Hiroshi Tsubokawa

A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of α‐pompilidotoxin (α‐PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that α‐PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action of α‐PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and α‐scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of α‐PMTX on Na+ channels by whole‐cell recordings from rat trigeminal neurons. We found that α‐PMTX slowed the Na+ channels inactivation process without changing the peak current–voltage relationship or the activation time course of tetrodotoxin (TTX)‐sensitive Na+ currents, and that α‐PMTX had voltage‐dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that α‐PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of α‐PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Stimulation of the caudate nucleus induces contraversive saccadic eye movements as well as head turning in the cat

The effects of stimulation of the caudate nucleus were investigated in alert cats, with special reference to the induction of eye and head movements. Stimulation of caudal portions of the caudate nucleus on one side with trains of current pulses induced gaze shifts towards the contralateral side. When the head of the animal was restrained, the majority of evoked eye movements were single conjugate saccades. The amplitude and direction of the evoked saccade varied depending on the initial eye position. The amplitude of the horizontal component tended to be larger for saccades initiated from more ipsilateral positions, and became gradually smaller as the initial eye position shifted to the contralateral side. If the eye was far into the contralateral positions, no saccades were induced. Furthermore, the saccades tended to have a downward component when the eye was initially focused upward, and an upward component when the eye was focused downward. When the head was made free to move, the same stimulation induced a sequence of contraversive staircase gaze shifts composed of coordinated eye and head movements. The eye movements in the orbit resembled nystagmus, consisting of contraversive saccades followed by reverse compensatory movements. The head turning, though smooth and continuous, was also suggested to consist of a series of movements coupled with saccadic eye movements. This study indicates a potential role of the caudate nucleus in the control of orienting reflexes.

GABAergic input contributes to activity-dependent change in cell volume in the hippocampal CA1 region

Swelling of brain cells is one of the physiological responses associated with neuronal activation. To investigate underlying mechanisms, we analyzed interactions between changes in cell volume and synaptic responses in the hippocampal slices from rodents. Swelling within the CA1 area was detected as increases in transmittance of near-infrared light (IR), and field excitatory postsynaptic potentials (fEPSPs) were recorded simultaneously. High frequency stimulation (HFS) of afferent fibers induced a transient increase in IR transmittance in both somatic and dendritic regions, which was temporally associated with fEPSPs. Stimulus-induced increases in transmittance were strongly reduced in the presence of DL-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, indicating involvement of glutamate receptors. Application of a GABA-A receptor antagonist, bicuculline, increased the amplitude and time course of the fEPSPs but rather decreased HFS-induced optical signals. When the extracellular Cl(-) was reduced to 10.5 mM, HFS induced a decrease in transmittance, which was also blocked by bicuculline. In hippocampal slices obtained from mice deficient in the 65 kDa isoform of glutamic acid decarboxylase, HFS-induced signals were significantly smaller than in the wild-type mice, although fEPSP profiles did not differ. These results suggest that Cl(-) influx through GABA-A receptors contributes to synaptically evoked swelling in the hippocampal CA1 region.

Single glutamate channels in CA1 pyramidal neurones after transient ischaemia

Patch clamp recordings were made from CA1 pyramidal neurones to study changes in the glutamate receptor subtypes in the gerbil hippocampus after transient ischaemia. In whole-cell recordings, the maximum chord conductances of AMPA currents in ischaemic neurones were increased over those of control neurones but NMDA-induced currents in the ischaemic neurones were smaller than the control. In AMPA-activated single channel currents, an open time histogram of the control neurones was well fitted by a single exponential function whereas in the ischaemic patches it was fitted by a double exponential function, indicating that currents consisted of at least two kinetically different types. These functional changes of the glutamate receptor channels may contribute to the abnormalities of the excitatory synaptic currents recorded in post-ischaemic CA1 neurones.

