Mineo Matsumoto

A novel neurological mutant mouse, yotari, which exhibits reeler-like phenotype but expresses CR-50 antigen/reelin.

We present yotari, a novel neurological mutant mouse whose mutation is transmitted in an autosomal recessive manner. The phenotype of yotari is very similar to that of reeler. yotari mutants are recognizable by their unstable gait and tremor and by their early deaths at around the time of weaning. The cerebella of homozygous yotari are hypoplastic and have no foliation. A molecular and a granular cell layer can be identified, but Purkinje cells are scattered throughout both the granular layer and white matter. The laminar structure of the cerebral cortex and the hippocampal formation are also distorted. To test whether the mutated gene in yotari is the reeler gene, reelin, yotari heterozygotes were mated with reeler homozygotes or heterozygotes. The absence of abnormal offspring indicated that the yotari gene is distinct from reelin. Furthermore, expression of mRNA and protein of reelin was verified by Northern blotting and immunohistochemistry using a CR-50 monoclonal antibody (mAb) which is specific to Reelin, the reelin gene product. Although the mutation of several genes, including cyclin-dependent kinase 5 (Cdk 5), p35 and LIS1, 45 kDa subunits of platelet-activating factor acetylhydrolase (PAF-AH) Ib, in Miller-Dieker lissencephaly syndrome (MDS) has been reported to cause abnormal laminar structure in the brain, no abnormality was found in yotari by Western blotting with antibodies (Abs) against these molecules. The close similarity of the phenotypes of yotari and reeler and the expression of reelin in yotari may suggest that the gene mutated in yotari encodes a molecule that is on the same signaling pathway as Reelin, the product of reelin. yotari will provide valuable clues to explore the molecular mechanism of neuronal migration and orderly laminar structure formation of the brain.

Motor discoordination in mutant mice heterozygous for the type 1 inositol 1,4,5-trisphosphate receptor.

In the brain, the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a major subtype of receptors for inositol 1,4,5-trisphosphate, which mediates the release of calcium from intracellular stores. The motor function of knockout mice heterozygous for IP3R1 (IP3R1+/-) was assessed. An impairment of motor coordination was observed in IP3R1+/- mice in the rotating rod test. There was no observable difference between genotypes in spontaneous motor activity, grip strength, the hanging test, or walking pattern. These results suggest that IP3R1 plays a substantial role in motor coordination.

Type 1 inositol 1,4,5-trisphosphate receptor knock-out mice: their phenotypes and their meaning in neuroscience and clinical practice

Abstract Cytoplasmic calcium, which acts as a second messenger, is derived not only from outside the cell but also from intracellular stores. A receptor for inositol 1,4,5-trisphosphate (IP3), an intracellular second messenger, is located on these internal calcium stores and functions as a calcium releasing channel. The ”type 1” IP3 receptor (IP3R1) is concentrated predominantly in cerebellar Purkinje cells and is also widely present in other neural and peripheral tissues, but many of its physiological roles in these cells are still unclear. We have previously succeeded in obtaining mice with disruption of this IP3R1 gene, in which brain IP3-induced calcium release was almost completely abolished. They were rarely born alive, indicating that IP3R1 has some functions during embryonic development. Animals exhibited severe neurological symptoms, ataxia and epilepsy, and were shown to be deficient in the cerebellar long-term depression. They give us promising clues regarding the physiological roles of calcium release from internal stores and serve as a model for the relevant human disease states.

Long-term potentiation and long-term depression in hippocampal CA1 neurons of mice lacking the IP3 type 1 receptor

To investigate the role in synaptic plasticity of Ca(2+) released from intracellular Ca(2+) stores, mice lacking the inositol 1,4,5-trisphosphate type 1 receptor were developed and the physiological properties, long-term potentiation, and long-term depression of their hippocampal CA1 neurons were examined. There were no significant differences in basic synaptic functions, such as membrane properties and the input/output relationship, between homozygote mutant and wild-type mice. Enhanced paired-pulse facilitation at interpulse intervals of less than 60 ms and enhanced post-tetanic potentiation were observed in the mutant mice, suggesting that the presynaptic mechanism was altered by the absence of the inositol 1,4,5-trisphosphate type 1 receptor. Long-term potentiation in the field-excitatory postsynaptic potentials induced by tetanus (100 Hz, 1 s) and the excitatory postsynaptic currents induced by paired stimulation in hippocampal CA1 pyramidal neurons under whole-cell clamp conditions were significantly greater in mutant mice than in wild-type mice. Homosynaptic long-term depression of CA1 synaptic responses induced by low-frequency stimulation (1 Hz, 500 pulses) was not significantly different, but heterosynaptic depression of the non-associated pathway induced by tetanus was blocked in the mutant mice. Both long-term potentiation and long-term depression in mutant mice were completely dependent on N-methyl-D-aspartate receptor activity. To rule out the possibility of an effect compensating for the lack of the inositol 1,4,5-trisphosphate type 1 receptor occurring during development, an anti-inositol 1,4,5-trisphosphate type 1 receptor monoclonal antibody that blocks receptor function was diffused into the wild-type cell through a patch pipette, and the effect of acute block of inositol 1,4,5-trisphosphate type 1 receptor on long-term potentiation was examined. Significant enhancement of long-term potentiation was observed compared with after control immunoglobulin G injection, suggesting that developmental redundancy was not responsible for the increase in long-term potentiation amplitude observed in the mutant mouse. The properties of channels that could be involved in long-term potentiation induction were examined using whole-cell recording. N-methyl-D-aspartate currents were significantly larger in mutant mice than in wild-type mice only between holding potentials of -60 and -80 mV. We conclude that inositol 1,4,5-trisphosphate type 1 receptor activity is not essential for the induction of synaptic plasticity in hippocampal CA1 neurons, but appears to negatively regulate long-term potentiation induction by mild modulation of channel activities.

