Satsuki Sawada

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.

A reliable method for culture of dissociated mouse cerebellar cells enriched for Purkinje neurons

The cerebellar Purkinje neuron (PN) serves as an important model in studies of neuronal development in the mammalian central nervous system. Dissociated PN preparations maintained in an in-vitro environment with simplified cellular and biochemical conditions can facilitate molecular analyses of neuronal development. Here we describe a reliable method to prepare dissociated cultures of mouse cerebellar neurons maintained with a serum-free, Dulbeccos modified Eagles medium/F-12 nutrient-based medium, which facilitates PN survival and dendritic differentiation. The survival of mouse PNs in this culture was maximized when cerebellar cells were (1) taken from prenatal animals, (2) dissociated with papain, and (3) seeded at a density of 5 000 000 cells/ml or higher. Dissociated PNs prepared by our method from mice of embryonic day 18 (E 18) reproduced several morphological and electrophysiological changes seen in intact postnatal rodents with similar time-courses. Therefore, our culture method offers a useful model for investigating molecular mechanisms underlying postnatal neuronal development.

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.

Quantal analysis of potentiating action of phorbol ester on synaptic transmission in the hippocampus.

The mechanism of the potentiating action of phorbol diacetate on synaptic transmission in the hippocampus was studied by the quantal analysis technique. Thin transverse sections were prepared from guinea pig hippocampus and intracellular potentials were recorded from CA3 neurons. Unitary excitatory postsynaptic potentials (EPSPs) were induced in the impaled neurons by brief glutamate pulses administered to granule cells. The amplitude of the unitary EPSPs fluctuated according to Poisson distribution. From the mean and variance of the amplitude of the unitary EPSPs, the mean quantal content (m) and the mean quantal amplitude (q) were calculated. Before phorbol diacetate administration, the values of m and q were 9.7 +/- 1.4 and 1.1 +/- 0.28 mV (mean +/- S.D.), respectively. Potentiation of synaptic transmission by phorbol diacetate was accompanied by increases in the value of m. The value of q remained unchanged in most neurons and decreased in some. These results indicate that the phorbol ester causes an increase in release of neurotransmitter and thereby potentiates synaptic transmission.

Insulin-like growth factor-I as a promoting factor for cerebellar Purkinje cell development

In the mammalian CNS, the peptide hormone insulin‐like growth factor‐I (IGF‐I) is synthesized in a certain subset of neurons and, it has been suggested, serves as a local neurotrophic factor. A postnatal increase in the expression of IGF‐I and the type‐1 IGF receptors (IGFR1) in the cerebellar cortex and its related brain regions indicates that developing cerebellar Purkinje cells (PC) may be an important target of IGF‐I. However, little is known about how IGF‐I influences PC development. Here we addressed this question, using a reduced environment of cerebellar neuron culture derived from perinatal mice. IGF‐I exogenously applied at a physiological concentration (10 nm) greatly promoted the dendritic growth and survival of the PCs. By contrast, IGF‐I only slightly promoted the somatic growth and little affected the maturation of the electrophysiological excitability of the PCs. The closely related hormone insulin had weaker promoting effects than did IGF‐I. IGF‐I appeared to at least bind to IGFR1 and to up‐regulate the signalling pathways involving the phosphoinositide 3‐kinase (PI3‐K), mitogen‐activated protein kinase (MAPK), p38 kinase (p38K), and an unknown signalling molecule(s). These signalling pathways may be coupled to the individual aspects of PC development in different manners and this may explain the difference in effects of IGF‐I among these aspects. These findings suggest that IGF‐I serves as a promoting factor for PC development, particularly postnatal survival and dendritic growth.