Regional differences in the cat caudate nucleus as to the effectiveness in inducing contraversive head-turning by electrical stimulation

An attempt was made to re-examine regional differences in the cat caudate nucleus as to the effectiveness in inducing contraversive head-turning by electrical stimulation and to analyze the time course of head-turning quantitatively. In 5 of the total 9 cats, the right sensorimotor cortex and its surrounding areas had been ablated chronically. While the awake, unrestrained cat maintained a stable standing posture facing forward, stimulation was applied systematically to various points in and around the caudate nucleus with a movable stimulating electrode. Trains of stimulating current pulses of less than 300 microA were given, mostly at a rate of 100 Hz for 5 s. In most experiments in which stimulation was given to the side of the intact cerebral cortex, stimulation of caudal portions of the head of the caudate nucleus was effective in inducing contraversive head-turning, but that of its rostral portions was ineffective. In experiments on the side of chronic cortical ablation, similar results were obtained. These results suggested that head-turning induced by stimulation of the caudate nucleus was brought about not by the activation of the corticofugal fibers from these cortical areas by a current spreading to the internal capsule, but by the activation of caudate neurons. Hence, it was demonstrated that there were regional differences in the cat caudate nucleus as to the effectiveness in inducing head-turning. The mean of the shortest latencies of the onset of head-turning for individual stimulation points was 396 ms (S.D., 210 ms) for the side of the intact cerebral cortex, and 454 ms (S.D., 289 ms) for the side of the cortical ablation. Statistically, there was no significant difference between them. Therefore, it was further revealed that the elimination of the sensorimotor cortex did not affect the caudate-induced head-turning in terms of the latency of its onset.

Modulation of synaptic transmission in hippocampal CA1 neurons by a novel neurotoxin (β-pompilidotoxin) derived from wasp venom

We examined the effects of beta-pompilidotoxin (beta-PMTX), a neurotoxin derived from wasp venom, on synaptic transmission in the mammalian central nervous system (CNS). Using hippocampal slice preparations of rodents, we made both extracellular and intracellular recordings from the CA1 pyramidal neurons in response to stimulation of the Schaffer collateral/commissural fibers. Application of 5-10 microM beta-PMTX enhanced excitatory postsynaptic potentials (EPSPs) but suppressed the fast component of the inhibitory postsynaptic potentials (IPSPs). In the presence of 10 microM bicuculline, beta-PMTX potentiated EPSPs that were composed of both non-NMDA and NMDA receptor-mediated potentials. Potentiation of EPSPs was originated by repetitive firings of the presynaptic axons, causing summation of EPSPs. In the presence of 10 microM CNQX and 50 microM APV, beta-PMTX suppressed GABA(A) receptor-mediated fast IPSPs but retained GABA(B) receptor-mediated slow IPSPs. Our results suggest that beta-PMTX facilitates excitatory synaptic transmission by a presynaptic mechanism and that it causes overexcitation followed by block of the activity of some population of interneurons which regulate the activity of GABA(A) receptors.

Differential effects of novel wasp toxin on rat hippocampal interneurons

We studied the effects of a wasp toxin beta-pompilidotoxin (beta-PMTX) on rat hippocampal CA1 interneurons by the current-clamp technique. The firing patterns of pyramidal neurons and pyramidale interneurons were not affected by beta-PMTX, but in oriens and radiatum interneurons, beta-PMTX converted the action potentials to prolonged depolarizing potentials by slowing the inactivation of Na(+) channels. In lacunosum moleculare interneurons, beta-PMTX induced initial bursting spikes followed by block of succeeding spikes. Comparison of beta-PMTX with a sea anemone toxin, ATX II, revealed that ATX II altered the firing properties of pyramidal neurons and pyramidale interneurons that were unchanged by beta-PMTX. Our results suggest that beta-PMTX modulates Na(+) currents in CA1 interneurons differently in various CA1 neurons and the toxin is useful to classify Na(+) channel subtypes.