Molecular analysis of structural protein genes of the yamagata-1 strain of defective subacute sclerosing panencephalitis virus. II. Nucleotide sequence of a cDNA corresponding to the P plus M dicistronic mRNA

The nucleotide sequence of a cloned cDNA corresponding to the P+M dicistronic mRNA of a subacute sclerosing panencephalitis (SSPE) virus was determined and compared with data of measles virus (MV). The dicistronic mRNA of the SSPE virus consisted of the 3′ proximal 626 nucleotides of P mRNA, intercistronic trinucleotides, a full length of M mRNA, and 75 poly A nucleotides. The part encoding the P protein had a high homology to MV, except at the noncoding region. The terminating consensus sequence of the P gene and the intercistronic trinucleotides of the SSPE virus were CTAC(A)6 and CCT; in MV they are TTAT(A)6 and CTT, respectively. In the M gene, the starting consensus sequence was exactly the same as MV, but at the 5′ proximal end, one third of this gene was different: The first ATG codon of the MV M gene signaling opening of the reading frame was changed to ACG in the SSPE virus and one long open reading frame started from the third ATG codon. The stop codon (TAG) of the MV M gene was also changed to CAG in the SSPE virus. Thus, the deduced SSPE-virus M protein lacked 50 amino acids at the amino terminal and had 15 extra amino acids at the carboxyl end when compared with the MV M protein.

Brainstem auditory evoked potentials during brainstem ischemia and reperfusion in gerbils

To evaluate the reversibility of neural function in the brainstem following ischemia, we investigated the effect of transient brainstem ischemia on the brainstem auditory evoked potential in gerbils. Brainstem ischemia was produced by bilateral extracranial occlusion of vertebral arteries. Local cerebral blood flow was measured by quantitative autoradiography after 5 min of ischemia and was reduced to less than 3 ml/100 g per min in the pons and lower midbrain, indicating severe and reproducible brainstem ischemia. During brainstem ischemia, brainstem auditory evoked potential waveforms disappeared completely. After a brief ischemic insult (5 min), all four brainstem auditory evoked potential components recovered to normal. After longer ischemic insults (10-30 min), brainstem auditory evoked potential components never recovered to normal. Microtubule-associated protein 2 immunoreactivity revealed differential vulnerability of the acoustic relay nuclei in the brainstem. Neurons in the lateral lemniscus were most vulnerable, followed in order by neurons in the trapezoid body, the superior olive and the cochlear nucleus. We also demonstrated a close relationship between the reversibility of ischemia-induced changes on brainstem auditory evoked potential and ischemic lesions of these relay nuclei. These data may be useful for evaluating the therapeutic window of thrombolytic therapy during acute vertebrobasilar occlusion.

Neurological aspects of mice heterozygous for type 1 inositol 1,4,5-trisphosphate receptor - electroencephalographic study

Seizure susceptibiltty of El mice increases bvith grolvth. Young El mice do not eshibit seizures, but 1OO’i; of’ adult El mice do. We have analyzed in previous experiments paired-pulse responses and frequent!, potentiation in the hlppocampal slices of young (Sw) and adult (10-15~) El mice (Ono et al., Brain Res. 745 ( 1997) 16% 172; Fucta et al., Bram Res. 779 (1998) 324-328, Fueta et al, Brain Res. in press). Hyperexcitability of El mice is classified Into t\vo categories, azeindependent and age-dependent ones: the disinhibition in CA1 and large frequent) potenttatlon m CA3 are age-Independent ones and the paired-pulse facilitation in CA3 and the dtsinhibltlon in the dentate gyrus are age-dependent. They are due to a decrease in GABAergic inhibition. Therefore, we tested the effects of tiagabme, a GABA transporter Inhibitor, expecting an increase in the GABA level in the synaptic cleft. Tlagabine (0.3-3mg/kg, I. p. ) suppressed the epileptic selzurcs of El mice, A perfusion of tiagabine (2OpM) on hippocampal shces suppressed only the age-dependent hypercscitabllic of El mice, Thus, the action of tiagabine IS selective in suppressing the hyperescitability responsible for generating seizures in adult El mice

