Published papers

The concentration of cytoplasmic free calcium (Ca2+) increases in various stimulated cells in a wave (Ca2+ wave) and in periodic transients (Ca2+ oscillations). These phenomena are explained by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (IICR) and Ca2+-induced Ca2+ release (CICR) from separate intracellular stores, but decisive evidence is lacking. A monoclonal antibody to the IP3 receptor inhibited both IICR and CICR upon injection of IP3 and Ca2+ into hamster eggs, respectively. The antibody completely blocked sperm-induced Ca2+ waves and Ca2+ oscillations. The results indicate that Ca2 release in fertilized hamster eggs is mediated solely by the IP3 receptor, and Ca2+-sensitized IICR, but not CICR, generates Ca2+ waves and Ca2+ oscillations.

In order to clarify the nature of N-methyl-D-aspartate (NMDA) receptors in cerebellum, where heterogeneity of the NMDA receptor has been suggested, we investigated the action of MK-801 on the NMDA-induced [Ca2+]i rise in cultured cerebellar neurons using video-assisted microfluorometry. MK-801 caused a potent and selective blockade of the NMDA-activated [Ca2+]i elevation. The blockade caused by MK-801 was dependent on the presence of NMDA, i.e., use-dependent. There was no difference in the mode of blockade between immunocytochemically identified Purkinje and non-Purkinje cells, although the relative size of the NMDA-induced [Ca2+]i rise was significantly less in Purkinje cells. These results indicate that the NMDA receptors in cultured cerebellar neurons are coupled with the same channels as those in other brain regions. © 1990.

Microfluorometry with fura-2 was applied to study the action of the anticonvulsant (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) on N-methyl-d-aspartate (NMDA)-induced increase in intracellular Ca2+ concentration ([Ca2+]1) in cultured mouse hippocampal neurons. MK-801 caused a potent and long-lasting blockade of the NMDA-activated [Ca2+]i elevation in a selective manner, not affecting the [Ca2+]i rise induced by quisqualate or kainate. Blockade and recovery from the blockade by MK-801 showed use dependency; the degree of blockade was dependent on the presence of NMDA. The use-dependent onset of antagonism was, however, highly sensitive to the bath temperature. MK-801 applied in the absence of NMDA had no effect on the response to subsequent application of NMDA at 22°C, whereas it reduced the subsequent response to NMDA significantly at 37°C. MK-801 interacted with the receptor-ion channel complex even when Mg2+, which is considered to block the open channel, had already blocked the NMDA-induced [Ca2+]i. The recovery from blockade by MK-801 was not accelerated by the application of 10 mM Mg2+ for 5 min. These results suggest that MK-801 can gain access to its binding site in the absence of NMDA at physiological temperature, and that this binding site is distinct from that for Mg2+. © 1990.

Muscle mitochondrial DNAs from two brothers with mitochondrial myopathy associated with peripheral neuropathy had multiple deletions, most of which started in non-D-loop regions, unlike in an autosomal dominant mitochondrial myopathy (Zeviani, M. et al., Nature 339, 309 (1989)). The non-D-loop regions with deletions were amplified by the polymerase chain reaction and the resulting fragments were subcloned and then sequenced. At least 12 deletions of different lengths in different sites were found. However, all the deletions were flanked by short direct repeats (4-12 base pairs). © 1989.

An in vitro autoradiographic technique has been used to localize [125I]ω-conotoxin GVIA binding sites in the brains of normal and cerebellar mutant mice. In the brains of normal mice, the highest densities of binding sites were observed at glomeruli of the olfactory bulb, cerebral cortex, caudate nucleus-putamen, hippocampus, and the nucleus of the solitary tract. Moderate densities of the silver grains occured on the granular layer of the olfactory bulb, the molecular layer of the dentate gyrus, the molecular layer of the cerebellum, and the cochlear nucleus. No specific binding appeared in the white matter or the deep nucleus of the cerebellum, the corpus callosum, the internal capsule and the external plexiform layer of the olfactory bulb. Autoradiographic studies of the cerebella of Purkinje cell degeneration (pcd) mice showed that the distribution of binding sites on the molecular layer of the cerebellum are not affected by the degeneration of Purkinje cells. However, only background levels of the silver grains occured on the cerebella of agranular weaver mutant mice, suggesting that the receptor for ω-conotoxin GVIA in the cerebellum are predominantly distributed on the parellel fibers of granule cells. © 1989.

The ionic mechanism of the membrane permeability changes caused by l-glutamate in hippocampal neurons prepared from 17- to 19-day-old fetal rat in dispersed cell cultures was studied with the whole-cell variation of the patch electrode voltage-clamp technique. The cultured hippocampal neurons became sensitive to glutamate 7 days after plating, and thereafter the sensitivity gradually increased. The conductance increase caused by glutamate was voltage-sensitive, decreasing with membrane hyperpolarization at potentials more negative than -40 mV. The relative permeability of glutamate-activated channels to alkali metal and alkaline earth cations was estimated by reversal potential measurements. The alkali metal cations, Li+, Na+, K+, Rb+ and Cs+ were permeant to the glutamate channels, and the selectively among them was weak. The alkaline earth cations, Ca2+, Sr2+ and Ba2+ were more permeant than the alkali metals. The permeability ratios of these divalent cations relative to Na+ were 2.4 (Ca2+), 2.4 (Sr2+) and 2.8 (Ba2+), respectively. Mg2+ was much less permeant and the permeability ratio (PMg/PNa) was only 0.1. Anion conductance made no contribution to the glutamate-induced current. Functional implications of the glutamate-induced increase in Ca2+-influx were discussed. © 1988.

