Chosaburo Yamamoto

Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro

Summary1.Transmission through the synapse between the mossy fiber and the CA3 neurons was studied in vitro in thin transverse sections from the hippocampus of the guinea-pig.2.Stimulation to the granular layer elicited in the pyramidal cell layer of the regio CA3 a mainly negative focal potential consisting of the early and late waves. Conditioning stimulation potentiated the late wave but not the early wave. The late wave was strongly suppressed by an excess of Mg++ or a reduction in Ca++ in the medium, but the early wave was affected only a little.3.The action potentials, EPSP, and IPSP recorded intracellularly differed little from those observed in vivo except that most of the action potentials lacked the depolarizing afterpotential. The EPSP was markedly potentiated during low frequency stimulation. At nearly threshold strength, it showed a wide fluctuation in amplitude.4.The latency and properties of the action potential triggered by the EPSP agreed with those of the late wave, which was therefore concluded to represent activation of the CA3 neurons by chemical transmission.5.Corresponding to the early wave in the field potential, some cells generated action potentials before the onset of the EPSP. Probable explanations were offered for the activation of the CA3 neurons without preceding EPSPs.

Presynaptic action of acetylcholine in thin sections from the guinea pig dentate gyrus in vitro

Effects of acetylcholine (Ach) upon electrical activities of the dentate gyrus were studied in vitro in thin sections prepared from the guinea pig brain. In the presence of physostigmine, Ach suppressed the potential evoked by direct stimulation in the slice including the intact granule cell layer, but it was without effect upon the potential elicited in the slice consisting of the molecular layer alone. The threshold concentration to cause such suppression was about 1 × 10−7m. Atropine blocked the action of Ach. The effects of atropine could not be removed by repeated washing of the slice. The suppressing effects of Ach were observed in the Cl-deficient medium. The single neuron discharge elicited in the granule cell by direct stimulation was suppressed during perfusion with the medium containing Ach, but the spontaneous neuron discharge was induced by Ach. From these findings, it was concluded that Ach affected the presynaptic terminals making synapses with the granule cell soma and proximal dendrites, while the synaptic terminals making synapses with the distal portion of the dendrites were free from the action of Ach.

Intracellular study of seizure-like afterdischarges elicited in thin hippocampal sections in vitro

Abstract Thin transverse sections of the guinea pig hippocampus generated a train of afterdischarges in the C1-deficient medium in response to electric stimulation of the granular layer. Corresponding to the afterdischarges, a large depolarizing shift (DS) of about 100-msec duration was recorded intracellularly from CA3 neurons. Abortive action potentials were superimposed on the DS. The DS occurred in an all-or-nothing manner at a threshold strength. A small hyperpolarization of about 400-msec duration followed the DS. Similar DS were evoked in the medium containing strychnine or thiosemicarbazide. These findings indicate that the afterdischarges elicited in vitro are akin to the interictal paroxysmal discharges.

Long-term potentiation in thin hippocampal sections studied by intracellular and extracellular recordings

To study the mechanism for long-term potentiation, extracellular and intracellular potential changes were examined in thin hippocampal sections in vitro. The field potential elicited by mossy fiber stimulation in the CA3 region (primary response) was augmented for observation periods of 15–60 min after conditioning tetanic stimulation. Tetanization of a group of mossy fibers potentiated responses induced by another group of presynaptic fibers. The augmented primary response was sometimes followed by a train of after-discharges. When long-lasting potentiation of the primary response was observed, an increase in excitatory postsynaptic potential amplitude or a depression of inhibitory postsynaptic potentials was found intracellularly. The afterdischarge train was associated with a large intracellular depolarization of long durations. These results suggest that long-term potentiation of the primary response is due to an enhanced excitatory synaptic transmission and a depression of the inhibitory circuit, and that the depression of inhibitory postsynaptic potentials may be related partly to paroxysmal activities of neurons.

Identification of an ATP-sensitive K+ channel in rat cultured cortical neurons

To determine whether membranes of mammalian central neurons contain an ATP-sensitive K+ (KATP) channel similar to that present in pancreatic β cells, the patch-clamp technique was applied to cultured neurons prepared from the neonatal rat cerebral cortex and hippocampus. In whole-cell experiments with hippocampal neurons, extracellular application of 0.5 mM diazoxide (a KATP channel activator) elicited a hyperpolarization concomitant with an increase in membrane conductance, whereas application of 0.5 mM tolbutamide (a KATP channel blocker) induced a depolarization with a decrease in conductance. Similar results were obtained with cortical neurons. In outside-out patch experiments with cortical neurons, a K+ channel sensitive to these drugs was found. The channel was completely blocked by 0.5 mM tolbutamide and activated by 0.5 mM diazoxide. The single-channel conductance was 65 pS under symmetrical 145 mM K+ conditions and 24 pS in a physiological K+ gradient. In inside-out patch experiments, this channel was demonstrated to be inhibited by an application of 0.2–1 mM ATP to the cytoplasmic surface of the patch membrane. These results indicate that the membranes of rat cortical neurons contain a KATP channel that is quite similar to that found in pancreatic β cells. It is also suggested that the same or a similar K+ channel may exist in membranes of hippocampal neurons.

