–15 μM) and high (50 μM) concentrations of CBZ inhibited the evoked and spontaneous epileptiform bursting in a concentration- and voltage-dependent manner. At all concentrations tested, CBZ produced inhibition of epileptiform activity without affecting the membrane resting potential or input resistance. However, at 50 μM, the drug increased the firing threshold of neurons. These results confirm the suitability of this model for testing sodium channel-dependent antiepileptic agents.

Bicuculline, pentobarbital and diazepam modulate spontaneous GABAA channels in rat hippocampal neurons

Spontaneously opening, chloride-selective channels that showed outward rectification were recorded in ripped-off patches from rat cultured hippocampal neurons and in cell-attached patches from rat hippocampal CA1 pyramidal neurons in slices. In both preparations, channels had multiple conductance states and the most common single-channel conductance varied. In the outside-out patches it ranged from 12 to 70 pS (Vp=40 mV) whereas in the cell-attached patches it ranged from 56 to 85 pS (-Vp=80 mV). Application of GABA to a patch showing spontaneous channel activity evoked a rapid, synchronous activation of channels. During prolonged exposure to either 5 or 100 microM GABA, the open probability of channels decreased. Application of GABA appeared to have no immediate effect on single-channel conductance. Exposure of the patches to 100 microM bicuculline caused a gradual decrease on the single-channel conductance of the spontaneous channels. The time for complete inhibition to take place was slower in the outside-out than in the cell-attached patches. Application of 100 microM pentobarbital or 1 microM diazepam caused 2 - 4 fold increase in the maximum channel conductance of low conductance (<40 pS) spontaneously active channels. The observation of spontaneously opening GABA(A) channels in cell-attached patches on neurons in slices suggests that they may have a role in neurons in vivo and could be an important site of action for some drugs such as benzodiazepines, barbiturates and general anaesthetics.

Distribution of spontaneous currents along the somato-dendritic axis of rat hippocampal CA1 pyramidal neurons

Excitatory and inhibitory pathways have specific patterns of innervation along the somato-dendritic axis of neurons. We have investigated whether this morphological diversity was associated with variations in the frequencies of spontaneous and miniature GABAergic and glutamatergic synaptic currents along the somato-dendritic axis of rat hippocampal CA1 pyramidal neurons. Using in vitro whole cell recordings from somata, apical dendrites and basal dendrites (for which we provide the first recordings) of CA1 pyramidal neurons, we report that over 90% of the spontaneous currents were GABAergic, <10% being glutamatergic. The frequency of spontaneous GABAergic currents was comparable in the soma and in the dendrites. In both somata and dendrites, the Na + channel blocker tetrodotoxin abolished more than 80% of the spontaneous glutamatergic currents. In contrast, tetrodotoxin abolished most dendritic (>90%) but not somatic (<40%) spontaneous GABAergic currents. Computer simulations suggest that in our experimental conditions, events below 40 pA are electrotonically filtered to such a degree that they are lost in the recording noise. We conclude that, in vitro, inhibition is massively predominant over excitation and quantitatively evenly distributed throughout the cell. However, inhibition appears to be mainly activity-dependent in the dendrites whereas it can occur in the absence of interneuron firing in the soma. These results can be used as a benchmark to compare values obtained in pathological tissue, such as epilepsies, where changes in the balance between excitation and inhibition would dramatically alter cell behaviour.

Effects of hemoglobin and its breakdown products on synaptic transmission in rat hippocampal CA1 neurons

During head injuries and hemorrhagic stroke, blood is released into the extravascular space. The pooled erythrocytes get lysed and hemoglobin is released into the intracranial cavities. Therefore, neurons may be exposed to hemoglobin and/or its breakdown products, hemin and iron, for long periods of time. In this study, the electrophysiological actions of these agents on synaptic transmission in rat hippocampal CA1 pyramidal neurons were studied using extracellular field- and whole cell patch-recordings. Previously our laboratory reported that commercially available hemoglobin produced a dose dependent suppression of synaptic transmission in hippocampal CA1 neurons. In the present study, however, we found that this depression was caused by impurities present in the hemoglobin samples. Commercially available hemoglobin and methemoglobin did not have a significant effect on synaptic transmission. Although, reduced-hemoglobin prepared using a method described by Martin et al. [J. Pharm. Exp. Ther. 232 (1985) 708], produced a significant depression of synaptic transients, these effects were due to contamination with bisulfite that was present due to the reducing procedure. Therefore, the technique of Martin et al. was inadequate in removing the reducing agents or their breakdown products. A number of studies in literature used commercial samples of hemoglobin or reduced hemoglobin prepared using the method of Martin et al. Our observations indicate that it would be important to determine if contaminants, rather than hemoglobin, are responsible for the observed effects in these studies. Unlike hemoglobin, its breakdown products, ferrous chloride and hemin, produced an irreversible and significant depression of field excitatory postsynaptic potentials. The relevance of these effects in neurological complications that follow head injuries and hemorrhagic stroke awaits further investigation.

Apamin-sensitive conductance mediates the K(+) current response during chemical ischemia in CA3 pyramidal cells.

Pyramidal cells typically respond to ischemia with initial transient hyperpolarization, which may represent a neuroprotective response. To identify the conductance underlying this hyperpolarization in CA3 pyramidal neurons of rat hippocampal organotypic slice cultures, recordings were obtained using the single-electrode voltage-clamp technique. Brief chemical ischemia (2 mM 2-deoxyglucose and 3 mM NaN(3), for 4 min) induced a response mediated by an increase in K(+) conductance. This current was blocked by intracellular application of the Ca(2+) chelator, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA), reduced with low external [Ca(2+)], and inhibited by a selective L-type Ca(2+) channel inhibitor, isradipine, consistent with the activation of a Ca(2+)-dependent K(+) conductance. Experiments with charybdotoxin (10 nM) and tetraethylammonium (TEA; 1 mM), or with the protein kinase C activator, phorbol 12,13-diacetate (PDAc; 3 microM), ruled out an involvement of a large conductance-type or an apamin-insensitive small conductance, respectively. In the presence of apamin (1 microM), however, the outward current was significantly reduced. These results demonstrate that in rat hippocampal CA3 pyramidal neurons an apamin-sensitive Ca(2+)-dependent K(+) conductance is activated in response to brief ischemia generating a pronounced outward current.

