Cellular and subcellular localization of necdin in fetal and adult mouse brain.

Necdin is a 325-amino-acid residue protein encoded by a cDNA clone isolated from neurally differentiated embryonal carcinoma cells. Ectopic expression of necdin induces growth arrest of proliferative cells. Necdin binds to major transcription factors E2F1 and p53, suggesting that necdin exerts its functions through the interactions with these cell-cycle-regulating factors. However, information about precise localization of endogenous necdin protein is currently lacking. A rabbit polyclonal antibody was raised against a bacterially expressed recombinant protein of necdin (amino acids 83-325). Immunoblot analysis revealed that necdin protein was expressed almost exclusively in the brain of adult mice. A relative molecular mass of endogenous necdin was estimated at approximately 43,000. In developing mouse brain, necdin was most abundant during fetal and neonatal periods. Necdin was highly enriched in the cytoplasm of hypothalamic neurons in fetal and adult mice. The subcellular fractionation analysis revealed that necdin was concentrated in the cytosol fraction of brain cells. These results suggest that endogenous necdin protein is localized predominantly in the cytoplasm of differentiated neurons and moves into the nucleus under specific conditions.

The postmitotic growth suppressor necdin interacts with a calcium-binding protein (NEFA) in neuronal cytoplasm.

Necdin, a growth suppressor expressed predominantly in postmitotic neurons, interacts with viral oncoproteins and cellular transcription factors E2F1 and p53. In search of other cellular targets of necdin, we screened cDNA libraries from neurally differentiated murine embryonal carcinoma P19 cells and adult rat brain by the yeast two-hybrid assay. We isolated cDNAs encoding partial sequences of mouse NEFA and rat nucleobindin (CALNUC), which are Ca(2+)-binding proteins possessing similar domain structures. Necdin interacted with NEFA via a domain encompassing two EF hand motifs, which had Ca(2+) binding activity as determined by (45)Ca(2+) overlay. NEFA was widely distributed in mouse organs, whereas necdin was expressed predominantly in the brain and skeletal muscle. In mouse brain in vivo, NEFA was localized in neuronal perikarya and dendrites. By immunoelectron microscopy, NEFA was localized to the cisternae of the endoplasmic reticulum and nuclear envelope in brain neurons. NEFA-green fluorescent protein (GFP) fusion protein expressed in neuroblastoma N1E-115 cells was retained in the cytoplasm and partly secreted into the culture medium. Necdin enhanced the cytoplasmic retention of NEFA-GFP and potentiated the effect of NEFA-GFP on caffeine-evoked elevation of cytosolic Ca(2+) levels. Thus, necdin and NEFA might be involved in Ca(2+) homeostasis in neuronal cytoplasm.

Regulation and deregulation of E2F1 in postmitotic neurons differentiated from embryonal carcinoma P19 cells.

Neurons withdraw from the cell cycle immediately after differentiation from their proliferative precursors. E2F1, a principal transcription factor that promotes cell cycle progression, must be silenced in neurons. We investigated the E2F1 system in postmitotic neurons derived from murine embryonal carcinoma P19 cells. P19 cells highly expressed the E2F1 gene during neural differentiation, and enriched neurons contained a high abundance of E2F1 mRNA. In contrast, postmitotic neurons possessed extremely low levels of E2F1 protein as assessed by the electrophoretic mobility shift assay and Western blotting. A recombinant E2F1 fusion protein was ubiquitinated in vitro when incubated with neuronal lysates. In addition, treatment with the proteasome inhibitor MG132 increased the endogenous level of E2F1 protein in neurons. These results suggest that the ubiquitin-proteasome pathway contributes, at least in part, to the downregulation of E2F1 protein in postmitotic neurons. Adenovirus-mediated transfer of E2F1 cDNA into postmitotic neurons induced both bromodeoxyuridine incorporation and chromatin condensation, suggesting that deregulated E2F1 expression causes both aberrant S-phase entry and apoptosis of postmitotic neurons. Thus, downregulation of endogenous E2F1 protein in postmitotic neurons may be indispensable for the prevention of their reentry into the cell cycle.

Activation of neuronal caspase-3 by intracellular accumulation of wild-type Alzheimer amyloid precursor protein.