Synthetic ω-conotoxin blocks synaptic transmission in the hippocampus in vitro

Abstract The effects of some organic calcium channel blockers and a toxin on synaptic transmission were examined in hippocampal slices from the guinea pig. ω-Conotoxin fraction GVIA, a novel peptide that blocks N and L type voltage-sensitive calcium channels, blocked synaptic transmission from mossy fibers to CA3 neurons at very low concentration (100 nM). However, organic calcium channel blockers such as verapamil (100 μM) and nifedipine (10 μM), which block L type calcium channels, had little effect on the synaptic transmission. Phenytoin (100 μM, T type calcium channel blocker) was also ineffective to block the synaptic transmission. These results suggest that presynaptic calcium channels which cause transmitter release may be N type calcium channel.

Blocking action of pentobarbital on receptors for excitatory amino acids in the guinea pig hippocampus

SummaryThe actions of pentobarbital sodium (Pent) on receptors for glutamate (Glu) and related compounds were studied in thin sections of the guinea pig hippocampus. Depolarizations induced by Glu and quisqualate (Quis) in CA3 neurons were reduced in amplitude during iontophoretic administration of Pent. This action of Pent was not accompanied by any noticeable changes in membrane potential or neuron input resistance. Depolarizations induced by N-methyl-D-aspartate were less sensitive to Pent. The fast kainate (KA) response was as susceptible as the Glu response, whereas the slow KA response was unaffected by Pent in three quarters of the neurons examined. Pent suppressed the Glu response at lower concentrations than required to potentiate responses to gamma-amino butyric acid. Excitatory postsynaptic potentials (EPSPs) elicited by stimulation of mossy fibers were suppressed by Pent. The EPSPs were a little more resistant to Pent than were the Glu responses. These results indicate that Pent blocks receptors for excitatory amino acids in the hippocampus. Of the three different populations of the receptors, Quis receptors are the most sensitive to Pent and KA receptors are the least sensitive. The suppression of the EPSPs is in accordance with the notion that Glu is the transmitter released from mossy fibers.

Suppressing action of 2-amino-4-phosphonobutyric acid on mossy fiber-induced excitation in the guinea pig hippocampus

SummaryThe action of 2-amino-4-phosphonobutyric acid (APB) on mossy fiber-induced excitation in CA3 neurons was studied in vitro with thin sections of guinea pig hippocampus. D- and DL-APB suppressed field potentials induced in regio CA3 by granular layer stimulation. Threshold concentration of DL-APB to induce the suppression was 2–5 μM. D-APB was about 10-fold less potent than DL-APB. Field potentials induced by fimbrial stimulation were much less affected. DL-APB was without effect on antidromic activation of granule cells. 2-Amino-phosphonovaleric acid had a similar but less potent action. Gamma-D-glutamylglycine and cis-2,3-piperidine dicarboxylic acid were almost ineffective. DL-APB suppressed excitatory postsynaptic potentials recorded intracellularly from CA3 neurons in response to granular layer stimulation but caused no marked changes in resting potentials, action potentials and membrane conductance. Single cell discharges induced by iontophoretic administration of glutamate (Glu) or aspartate (Asp) were unaffected when mossy fiber-induced excitation was suppressed by D- or DL-APB. DL-APB had no suppressing action on Glu- or Asp-induced depolarizing potentials. These results indicate that APB is a relatively specific blocker of synaptic transmission between mossy fibers and CA3 neurons, and that this action does not result from blockade of receptors for Glu or Asp.

Potentiation of excitatory postsynaptic potentials during and after repetitive stimulation in thin hippocampal sections

SummaryExcitatory postsynaptic potentials (EPSPs) elicited by mossy fiber stimulation were recorded intracellularly from neurons in the CA3 region in thin hippocampal sections in vitro and potentiation of the EPSPs was examined during and after repetitive stimulation. Inhibitory postsynaptic potentials (IPSPs) and seizure discharges were blocked by bicuculline and high concentrations of Mg2+. When two shocks were applied at short intervals, the second EPSP was markedly potentiated. This potentiation declined exponentially with a time-constant of about 180 ms and was unaffected by changes in ambient temperature. The amount of potentiation during a pulse train was explained by summation of potentiation by individual pulses. Post tetanic potentiation lasted longer in media containing Ca2+ at higher concentrations and Mg2+ at lower concentrations. At high Ca2+ concentrations, tetanic stimulation induced long-term potentiation which was occasionally preceded by a long-lasting suppression. Tetanus to a bundle of mossy fibers potentiated EPSPs elicited by stimulation of a separate bundle of mossy fibers (heterosynaptic potentiation) but did not augment EPSPs elicited by fimbrial stimulation.

Postsynaptic inhibitory actions of catecholamines and opioid peptides in the bed nucleus of the stria terminalis

SummaryEffects of catecholamines, enkephalins and related compounds on electrical activity of the bed nucleus of stria terminalis (BST) were studied in vitro on thin BST sections prepared from guinea pig brains. Norepinephrine (NE) and epinephrine (E) suppressed field potentials elicited by a single shock to the stria terminalis (ST). The effects of NE and E were mimicked by phenylephrine and blocked by phenoxybenzamine. Isoproterenol and dichloroisoproterenol were without effect. NE and E suppressed the spontaneous firing of BST neurons and discharges elicited by ST stimulation. Dopamine was a less potent depressant. [D-Ala2]-Met-enkephalinamide (EKA) suppressed the field potentials and spike discharges elicited by ST stimulation. Spikes occurring spontaneously or during administration of glutamate were also suppressed by EKA. The action of EKA was blocked by naloxone. Late inhibition induced by stimulation of the lateral division of the ST was blocked by naloxone in about a third of the neurons examined. These results indicate that norepinephrine suppresses the activity of BST neurons by activating postsynaptic α-receptors. It is also suggested that opioid peptides mediate inhibitory control of the amygdala over the BST.