Yasuyoshi Arimatsu

Monoclonal antibodies to tyrosine hydroxylase from rat pheochromocytoma PC12h cells with special reference to nerve growth factor-mediated increase of the immunoprecipitable enzymes

Twelve hybridomas, which secrete the monoclonal antibodies to rat pheochromocytoma tyrosine hydroxylase (TH), were obtained. All of these antibodies immunoprecipitated the TH molecules from rat pheochromocytoma PC12h cells, adrenal medulla and brain. Two antibodies, namely PCTH-3 and -7 (both IgG1), directly inhibited the catalytic activity of TH. Another antibody, PCTH-4 (IgG1), bound to the enzyme without inhibition of the catalytic activity. The antibodies PCTH-3 and -4 immunostained the 60 K bands on a nitrocellulose sheet, which were electrotransfered from the gel after the SDS-polyacrylamide gel electrophoresis of PC12h cell and rat adrenal medulla homogenates. The antibody PCTH-4 immunocytochemically labeled specific neurons in the locus ceruleus, hypothalamus and substantia nigra of rat brain. Immunotitration using PCTH-4 antibody revealed that the nerve growth factor-mediated increase of TH activity in PC12h cells is due to the increase of the enzyme molecules.

Protein kinase C and Ca2+/calmodulin-dependent protein kinase II phosphorylate a novel 58-kDa protein in synaptic vesicles

A monoclonal antibody was made using the spleen cells of a mouse immunized with chick synaptic membranes and designated as mAb 1D12. It immunoprecipitated 25% of the omega-conotoxin binding protein but no dihydropyridine binding protein solubilized from chick brain membranes. By immunoblotting, a polypeptide of 58-kDa was identified as the antigen of this antibody in chick, rat, rabbit and guinea pig brain. Immunohistochemical observation indicated the immunoreactivity of mAb 1D12 to be localized in the synaptic regions of central and peripheral neurons. In peripheral organs, there was additional staining in the distal portions of nerve fibers. Immunoelectron microscopy showed immunoreactivity to be located in synaptic vesicle and presynaptic plasma membranes. In the subcellular fractionation of rat brain, 58-kDa protein was recovered in the fractions of synaptic vesicles and plasma membranes but not soluble proteins. This protein could be extracted from membranes by Triton X-100 but treatment with EDTA, acid, base or high salt failed to have such effect. Solubilized 58-kDa protein of rat brain was purified by immunoaffinity chromatography using mAb 1D12. Both protein kinase C and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated purified 58-kDa protein, and maxima of 0.47 and 0.94 mol of phosphates, respectively, were incorporated per mol of 58-kDa protein. 58-kDa protein was not phosphorylated by either cAMP-dependent or cGMP-dependent protein kinase. When present in membranes, it was also phosphorylated by protein kinase C and CaM kinase II. Possible involvement of 58-kDa protein in the protein kinase C and CaM kinase II-mediated regulation of synaptic transmission in central and peripheral neurons is discussed.

Organization and development of corticocortical associative neurons expressing the orphan nuclear receptor Nurr1

The developmental mechanism that contributes to the highly organized axonal connections within the cerebral cortex is not well understood. This is partly due to the lack of molecular markers specifically expressed in corticocortical associative neurons during the period of circuit formation. We have shown previously that latexin, a carboxypeptidase A inhibitor, is expressed in intrahemispheric corticocortical neurons from the second postnatal week in the rat (Arimatsu et al. [1999] Cereb. Cortex 9:569–576). In the present study, we first demonstrate in the adult rat that the orphan nuclear receptor Nurr1 is coexpressed in latexin‐expressing neurons located in layer V, sublayer VIa, and the white matter of the lateral sector of the neocortex, and also in latexin‐negative early born neurons in sublayer VIb of the entire neocortex. Virtually all Nurr1‐expressing neurons exhibit immunoreactivity for phosphate‐activated glutaminase but not for γ‐aminobutyric acid, suggesting that they are glutamatergic‐excitatory neurons. By combining Nurr1 immunohistochemistry and 5‐bromo‐2′‐deoxyuridine‐birthdating, we then show that Nurr1 is expressed in (early born) subplate neurons and (later born) presumptive latexin‐expressing neurons from embryonic day 18 onward. Finally, by combination of Nurr1 immunohistochemistry and retrograde tracing, we show that Nurr1‐expressing neurons, including those in sublayer VIb, contribute predominantly to long‐range intrahemispheric corticocortical projections. These results raise the possibility that Nurr1 plays a role in the establishment and maintenance of normal corticocortical circuitry and function. J. Comp. Neurol. 466:180–196, 2003.