Forced overexpression of wild-type Alzheimer amyloid precursor protein (APP) causes postmitotic neurons to degenerate. Caspase-3 (CPP32) is a principal cell death protease involved in neuronal apoptosis during physiological development and under pathological conditions. Here, we investigated whether APP overexpression activates caspase-3 in human postmitotic neurons using adenovirus-mediated gene transfer. When a recombinant adenovirus vector expressing human wild-type APP695 was infected in vitro into neurally differentiated embryonal carcinoma NT2 cells, only postmitotic neurons underwent severe degeneration. Before neurodegeneration, full-length APP- and Abeta-immunoreactive peptides were accumulated in infected neurons, and caspase-3-like protease activity was markedly elevated. Western blot analysis revealed that activated caspase-3 subunits were generated in APP-accumulating neurons. Such neuronal caspase-3 activation was undetectable in NT2 neurons infected with beta-galactosidase-expressing adenovirus. Addition of the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde to the culture medium significantly reduced the severity of degeneration exhibited by APP-overexpressing neurons. Immunocytochemical analyses revealed that some APP-accumulating neurons contained activated caspase-3 subunits and exhibited the characteristics of apoptosis, such as chromatin condensation and DNA fragmentation. Activation of caspase-3 was also observed in vivo in rat hippocampal neurons infected with the APP-expressing adenovirus. These results suggest that wild-type APP is an intrinsic activator of caspase-3-mediated death machinery in postmitotic neurons.

Regulation of AP-2-synaptotagmin interaction by inositol high polyphosphates.

The inositol high-polyphosphate series (IHPS) inhibits neurotransmission through binding to the second C2 domain of synaptotagmins I and II(Syt), synaptic vesicle membrane proteins. We have revealed that several proteins, including alpha adaptins which are specific subunits of clathrin assembly protein, AP2, were eluted from mouse brain by affinity elution chromatography from the C2 domains of Syt II-immobilized Sepharose using 50 microM of InsP6. The interaction between Syt II and AP2 was more markedly inhibited by IHPS than by the same concentration of InsP3. Limited digestion of mouse crude synaptosomal fractions with trypsin revealed different cleavage patterns in the presence and absence of 50 microM InsP6. These results suggest that IHPS-binding to the C2B domain of synaptotagmin alters the state of protein-protein interaction including the synaptotagmin-AP2 interaction, possibly resulting in the inhibition of events involved in the synaptic vesicle trafficking.

Roles of synaptotagmin C2 domains in neurotransmitter secretion and inositol high-polyphosphate binding at mammalian cholinergic synapses.

To determine the functional role of synaptotagmin (Syt) regulatory domains, affinity-purified antibodies specific for C2A or C2B domains were injected into presynaptic neurons of cholinergic synapses formed between rat sympathetic neurons in culture. Following injection of anti-C2A antibody, postsynaptic responses evoked by presynaptic action potentials at a frequency of 0.05 Hz decreased rapidly, while anti-C2B antibody slowly decreased synaptic transmitter release. The inhibitory effect of anti-C2B antibody depended on the amount of synaptic activity. Asynchronous release induced by hypertonic solution was also affected by the antibodies. Anti-C2A antibody showed a dual action on miniature excitatory postsynaptic potentials, a decrease and following increase in the frequency, while synapses loaded with anti-C2B antibody showed a decrease in the frequency after long repetitive stimulation (0.05 Hz for more than 60 min). Anti-C2B antibody prevented the inhibition of acetylcholine release induced by injection of inositol 1,3,4,5-tetrakisphosphate (IP4), indicating that C2B domain may down-regulate transmitter release by IP4 binding. These results confirm similar experiments in the glutamatergic squid giant synapses and suggest a model in which Syt C2A and C2B domains differentially control synaptic vesicle trafficking in mammalian cholinergic terminals; C2A domain may act on the fusion step as a calcium sensor in synaptic vesicle exocytosis evoked by action potentials in addition to controlling spontaneous transmitter release, while C2B domain is involved in exo- and endocytosis.

Distinct roles of C2A and C2B domains of synaptotagmin in the regulation of exocytosis in adrenal chromaffin cells.