Inhibitors of high-affinity uptake augment depolarizations of hippocampal neurons induced by glutamate, kainate and related compounds

SummaryActions of dihydrokainate (DHKA) and 3-hydroxy-DL-aspartate (HAsp), inhibitors of high-affinity uptake for L-glutamate (Glu), were studied in vitro in thin hippocampal slices of the guinea pig. The amplitude of the depolarizations induced by Glu and by L-aspartate (Asp) in CA3 neurons are markedly augmented by DHKA and HAsp. Depolarizations induced by D-homocysteate (DH) were unaffected by the inhibitors. In about half of the neurons, depolarizations induced by L-homocysteate (LH) and by quisqualate (Quis) were slightly augmented by the inhibitors. Fast responses to kainate (KA) were augmented by the inhibitors to a similar extent as were Glu responses whereas slow KA responses were insensitive to HAsp. HAsp was without effect on excitatory postsynaptic potentials elicited by stimulation of granular layer. These findings are in general agreement with the biochemical data on amino acid uptake processes and are also consistent with the slow time-courses of depolarizations induced by DH, LH and Quis. Augmentation of fast KA responses provides strong evidence for the hypothesis that an KA pulse causes a liberation of Glu and/or Asp from the tissue and the liberated amino acid(s) induces the fast KA response in neurons nearby.

Electrical activity recorded from thin sections of the bed nucleus of the stria terminalis, and the effects of neurotensin

Effects of neurotensin on neurons in the bed nucleus of the stria terminalis (BST) were studied in vitro in thin brain sections of the guinea pig. Electrical stimulation to the stria terminalis (ST) elicited a negative field potential in the BST. The field potential was markedly suppressed in the medium containing Ca2+ at a low concentration of Mg2+ at a high concentration. BST neurons discharged in response to ST stimulation. Some of them also fired spontaneously. Neurotensin excited about two-thirds of BST neurons at concentrations of 0.35-35 nM. This excitation was not blocked when synaptic transmission was blocked in a medium containing Ca2+ in a low concentration and Mg2+ in a high concentration. These observations suggest that neurotensin is an excitatory transmitter or modulator in the amygdalo-BST projection.

Long-term potentiation and depression in the dentate gyrus, and effects of nicotine

Effects of a brief high-frequency stimulation or a prolonged low-frequency stimulation in the presence and absence of nicotine were studied in thin transverse slices of the dentate gyrus prepared from the guinea pig. Test and conditioning stimulations were delivered to the middle one-third of the molecular layer, and the slope of the population excitatory postsynaptic potentials (EPSPs) elicited by the test stimulation was taken as an indicator of potentiation or depression. Nicotine was without effects on the N-methyl-D-aspartate (NMDA) component of EPSPs at 50 or 100 microM. A brief high-frequency stimulation induced long-term potentiation (LTP) in the presence of bicuculline. Nicotine (50 microM) almost doubled the magnitude of LTP. In the absence of bicuculline, an identical high-frequency stimulation induced a brief depression, the duration and magnitude of which were increased by nicotine. The increase was not statistically significant, however. In contrast to observations in the region CA1, 720 pulses at 1 Hz delivered after induction of LTP failed to induce long-term depression. Nicotine did not modify the after-effect of the low-frequency stimulation. It was discussed that the facilitation of LTP by nicotine probably resulted from suppression of inhibitory processes.

Quantal analysis of suppressing action of baclofen on mossy fiber synapses in guinea pig hippocampus

Baclofen, a selective agonist at the gamma-aminobutyric acidB (GABAB) receptor, has been considered to reduce the release of transmitter from nerve terminals by acting on presynaptic GABAB receptors, in addition to its postsynaptic action. The purpose of this study has been to re-examine quantitatively the action of baclofen in the hippocampus by a rigorous quantal analysis. (+/-)-Baclofen suppressed field potentials and intracellularly-recorded synaptic potentials induced in the subfield CA3 by mossy fiber stimulation in thin transverse sections of the guinea pig hippocampus. The amplitude distribution of excitatory postsynaptic potentials (EPSPs) induced monosynaptically by a granule cell could be described by the Pascal statistics. Suppression of the unitary EPSPs by baclofen (1 and 5 microM) was accompanied by decreases both in mean quantal content (m) and by mean quantal amplitude (q). The reduction in q was smaller than expected from a decrease in the input resistance of the postsynaptic neuron. It was suggested that the presynaptic and postsynaptic actions of baclofen contribute almost equally to suppression of the transmission at 1 microM, whereas the presynaptic action predominates at 5 microM.