Estrogen treatment enhances survival of cultured fetal rat amygdala neurons in a defined medium

Effects of estradiol on the survival of cultured fetal rat amygdala neurons were estimated to assess a possible organizational action of the sex steroid on the developing amygdala tissue. Dissociated 17-day fetal amygdala cells were cultivated initially in a serum-containing and then in a serum-free defined medium. The survival of the cells in the serum-free medium was highly enhanced when supplemented with estradiol at the concentration of 10 ng/ml. Predominant cell populations of the culture were identified as neuronal cells by the tetanus toxin labeling method. The results support the idea that sex steroids play a role in the brain sexual differentiation by enhancing the neuronal survival in the developing amygdala tissue.

Cogeneration of neurons with a unique molecular phenotype in layers V and VI of widespread lateral neocortical areas in the rat

Monoclonal antibody PC3.1 detects a unique subpopulation of neurons located mainly in layer VI and, to a lesser extent, in layer V within the lateral neocortical areas in the rat. In an attempt to characterize these neurons, we determined the time of their generation in selected neocortical areas by a double-labeling experiment combining quantitative long-survival 3H-thymidine autoradiography and immunohistochemistry for the PC3.1 antigen. We found that the vast majority of PC3.1-positive neurons in both layers V and VI were generated concurrently at embryonic day 15 in all areas examined, demonstrating a strict correlation between the molecular identity of neurons and the time of their generation, irrespective of their final positions along the radial and tangential axes. In contrast, PC3.1- negative neurons, which should represent more diverse phenotypic identities, were generated during a more extended period of cortical development and tended to exhibit radial (inside-to-outside) and tangential (ventral-to-dorsal and rostral-to-caudal) neurogenetic gradients. Our findings indicate that laminar and tangential locations of cortical neurons are not established solely by a combination of mechanisms for the inside-out movement of newly generated neurons in each cortical area and for the broad tangential neurogenetic gradients. The results of this study suggest a distinct way of cortical development in which neurons with a common molecular phenotype are generated concurrently and migrate toward their eventual positions, which are not necessarily located in a single lamina. In addition, our results suggest some kind of tangential heterogeneity in the mechanism involved in neocortical histogenesis, supporting the concept of early regional specification within the neocortex.

INTRACORTICAL REGIONALITY REPRESENTED BY SPECIFIC TRANSCRIPTION FOR A NOVEL PROTEIN, LATEXIN

The monoclonal antibody (mAb) PC3.1 recognizes a subset of neurons distributed in the infragranular layers of the lateral neocortex of the rat. Immunoaffinity chromatography with mAb PC3.1 showed that this antibody specifically binds a peptide epitope on a 29 kDa protein named latexin. To study the molecular details of the protein, we isolated four independent cDNA clones for latexin from cDNA libraries of the rat cerebral cortex and whole brain using the amino acid sequences of latexin fragments. Analysis of these cDNA clones showed that the predicted primary structure of latexin consists of 223 amino acids, and has no strict homology to any sequences so far known. Western and Northern blots demonstrated that the latexin and its mRNA were expressed predominantly in neural tissues with some expression in non‐neural tissues. The gene that encodes latexin in the rat appeared to have homologues in other mammalian species and in the chick. In situ hybridization showed that latexin mRNA is synthesized in a subset of neurons in the lateral but not the dorsal neocortex, and that the distribution profile of these neurons is quite similar to that of neurons expressing latexin. These results indicate that latexin is a novel class of neuronal protein which represents intracortical regionality, and suggest that the regional specification of the neocortex involves selective parcellation of neurons which express a particular gene.

Area- and lamina-specific organization of a neuronal subpopulation defined by expression of latexin in the rat cerebral cortex.