L-Homocysteic acid (HCA), an endogenous excitatory amino acid in the mammalian CNS, potently activates N-methyl-D-aspartate (NMDA) receptors in hippocampal neurons. However, the responses to HCA in Purkinje cells, which lack functional NMDA receptors, have been largely unexplored: HCA may activate conventional non-NMDA receptors by its mixed agonistic action on both NMDA and non-NMDA receptors, or it may activate a novel non-NMDA receptor that has high affinity for HCA. To test these possibilities, we compared the responses to HCA in cultured Purkinje cells with those in hippocampal neurons by using the whole cell patch-clamp technique. To clearly isolate HCA responses mediated by non-NMDA receptors, we complemented pharmacological methods by using neurons from mutant mice (NR(-/-)) that lack functional NMDA receptors. A moderate dose of HCA (100 μM) induced substantial responses in Purkinje cells. These responses were blocked by non- NMDA receptor antagonists but were insensitive to NMDA receptor antagonists. HCA also activated responses mediated by non-NMDA receptors in both wild-type and NR1(-/-) hippocampal neurons. HCA responses in Purkinje cells had a pharmacological profile (EC50 and Hill coefficient) very similar to that of non-NMDA receptor components of HCA responses in hippocampal neurons. Moreover, the amplitude of the non-NMDA receptor component of HCA responses was directly correlated with that of α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA)- and kainate-induced responses in both types of neurons. Finally, in both types of neurons, HCA currents mediated by non- NMDA receptors were potently blocked by the AMPA receptor antagonist GYKI52466. These findings indicate that HCA-activated AMPA receptors in Purkinje cells are similar to those in hippocampal neurons and that there is no distinct HCA-preferring receptor in Purkinje cells. We also found that in hippocampal neurons, the EC50s of HCA for non-NMDA receptors and for NMDA receptors were more similar than originally reported; this finding indicates that HCA is similar to other mixed agonists, such as glutamate. HCA responses may appear to be selective at NMDA receptors in cells that express these receptors, such as hippocampal neurons; in cells that express few functional NMDA receptors, such as Purkinje cells, HCA may appear to be selective at non-NMDA receptors.

Interleukin (IL)-16 is a proinflammatory cytokine that has attracted widespread attention because of its ability to block HIV replication. We describe the identification and characterization of a large neuronal IL-16 precursor, NIL-16. The N-terminal half of NIL-16 constitutes a novel PDZ domain protein sequence, whereas the C terminus is identical with splenocyte- derived mouse pro-IL-16. IL-16 has been characterized only in the immune system, and the identification of NIL-16 marks a previously unsuspected connection between the immune and the nervous systems. NIL-16 is a cytosolic protein that is detected only in neurons of the cerebellum and the hippocampus. The N-terminal portion of NIL-16 interacts selectively with a variety of neuronal ion channels, which is similar to the function of many other PDZ domain proteins that serve as intracellular scaffolding proteins. Among the NIL-16-interacting proteins is the class C α1 subunit of a mouse brain calcium channel (mbC α1). The C terminus of NIL-16 can be processed by caspase-3, resulting in the release of secreted IL-16. Furthermore, in cultured cerebellar granule neurons undergoing apoptosis, NIL-16 proteolysis parallels caspase-3 activation. Cerebellar granule neurons express the IL-16 receptor CD4. Exposure of these cells to IL-16 induces expression of the immediate-early gene, c-fos, via a signaling pathway that involves tyrosine phosphorylation. This suggests that IL-16 provides an autocrine function in the brain. Therefore, we hypothesize that NIL-16 is a dual function protein in the nervous system that serves as a secreted signaling molecule as well as a scaffolding protein.

Glutamate and aspartate are endogenous excitatory amino acid neurotransmitters widely distributed in the mammalian central nervous system. Aspartate was shown to induce a large membrane current sensitive to N- methyI-D-aspartate (NMDA) and non-NMDA receptor antagonists in Purkinje cells from mice lacking functional NMDA receptors (NR1(-/-)). This response was accompanied by high permeability to calcium. In contrast, no current was induced by aspartate in hippocampal neurons and cerebellar granule cells from NR1-/- mice. Several other glutamate receptor agonists failed to evoke this response. Thus, in Purkinje cells, aspartate activates a distinct response capable of contributing to synaptic plasticity through calcium permeability.