EMG activities of neck muscles underlying lateral flexion of the neck during head-turning induced by electrical stimulation of the caudate nucleus in cats

Patterns of EMG activities of neck muscles underlying the initiation of head-turning, induced by stimulation of the caudate nucleus, were analyzed with special reference to temporal relations between the onset of head-turning and that of changes in EMG activities. These patterns were compared with those associated with the initiation of lateral flexion of the neck which occurred without electrical stimulation of the caudate nucleus in order to examine whether the caudate-induced head-turning was initiated via the same muscular system as that used in non-caudate-induced head movements. Experiments were carried out using 5 awake, unrestrained cats which were trained to stand still with one limb on each of 4 footplates. Trains of stimulating current pulses were applied to several stimulation points in the caudate nucleus while the animal maintained a stable standing posture with its neck extended. Head movements in the horizontal plane and EMGs of 6 neck muscles (splenius, longissimus cervicis, obliquus capitis caudalis, biventer cervicis, complexus and cervical multifidus) were recorded. Patterns of EMG activities of neck muscles around the onset of the caudate-induced head-turning were characterized by an increase in activity of the splenius, the longissimus cervicis and the obliquus capitis caudalis muscles, and by a decrease in activity of the complexus, the biventer cervicis and the cervical multifidus on the side of flexion. It is suggested that an increase in activity of the splenius, the longissimus cervicis and the obliques capitis caudalis muscles was responsible for the initiation of this evoked response. In non-caudate-induced lateral flexion of the neck, patterns of activities of neck muscles were similar to those in caudate-induced head-turning. It is therefore concluded that the caudate-induced head-turning as an evoked behavioral response was initiated through a muscular system similar to that utilized for similar head movements occurring without electrical stimulation of the caudate nucleus, although the pathways involved are thought to be different.

Spontaneous excitatory postsynaptic currents in hippocampal CA1 pyramidal neurons of the gerbil after transient ischemia

The changes in the spontaneous excitatory postsynaptic currents (sEPSCs) after transient cerebral ischemia were studied using whole-cell recording from CA1 pyramidal neurons in the gerbil. In neurons recorded 1-2 days after ischemia, sEPSCs had a slowed time course with the decay time constant fitted by a single exponential and it progressively increased after ischemia. Frequency and amplitude distribution of sEPSCs in ischemic neurons were not significantly different from those in the control neurons. The results support the view that abnormal non-N-methyl-D-aspartic acid currents originate at the degenerated postsynaptic site, unrelated to the presynaptic releasing mechanisms.

Cyclic changes in NMDA receptor activation in hippocampal CA1 neurons after ischemia.

We studied N-methyl-D-aspartate (NMDA) receptor-mediated synaptic potentials in CA1 pyramidal neurons using hippocampal slices of gerbils after transient forebrain ischemia. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and bicuculline, stimulation of Schaffer collateral/commissural fibers induced field excitatory postsynaptic potentials (fEPSP) activated by NMDA receptors. We found that in many slices after ischemia, prolonged low-frequency stimulation (0.1-10 Hz) caused repeated depression and potentiation of the NMDA-mediated fEPSP. Changes in fEPSP amplitude were dependent on stimulus frequency and the cycle frequency ranged from 0.08 to 2.5 cycles/min. These cyclic changes were blocked by application of BAPTA-AM, a membrane-permeable Ca2+ chelator, but were little affected by application of verapamil or by lowering the Ca2+ in bathing solution. Intracellular recordings from CA1 neurons revealed that low-frequency stimulation caused periodic depolarizations of membrane potential accompanied by depression of the excitatory postsynaptic potentials. The cyclic changes of fEPSPs were blocked by inhibitors of protein kinase C (PKC) but were unaffected by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) or myosin light-chain kinase (MLCK). These results suggest that stimulus-dependent NMDA-receptor activation, mediated by PKC, takes place in the postischemic CA1 neurons and that the cyclic change may reflect abnormal intracellular Ca2+ signaling processes leading to neuronal degeneration.