The aim of the present study was to investigate the density, laminar distribution, size, morphology, and neurotransmitter phenotype of rat cortical neurons expressing latexin, an inhibitor of carboxypeptidase A. Immunohistochemical analyses established that latexin-immunoreactive neurons are restricted essentially to the infragranular layers of lateral cortical areas in the rat. The overall density, laminar or sublaminar localization, and cell size distribution of latexin-positive neurons differed substantially across cytoarchitectonic areas within lateral cortex. Numerous latexin-positive neurons had the morphology of modified pyramidal cells especially of layer VI. The vast majority of latexin-positive neurons were glutamate-immunoreactive in the six lateral neocortical areas examined, while neurons immunoreactive for both latexin and GABA were virtually absent. Thus the majority of latexin-positive neurons are likely to be excitatory projection neurons. The area- and lamina-specific distribution of the latexin-expressing subpopulation of glutamate-immunoreactive neurons is a distinctive feature that may contribute to the functional specialization of the lateral cortical areas.

LATEXIN : A MOLECULAR MARKER FOR REGIONAL SPECIFICATION IN THE NEOCORTEX

It largely remains to be elucidated how the mammalian neocortex is regionally specified during development. In an attempt to obtain molecular markers in the neocortex, we have generated a monoclonal antibody PC3.1 which recognizes a subset of neurons located in lateral, but not dorsal, neocortical areas. The antigen is a novel class of protein, named latexin, having a molecular weight of 29,000. Our in vitro studies have revealed that the neocortical regional specification for the production of latexin-positive neurons occurs very early prior to thalamocortical interactions and the completion of neurogenesis, indicating that elements intrinsic to the neocortex play important roles in the neocortical specification. Furthermore, our recent analyses have suggested that this regional specification is attributable, at least in part, to an early restriction of developmental potential in neocortical progenitor cells to become latexin-positive neurons.

Sexual dimorphism in α-bungarotoxin binding capacity in the mouse amygdala

A sex difference in alpha-bungarotoxin binding capacity in the mouse amygdala has been demonstrated by quantitative light microscopic autoradiography. The difference persisted even under widely different steroid-hormonal environment. In addition, it was observed that the binding capacities in both sexes were reversibly activated by administration of either testosterone or estradiol. Neonatal castration, on the other hand, permanently altered the toxin binding capacity in the adult male mouse. These data suggest the possibility that neonatal sex steroids irreversibly modify the cholinergic nicotinic mechanism in the developing mouse amygdala, while the hormones reversibly modulate the mechanism when applied in adulthood.

Latexin, a carboxypeptidase A inhibitor, is expressed in rat peritoneal mast cells and is associated with granular structures distinct from secretory granules and lysosomes.

Latexin, a protein possessing inhibitory activity against rat carboxypeptidase A1 (CPA1) and CPA2, is expressed in a neuronal subset in the cerebral cortex and cells in other neural and non-neural tissues of rat. Although latexin also inhibits mast-cell CPA (MCCPA), the expression of latexin in rat mast cells has not previously been confirmed. In the present study we examined the expression and subcellular localization of latexin in rat peritoneal mast cells. Western blot and reverse-transcriptase-mediated PCR analyses showed that latexin was contained and expressed in the rat peritoneal mast cells. Immunocytochemically, latexin immunofluorescence was localized on granular structures distinct from MCCPA-, histamine- or cathepsin D-immunopositive granules. Immunoelectron microscopy revealed that latexin was associated with a minority population of granules. The latexin-associated granules were separated from MCCPA- or histamine-containing granules on a self-generating density gradient of polyvinylpyrrolidone-coated silica-gel particles (Percoll). Treatments with high ionic strength and heparinase released latexin from the granules, suggesting that latexin is non-covalently associated with a heparin-like component of the granules. MCCPA and histamine were released from the mast cells after non-immunological and immunological stimulation with compound 48/80, A23187 and anti-IgE antibody, whereas latexin was not released. These results show that latexin is synthesized in rat peritoneal mast cells and suggest that it is associated with a unique type of intracellular granules distinct from MCCPA- and histamine-containing secretory granules and lysosomes.