710 Yotari, a novel neurological mutation of mouse

Kazunori Sango’ , Shoji Yamanaka2, Richard L. Proia3, Shuji Inoue’ Tay-Sachs and Sandhoff diseases are clinically similar neurodegenerative disorders. These two sphingolipidoses are characterized by a heritable absence of &hexosaminidase A resulting in defective GM2 gangiioside degradation. Through disruption of the Hexa and Hexb genes in murine embryonic stem cells, we have established mouse models corresponding to each disease. Unlike the two human disorders, the two mouse models showed very different neurologic phenotypes. Although exhibiting biochemical and pathologic features of the disease, the Tay-Sachs model showed no neurological abnormalities. In contrast, the Sandhoff model showed profound neurologic disturbances with storage in brain that is increased in amount and distribution relative to the Tay-Sachs model. The phenotypic difference between the two mouse models is the result of differences in the ganglioside degradation pathway between mice and humans.

810 Morphological and neurological evaluations in mice lacking type 1 inositol 1,4,5-trisphosphate receptor (I)

809 Electrophysiological analysis of cerebellar Purkinje neuron in mice lacking type I inositol 1,4,5trisphosphate receptor (II). Takafumi Inoue’, Mineo Matsumoto”‘, Toshiyuki Nakagawa’, Eiichiro Nagata’, Kortaro Tanaka’, Maki Yamada’, Hiroyuki Yoneshima’, Atsushi Miyawaki’, Shinichi Sakakibara”4, Yasuo Fukuuchi’, Teiichi Furuichi’, Hideyuki Okano’V4, Tetsuo Noda3, Katsuhiko Mikoshiba’. Dept. of Molecular Neurobiology, Inst. Medical Science, Univ. of Tokyo’, Dept. of Neurology, Keio Univ. Sch. of Med.‘, Dept. of Cell Biology, Cancer Inst.3, Dept. of Molecular Neurobiology, Inst. of Basic Medical Sciences and Center of TARA, Univ. of Tsukuba4 The inositol 1,4,5trisphosphate (IP3) receptor acts as an IP3-gated Ca” release channel in a variety of cell types. Type 1 IP3 receptor (IP3Rl) is the major neuronal member of the IP3R family in the central nervous system, predominantly enriched in cerebellar Purkinje cells. We generated IP3Rl-deficient mice (IP3RL/-) by gene targeting to analyze the physiological function of IP3Rl. There was no abnormality in the haematoxylin-eosin staining pattern in the IP3Rl-/cerebellar cortex and Purkinje cells, though the IP3 binding and Ca2’ release activity of the cerebellum was apparently reduced. Electrophysiological measurements in the cerebellar slice from IP3Rl-/mice revealed that the unique electrophysiological properties of the Purkinje cell (e.g. Na and Ca spike complex, synaptic input from parallel and climbing fibers) were not severely impaired.

809 Electrophysiological analysis of cerebellar Purkinje neuron in mice lacking type I inositol 1,4,5-trisphosphate receptor (II)

809 Electrophysiological analysis of cerebellar Purkinje neuron in mice lacking type I inositol 1,4,5trisphosphate receptor (II). Takafumi Inoue’, Mineo Matsumoto”‘, Toshiyuki Nakagawa’, Eiichiro Nagata’, Kortaro Tanaka’, Maki Yamada’, Hiroyuki Yoneshima’, Atsushi Miyawaki’, Shinichi Sakakibara”4, Yasuo Fukuuchi’, Teiichi Furuichi’, Hideyuki Okano’V4, Tetsuo Noda3, Katsuhiko Mikoshiba’. Dept. of Molecular Neurobiology, Inst. Medical Science, Univ. of Tokyo’, Dept. of Neurology, Keio Univ. Sch. of Med.‘, Dept. of Cell Biology, Cancer Inst.3, Dept. of Molecular Neurobiology, Inst. of Basic Medical Sciences and Center of TARA, Univ. of Tsukuba4 The inositol 1,4,5trisphosphate (IP3) receptor acts as an IP3-gated Ca” release channel in a variety of cell types. Type 1 IP3 receptor (IP3Rl) is the major neuronal member of the IP3R family in the central nervous system, predominantly enriched in cerebellar Purkinje cells. We generated IP3Rl-deficient mice (IP3RL/-) by gene targeting to analyze the physiological function of IP3Rl. There was no abnormality in the haematoxylin-eosin staining pattern in the IP3Rl-/cerebellar cortex and Purkinje cells, though the IP3 binding and Ca2’ release activity of the cerebellum was apparently reduced. Electrophysiological measurements in the cerebellar slice from IP3Rl-/mice revealed that the unique electrophysiological properties of the Purkinje cell (e.g. Na and Ca spike complex, synaptic input from parallel and climbing fibers) were not severely impaired.