Synaptotagmin that contains two repeats of C2 regulatory domains is considered to be involved in neurotransmitter release. To reveal the roles of synaptotagmin in the regulation of exocytosis, we examined the effects of antibodies against C2A and C2B domains on Ca2+-evoked catecholamine (CA) release from digitonin-permeabilized adrenal chromaffin cells, resolving the Ca2+-evoked release into ATP-dependent priming and ATP-independent Ca2+-triggered steps. Anti-C2A antibody clearly reduced the ATP-independent release, suggesting that the C2A domain directly facilitate or promote Ca2+-triggered step, vesicular fusion. In contrast, anti-C2B antibody did not affect Ca2+-evoked release by itself, but significantly increased the spontaneous Ca2+-independent release. In addition, inositol high-polyphosphate series (IHPS) that bind the C2B domain inhibited both the ATP-independent Ca2+-evoked release and the spontaneous release in a dose-dependent manner. The inhibition by IHPS was totally reversed by anti-C2B antibody and significantly reversed by high concentration of Ca2+. These results suggest that IHPS binding to C2B domain arrests membrane fusion by presumably preventing interaction of synaptotagmin with phospholipids or with proteins of plasma membrane. Thus, IHPS binding to the C2B domain might keep the docked or primed vesicles away from spontaneous fusion at resting level of intracellular Ca2+. Binding of the increased intracellular Ca2+ to the C2A domain may facilitate or trigger the vesicular fusion by releasing this suppression by IHPS.

Myelin proteolipid protein (PLP), but not DM-20, is an inositol hexakisphosphate-binding protein.

Myelin proteolipid protein (PLP) and its alternatively spliced isoform, DM-20, are the major integral membrane proteins of central nervous system myelin. It is known that PLP and DM-20 are delivered to myelin by a finely regulated vesicular transport system in oligodendrocytes. Evolutionarily, it is believed that ancestral DM-20 acquired a PLP-specific exon to create PLP, after which PLP/DM-20 became a major component of central nervous system myelin. We purified PLP as an inositol 1,3,4,5-tetrakisphosphate-binding protein after solubilization in a non-organic solvent. However, under the isotonic condition, PLP binds inositol hexakisphosphate (InsP6) significantly, not inositol 1,3,4,5-tetrakisphosphate. Most of the InsP6-binding proteins are involved in vesicular transport, suggesting the involvement of PLP in vesicular transport. We separated DM-20 from PLP by CM-52 chromatography and showed that DM-20 has no InsP6 binding activity. These findings indicate that the PLP-specific domain confers the InsP6 binding activity and this interaction may be important for directing PLP transport to central nervous system myelin.

Functional diversity of C2 domains of synaptotagmin family. Mutational analysis of inositol high polyphosphate binding domain.

Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca(2+)-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.

Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

The inositol high-polyphosphate series blocks synaptic transmission by preventing vesicular fusion: a squid giant synapse study.

Presynaptic injection of inositol 1,3,4,5-tetraphosphate, inositol 1,3,4,5,6-pentakisphosphate, or inositol 1,2,3,4,5,6-hexakisphosphate--which we denote here the inositol high-polyphosphate series (IHPS)--is shown to block synaptic transmission when injected into the preterminal of the squid giant synapse. This effect is not produced by injection of inositol 1,4,5-trisphosphate. The synaptic block is characterized by a time course in the order of 15-45 min, depending on the injection site in the preterminal fiber; the fastest block occurs when the injection is made at the terminal release site. Presynaptic voltage clamp during transmitter release demonstrates that IHPS block did not modify the presynaptic inward, calcium current. Analysis of synaptic noise at the postsynaptic axon shows that both the evoked and spontaneous transmitter release are blocked by the IHPS. Tetanic stimulation of the presynaptic fiber at frequencies of 100 Hz indicates that block is accompanied by gradual reduction of the postsynaptic response, demonstrating that the block interferes with vesicular fusion rather than with vesicular docking. These results, in combination with the recently demonstrated observation that the IHPS bind the C2B domain in synaptotagmin [Fukada, M., Aruga, J., Niinobe, M., Aimoto, S. & Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211], suggest that IHPS elements are involved in vesicle fusion and exocytosis. In addition, a scheme is proposed in which synaptotagmin triggers transmitter release directly by promoting the fusion of synaptic vesicles with the presynaptic plasmalemma, in agreement with the very rapid nature of transmitter release in chemical synapses.

Synaptotagmin is an inositol polyphosphate binding protein: isolation and characterization as an Ins 1,3,4,5-P4 binding protein.

We isolated a binding protein for inositol 1,3,4,5-tetrakisphosphate (InsP4) from detergent-solubilized mouse cerebellar membrane fractions by sequential column chromatographies. Partial amino acid sequencing of the purified sample revealed that the protein is essentially identical to rat synaptotagmin II, an integral membrane protein of synaptic vesicles. Immunoprecipitation experiment of [3H]InsP4 binding activity of the purified protein using polyclonal antibody against the C2A domain of rat synaptotagmin II also revealed that mouse synaptotagmin II is the InsP4 binding protein (IP4BP). Scatchard analysis of InsP4 binding to the IP4BP/synaptotagmin indicates a single binding site with a Kd of 30 nM. The present finding that InsP4 binds strongly to synaptotagmin II suggests an important role for inositol polyphosphates in the regulation of neurotransmitter release.

Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II.

IP4BP/Synaptotagmin II is an inositol-1,3,4,5-tetrakisphosphate (IP4) or inositol polyphosphate-binding protein, which is accumulated at nerve terminals. Here we report a novel function of the C2B domain, which was originally thought to be responsible for Ca(2+)-dependent binding to phospholipid membranes. A study of deletion mutants showed that about 30 amino acids of the central region of the C2B domain of mouse IP4BP/synaptotagmin II (315 IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF 346) are essential for inositol polyphosphate binding. This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is also known to be essential for neurotransmitter release (Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165), suggesting that inositol polyphosphate has some effect on neurotransmitter release. Rabphilin 3A, another neuronal protein containing C2 domains, cannot bind IP4, indicating that the IP4 binding property is specific to the C2B domain of synaptotagmin. Phospholipid and IP4 binding experiments clearly indicated that the C2A and C2B domains have different functions. The C2A domain binds phospholipid in a Ca(2+)-dependent manner, but the C2B domain binds inositol polyphosphate and phospholipid irrespective of the presence of Ca2+. Our data suggest that the C2B domain of synaptotogamin is the inositol polyphosphate sensor at the synaptic vesicle and may be involved in synaptic function.

A new gerbil model of hindbrain ischemia by extracranial occlusion of the bilateral vertebral arteries.

A new gerbil model of hindbrain ischemia was induced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Carbon black studies, performed at 5 min after occlusion, revealed that the pons-medulla oblongata, and the cerebellum were quite ischemic in all animals. Cardiovascular changes in mean arterial blood pressure (MABP) and heart rate were recorded until 30 min after occlusion, and revealed that the typical cerebral ischemic response (i.e., abrupt increase in MABP, bradycardia, and apnea) was elicited in all animals (n = 10). Thirty minutes after occlusion, animals (n = 4) were decapitated and immersion-fixed. Brain sections were stained with hematoxylin-eosin (HE) and also immunostained for microtubule-associated protein 2 in order to evaluate ischemic neuronal damage from 30 min of ischemia. By HE staining, ischemic lesions were detected bilaterally in the oculomotor, the trigeminal motor, the lateral vestibular, and the cerebellar interpositus nucleus. In addition, immunostaining revealed ischemic lesions in several other hindbrain areas. In conclusion, we could successfully establish a new gerbil model of hindbrain ischemia. Carbon black perfusion and hemodynamic studies revealed that severe and reproducible hindbrain ischemia was produced. By histopathological examination, we could also clearly demonstrate symmetrical ischemic lesions in several hindbrain areas.

Differential vulnerability in the hindbrain neurons and local cerebral blood flow during bilateral vertebral occlusion in gerbils.

Differential vulnerability in the hindbrain neurons was examined immunohistochemically during hindbrain ischemia in the gerbil. Hindbrain ischemia was produced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Local cerebral blood flow was measured by quantitative autoradiographic technique after 5 min of ischemia and was reduced to less than 5 ml/100 g per min in the cerebellum, the pons, and the medulla, indicating that severe and reproducible hindbrain ischemia was induced immediately after occlusion. For immunohistochemical investigation, four gerbils each were used for each ischemic period of 5, 10, 15, and 30 min. Immunohistochemical lesions, detected by the reaction for microtubule-associated protein 2, were visible in the lateral vestibular nucleus and the cerebellar interpositus nucleus even after 5 min of ischemia. These results suggested that these areas were more vulnerable than others, although blood flow was markedly reduced in various regions of the hindbrain. In contrast, areas related to respiratory or cardiovascular control were rather resistant to ischemia. The present study suggests that selective vulnerability during hindbrain ischemia depends mainly on different metabolic characteristics inherent to various neurons in the hindbrain.

Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture.

