Nobufumi Kawai

Inhibitory control of intracerebellar nuclei by the Purkinje cell axons

SummaryIn anaesthetized cats, synaptic events in cerebellar nuclei neurones were investigated with intracellular microelectrode techniques. These cells were identified by their antidromic activation along their axons and/or by their location in histological sections. In the cells of lateral nucleus IPSPs were induced monosynaptically during stimulation of the overlying hemispheral cortex of the cerebellum. In the cells of nuclei interpositus and fastigii, similar IPSPs were produced from the paravermal and vermal cortices, respectively. The postulate that the Purkinje cells exert an inhibitory action upon their target neurones thus applies not only to Deiters neurones, as previously proposed, but also to cells in the cerebellar nuclei. Stimulation of the cerebellar afferents at the inferior olive, the pontine nucleus and the lateral reticular nucleus produced EPSPs in cerebellar nuclei cells with relatively brief latencies, probably through axon collaterals of these afferents. The EPSPs were followed by IPSPs and slow depolarizations of disinhibitory nature, which, as studied previously in Deiters neurones, might be caused respectively by activation and subsequent depression of Purkinje cells through the cerebellar intracortical mechanisms.

Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.

A novel technique was developed for injecting a time-varying conductance into a neuron, to allow quantitative measurement of the processing of synaptic inputs. In current-clamp recording mode, the membrane potential was sampled continuously and used to calculate and update the level of injected current within 60 microseconds, using a real-time computer, so as to mimic the electrical effect of a given conductance transient. Cellular responses to synthetic conductance transients modelled on the fast (non-N-methyl-D-aspartate) phase of the glutamatergic postsynaptic potential were measured in cultured rat hippocampal neurons.

Increase in bcl-2 oncoprotein and the tolerance to ischemia-induced neuronal death in the gerbil hippocampus

We studied the expression of bcl-2 oncoprotein in the gerbil hippocampus after transient ischemia. Immunostaining using monoclonal antibody raised against bcl-2 oncoprotein revealed intense immunoreactivity in the CA1 area following 2 min of ischemia, which induced tolerance to subsequent ischemia and prevented delayed neuronal death (DND). Following ischemia for 5 min, however, bcl-2 oncoprotein immunoreactivity was decreased, reflecting neuronal death in the CA1 area. However, pretreatment with ischemia of 2 min that prevented DND due to subsequent ischemia for 5 min, showed increased immunoreactivity. On the other hand, following 1 min of ischemia which failed to induce tolerance, no increase in the bcl-2 oncoprotein was observed. The results evidenced that expression of bcl-2 oncoprotein in the CA1 area following brief ischemia is closely related to the acquisition of resistance to DND.

Spider venom contains specific receptor blocker of glutaminergic synapses

Abstract Effects of a toxin separated from a spider (Nephila clavata) venom on glutaminergic synapses were studied using lobster neuromuscular junctions. The spider toxin selectively and irreversibly blocked excitatory postsynaptic potentials without affecting the inhibitory postsynaptic potentials. The toxin had no effect on the nerve terminal spikes nor on the resting membrane conductance. Glutamate potential in the postsynaptic membrane was abolished by the toxin but aspartate-induced depolarization was unaffected.

The origin of cerebellar-induced inhibition of Deiters neurones III. Localization of the inhibitory zone

SummaryBy recording intracellularly from Deiters neurones of cats, there was a survey of those cerebellar areas that, when stimulated, produced inhibitory postsynaptic potentials (IPSPs) monosynaptically in Deiters neurones. The monosynaptic inhibitory area expanded longitudinally mainly along the ipsilateral vermal cortex of the anterior lobe. The ipsilateral cortex of the posterior lobe was also effective in inhibiting Deiters neurones though less prominently than the anterior lobe. The inhibitory fibers could be stimulated in the white matter of the cerebellum, predominantly in the ipsilateral side at rostral regions of nuclei fastigii and interpositus. It was further shown that the monosynaptic inhibition from the anterior and posterior lobes occurs chiefly in the dorsal portion of Deiters nucleus. Since in both the cerebellum and Deiters nucleus the spatial pattern of distribution of the inhibitory fibers conforms to that of the corticovestibular fibers as histologically defined, the experimental findings are in accord with the hypothesis that the cerebellar Purkinje cells are inhibitory in nature.