Valproic acid intensifies epileptiform activity in the hippocampal pyramidal neurons

The effects of large concentrations of valproic acid (VPA) on veratridine-induced epileptiform activity (veratridine model) were investigated in rat hippocampal CA1 pyramidal neurons. Studies were performed on the veratridine model in rat brain slices using conventional electrophysiological intracellular techniques. Large concentrations of VPA (5 mM or more) enhanced rather than inhibited epileptiform activity induced by veratridine. During the proepileptic phase of VPA, a membrane depolarization accompanied by a decrease in membrane input resistance were evident. The voltage-dependent proepileptic effect of VPA was blocked by tetrodotoxin (TTX; 100 nM) but not by the calcium channel blockers, diltiazem (5 microM) or omega-conotoxin GVIA (5 microM). VPA did not induce a proepileptic effect when it was superfused at high concentration (0.5-10 mM) on sodium channel-independent models such as the bicuculline or magnesium-free artificial cerebrospinal fluid. Large concentrations of VPA had no significant effect on untreated neurons. The VPA-enhanced veratridine bursting is probably related to the reported proepileptic activities observed in patients taking high doses of this drug. These data also suggest the involvement of sodium channels in the proepileptic effect of VPA.

Calcium dependence of depolarization-induced suppression of inhibition in rat hippocampal CA1 pyramidal neurons.

1. We made whole-cell recordings from CA1 pyramidal cells in the rat hippocampal slice preparation to study the calcium (Ca2+) dependence of depolarization-induced suppression of inhibition (DSI). DSI is a retrograde signalling process in which voltage-dependent Ca2+ influx into a pyramidal cell leads to a transient decrease in the release of GABA from interneurons. 2. To investigate the Ca2+ dependence of DSI without altering extracellular divalent cations, we varied the type and amount of Ca2+ chelator (EGTA or BAPTA) in the recording pipette (keeping the chelator : Ca2+ ratio constant). Evoked inhibitory postsynaptic currents (IPSCs) were induced in the presence of antagonists of ionotropic glutamate receptors. DSI was induced by depolarizing voltage steps, lasting from 0.025 to 5 s, to 0 mV. 3. DSI was directly dependent on the duration of the voltage step used to induce it, from threshold up to a maximal value of IPSC suppression, whether EGTA or BAPTA was used, and whether their concentrations were 0.1, 0.5 or 2 mM. For instance, a voltage step lasting 1.37 s produced half-maximal DSI with 2 mM BAPTA, but with 0. 1 mM BAPTA, half-maximal DSI was achieved with a step lasting 0.186 s. Peak DSI was the same in all cases, and DSI was blocked with either 10 mM EGTA or BAPTA in the pipette. Bath application of carbachol could overcome the block of DSI by 10 mM EGTA but not by 10 mM BAPTA. 4. We calculated that a voltage step lasting approximately 100 ms would be necessary to activate half-maximal DSI in the absence of exogenous Ca2+ buffers. 5. Log-log plots of calculated total Ca2+ influx, estimated from time integrals of Ca2+ currents, versus DSI yielded a straight line with a slope of approximately 1, and increasing extracellular [Ca2+] from 2.5 to 5 mM did not change the slope. 6. The time course of decay of DSI was well described by an exponential function with a time constant of approximately 20 s and was not affected by changes in either concentration or type of Ca2+ buffer. 7. The data suggest that, in its Ca2+ dependence, DSI more closely resembles the slow release of neuropeptides and hormones than it does the process of fast release of many neurotransmitters.

Ethanol inhibition of synaptically evoked kainate responses in rat hippocampal CA3 pyramidal neurons.

Many studies have demonstrated that intoxicating concentrations of ethanol (10-100 mM) can selectively inhibit the component of glutamatergic synaptic transmission mediated by N-methyl-D-aspartate (NMDA) receptors while having little or no effect on excitatory synaptic transmission mediated by non-NMDA receptors [i.e., alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and/or kainate (KA) receptors]. However, until the recent development of highly selective AMPA receptor antagonists, it was not possible to assess the relative contribution of AMPA and KA receptors to non-NMDA receptor-mediated synaptic transmission or to determine whether these glutamate receptor subtypes differed in their sensitivity to ethanol. In the present experiments, we used the highly selective AMPA receptor antagonist LY 303070 to pharmacologically isolate KA receptor-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal CA3 pyramidal neurons and tested their sensitivity to ethanol. Concentrations of ethanol as low as 20 mM significantly and reversibly depressed KA EPSCs. Ethanol also inhibited KA currents evoked by direct pressure application of KA in the presence of LY 303070, suggesting that this inhibition was mediated by a postsynaptic action. In contrast, ethanol had no effect on AMPA EPSCs in these cells, even at the highest concentration tested (80 mM). Ethanol significantly inhibited NMDA EPSCs in these neurons, but these responses were less sensitive to ethanol than KA EPSCs. These results suggest that in addition to its well-described depressant effect on NMDA receptor-mediated synaptic transmission, ethanol has an even greater inhibitory effect on glutamatergic synaptic transmission mediated by KA receptors in rat hippocampal CA3 pyramidal neurons.