In the present study, we cultured rat CNS neurons and tested the neurotrophic support provided by basic fibroblast growth factor (bFGF) to prevent the oxygen-induced neuronal cell death. When rat basal forebrain (septum and vertical limb of diagonal band of Broca) cells of embryonic day 20 were cultured in a serum-free medium containing 5 microM cytosine arabinoside in a 50% oxygen atmosphere, the neuronal cells, which were immunostained by an anti-microtubule-associated protein 2 (MAP2) antibody, gradually died after 1 day in culture. After 3.5 days in culture, only 2-5% of neuronal cells survived. This oxygen-induced cell death of cultured basal forebrain neurons was reversed by the addition of bFGF at a concentration of 100 ng/ml. This cell-saving effect was dose-dependent, and the ED50 value was 12 ng/ml. Nerve growth factor (NGF) and insulin-like growth factor II could not prevent cell death. The activity of choline acetyltransferase was also maintained when bFGF was present in the basal forebrain culture. Viable astroglial cells, which were immunostained by an anti-glial fibrillary acidic protein, accounted for a few percent of the total number of cells after 3 days in culture both with and without 100 ng/ml of bFGF. The survival-enhancing effect of bFGF was observed not only in basal forebrain neurons but also in neocortical and hippocampal neurons. However, the sensitivity to oxygen toxicity of cultured neurons from the 3 CNS regions varied greatly. The neocortical neurons were the most sensitive to oxidative stress, while the hippocampal neurons were the most resistant. These results suggest that bFGF plays an important role in saving neuronal cells from oxidative stress during their long life without division.

Two types of ryanodine receptors in mouse brain: skeletal muscle type exclusively in Purkinje cells and cardiac muscle type in various neurons.

Two types of ryanodine receptors, channels for Ca2+ release from intracellular stores, are known. We detected the skeletal muscle type only in cerebellum by immunoblot analysis of microsomes and partially purified proteins. The cardiac muscle type was found in all parts of the mouse brain. Immunohistochemical study showed that the cardiac muscle type was localized mainly at the somata of most neurons. Analysis of mutant cerebella suggested that the skeletal muscle type was present exclusively in Purkinje cells. These results suggest that Ca(2+)-induced Ca2+ release, probably mediated by the cardiac muscle receptor, functions generally in various neurons, whereas depolarization-induced Ca2+ release, probably mediated by the skeletal muscle receptor, functions specifically in Purkinje cells.

The synapsin I brain distribution in ischemia.

We examined the distribution of synapsin I in the gerbil brain and investigated ischemic damage of presynaptic terminals immunohistochemically by using this protein as a marker protein of synaptic vesicles. The reaction for synapsin I in normal gerbil brain is exclusively localized in the neuropil, and other brain structures such as neuronal soma, dendrites, axon bundles, glia and endothelial cells exhibited little immunoreactivity. In a reproducible gerbil model of unilateral cerebral ischemia, ischemic loss of synapsin I immunoreactivity in the affected hemisphere was confined to the area exhibiting overt infarction, where the breakdown of this protein was also confirmed by the immunoblot analysis, and noted much later than that of microtubule-associated protein 2 immunoreactivity, which was demonstrated in neuronal soma and dendrites. In the non-affected hemisphere, selective damage of presynaptic terminals due to Wallerian degeneration and subsequently occurring resynaptogenesis at the molecular layer of the dentate gyrus were clearly demonstrated as a loss and recovery of immunoreaction for synapsin I, respectively. In a gerbil model of bilateral cerebral ischemia, immunoreaction for synapsin I was persistently preserved after seven days to two months recirculation following a brief period of global forebrain ischemia in the CA1 region of the hippocampus, where delayed neuronal death was consistently observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The inositol 1,4,5-trisphosphate receptor.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger that releases Ca2+ from its intracellular stores. The InsP3 receptor has been purified and its cDNA has been cloned. We have found that the InsP3 receptor is identical to P400 protein, first identified as a protein enriched in cerebellar Purkinje cells. We have generated an L-fibroblast cell transfectant that produces cDNA-derived InsP3 receptors. The protein displays high affinity and specificity for InsP3. InsP3 induces greater Ca2+ release from membrane vesicles from transfected cells than from those from control L-fibroblasts. After incorporation of the purified InsP3 receptor into lipid bilayers InsP3-induced Ca2+ currents were demonstrated. These results suggest that the InsP3 receptor is involved in physiological Ca2+ release. Immunogold labelling using monoclonal antibodies against the receptor showed that it is highly concentrated on the smooth-surfaced endoplasmic reticulum and slightly on the outer nuclear membrane and rough endoplasmic reticulum; no labelling of Golgi apparatus, mitochondria and plasmalemma was seen. Cross-linking experiments showed that the receptor forms a homotetramer. The approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila, and this region contains the critical sequences for InsP3 binding. We have investigated the heterogeneity of the InsP3 receptor using the polymerase chain reaction and have found novel subtypes of the mouse InsP3 receptor that are expressed in a tissue-specific and developmentally specific manner.