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

Synaptic action of the fastigiobulbar impulses upon neurones in the medullary reticular formation and vestibular nuclei

SummaryIn anaesthetized cats, the fastigial nucleus of cerebellum was stimulated with electric pulse currents, and the effects thereby induced were investigated by recording intracellularly from cells in the medullary reticular formation, the nucleus of Deiters and the descending vestibular nucleus. The early effect commonly seen in these cells was initiation of excitatory postsynaptic potentials (EPSPs) with monosynaptic latencies from both sides of the fastigial nuclei. These EPSPs appeared to be produced in part by a kind of axon reflex through cerebellar afferent fibres, but a certain portion of them was ascribable to the crossed fastigiobulbar axons, as they were influenced by stimulation of the cerebellar cortex in the manner to be expected from the previous study on cerebellar nuclei. These EPSPs were followed by a sequence of a prolonged disfacilitatory hyperpolarization and a late facilitatory depolarization, which apparently reflected the inhibition and disinhibition, respectively, produced in fastigial neurones via Purkinje cell axons of the corticonuclear projection. Either EPSPs or IPSPs were also induced in both reticular and vestibular neurones through polysynaptic pathways in which the fastigiobulbar projection might have been involved.

Disturbance of Membrane Function Preceding Ischemic Delayed Neuronal Death in the Gerbil Hippocampus

Slice preparations were made from the hippocampus of gerbils after 5 min of ischemia by carotid artery occlusion and the membrane properties of pyramidal neurons were examined. A majority of CA1 neurons lost the capacity for long-term potentiation following tetanic stimulation of the input fibers. CA3 pyramidal neurons, in contrast, preserved responses similar to those in the normal gerbil. Following ischemia, CA1 pyramidal neurons showed increased spontaneous firing that was highly voltage dependent and was blocked by intracellular injection of the Ca2+ chelator, EGTA. Thirty-five percent of CA1 neurons showed an abnormal slow oscillation of the membrane potential after 24 h following ischemia. Intracellular injection of GTPγS or IP3 produced facilitation of the oscillations followed by irreversible depolarization. Our results indicate that ischemia-damaged CA1 neurons suffer from abnormal Ca2+ homeostasis, involving IP3-induced liberation of Ca2+ from internal stores.

Adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: prospects for gene therapy of ischemia-induced neuronal death.

The proto-oncogene bcl-2 is known as an anti-apoptotic gene that confers the ability to block neuronal cell death after transient ischemia. In order to examine whether the bcl-2 gene can be used for protection of ischemic brain injury, we generated adeno-associated virus (AAV) vectors capable of expressing human bcl-2. Replication-defective AAV vectors were found effectively to transfer and express bcl-2 gene in the gerbil hippocampal neurons. Transduction with AAV bcl-2 5 days before forebrain ischemia prevented the DNA fragmentation in the CA1 neurons that is commonly associated with ischemia-induced cell death. Furthermore, the application of AAV bcl-2 as late as 1 h following an ischemic insult also prevented DNA fragmentation in CA1 neurons. These results suggest that the bcl-2 protein has neuroprotective functions that inhibit ischemic cell death and demonstrate the potential of AAV bcl-2 for use in post-ischemic gene therapy in the brain.

Nonstationary fluctuation analysis and direct resolution of single channel currents at postsynaptic sites

In order to measure unitary properties of receptor channels at the postsynaptic site, the noise within the decay phases of inhibitory postsynaptic currents (IPSCs) and of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents (EPSCs) in rat hippocampal neurons was studied by nonstationary fluctuation analysis. Least squares scaling of the mean current was used to circumvent the wide variation in amplitude of postsynaptic currents. The variance of fluctuations around the expected current was analyzed to calculate single channel conductance, and fluctuation kinetics were studied with power spectra. The single channel conductance underlying the IPSC was measured as 14 pS, whereas that underlying the EPSC was 42 pS. Openings of the EPSC channel could also be resolved directly in low-noise whole-cell recordings, allowing verification of the accuracy of the fluctuation analysis. The results are the first measurements of the properties of single postsynaptic channels activated during synaptic currents, and suggest that the technique can be widely applicable in investigations of synaptic mechanism and plasticity.