Conflicting evidence exists concerning the activity of NMDA receptors (NMDARs) in cerebellar Purkinje cells and their possible functions. To investigate the activity of NMDARs, we used whole-cell recording on immunocytochemically identified Purkinje cells in primary culture. In addition, we used mice with a disrupted NMDAR1 gene that lack functional NMDARs (NR1-/-) to assess the physiological role of NMDARs. In cultures from normal mice, NMDA-mediated currents were detected in all identified Purkinje cells at 4 d in vitro (div). After 14 d, however, NMDA responses were reduced in amplitude, whereas the responses to kainate and glutamate increased steadily in amplitude. In addition, the NMDA-induced current displayed a pronounced desensitization at these later stages; peak current declined to zero during steady application of NMDA. At 7 div, the number of surviving Purkinje cells was less in cultures treated with NMDA antagonists, and their survival was dose-dependent. Purkinje cell survival was correspondingly poorer in cultures from the NR1-/- mice than in wild-type controls, suggesting that NMDAR activity enhances the survival of Purkinje cells in vitro. The addition of moderate doses of NMDA promoted the survival of wild- type Purkinje cells in the presence of tetrodotoxin. Feeder layers of cerebellar granule cells derived from wild-type or NR1-/- mice promoted survival of Purkinje cells to a similar degree, suggesting that the NMDAR in Purkinje cells, but not in other cells, is directly involved in Purkinje cell viability. The results demonstrate that NMDARs transiently produce membrane current in Purkinje cells and may serve as one of the epigenetic factors that support the survival of Purkinje cells in vitro.

In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR I allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1-/- mice, demonstrating that there is an interdependence of subunit expression. NR1-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death. © 1994.

Long-term desensitization of AMPA receptors (LTDA) is a core mechanism of long-term depression, a model of motor learning in the cerebellum. In this study we investigated the expression of neurotrophic factor genes after induction of LTDA in cultured cerebellar slices. LTDA was induced by application of quisqualate and monitored as a population response with a wedge recording technique. The levels of mRNA were quantified by reverse transcription followed by polymerase chain reaction. Quisqualate, at a dose and duration that reliably induced LTDA, elicited a significant and specific increase in BDNF mRNA with a peak at four hours after the application. By cell fractionation, the major source of BDNF mRNA increase was found to be in granule cells. In addition, a small but significant increase of transcripts with specific exon usage was observed in a Purkinje cell fraction. These results indicate that BDNF may be coinduced with LTDA and suggest that the slow and sustained increase of BDNF mRNA might play a role in later phases of synaptic plasticity in the cerebellum.

As a first step toward identifying the factor(s) that is/are produced by astrocytes and support(s) the survival of cerebellar Purkinje cells in dissociated culture, we compared the effect of astrocytes of cerebellar, hippocampal, and cerebral origin. A feeder coverslip of cerebellar astrocytes, which did not have cell-to-cell contact to neuronal culture, increased the percentage of Purkinje cells to about 8-9 fold with no change in the percentage of astrocytes. On the other hand, astrocytes of hippocampal or cerebral origin did not increase the percentage of Purkinje cells at low plating density, whereas they increased the number of astrocytes in neuronal culture. These results indicate that the factor(s), tentatively named as Purkinje-cell suvival factor was/were specifically produced by cerebellar astrocytes and affected directly on Purkinje cells. © 1993 Academic Press. All rights reserved.

Metabotropic glutamate receptor (mGluR) is highly expressed in cerebellar Purkinje cells. The purpose of this study was pharmacological and immunocytochemical characterization of the mGluR in single cerebellar neurons, especially Purkinje cells. Ca2+ imaging with fura-2 in cultured cerebellar neurons, identified immunocytochemically, was used to record the direct effects of drugs in stable conditions. In addition, the expression of mGluR was examined, and expression of the intracellular receptor for inositol trisphosphate (IP3) produced by mGluR activation was studied immunocytochemically with specific antibodies. Purkinje neurons and some other neurons showed Ca2+-mobilizing responses to mGluR agonists. These responses were mediated by mGluR because they were not blocked by ionotropic GluR antagonists, were independent of the caffeine-sensitive Ca2+ pool, and were blocked by inhibitors of IP3-induced Ca2+ release. This is the first pharmacological characterization of mGluR at single Purkinje cells. The results differed as follows from those in earlier studies in which phosphoinositide turnover of the entire population of cerebellar cells was monitored: (1) the mGluR responses were not blocked by pertussis toxin or D,L-2-amino-3-phosphonopropionic acid; (2) glutamate was a potent agonist, whereas L-aspartate was ineffective; and (3) the dose-response relationship showed an all-or-none tendency. The metabotropic response of Purkinje cells changed markedly during development, with a sharp peak after day 4 of culture, whereas mGluR and IP3 receptor proteins increased steadily during maturation. This apparent desensitization of mGluR was not blocked by inhibitors of protein kinase C (PKC) or ADP-ribosyltransferase. The metabotropic responses were mainly localized to the center of the somata of Purkinje cells even on day 4, whereas both receptor proteins were expressed throughout the cell. These results suggest that the function of mGluR is spatially and developmentally controlled by a posttranslational mechanism involving a mechanism other than phosphorylation by PKC or ADP-ribosylation.

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca2+-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools. © 1992.