Hidetoshi Ino

Innervation of the lumbar intervertebral disc by nerve growth factor-dependent neurons related to inflammatory pain.

Study Design. We used anatomic tracers and immunoreactivity in rats to define dorsal root ganglion neuron populations innervating the lumbar discs in physiologic and inflammatory states. Objectives. To investigate the percentages of calcitonin gene-related peptide-immunoreactive (CGRP-ir) and isolectin B4 (IB4)-binding neurons innervating lumbar discs. Summary of Background Data. Small neurons are classified into two types. One contains CGRP and expresses the nerve growth factor receptor. The other binds IB4 and expresses the glial cell line-derived neurotrophic factor receptor. Methods. A neurotracer, Fluoro-Gold, was applied to the L5–L6 disc in rats. Five days later, 50-μL saline (control group: n = 8) or Complete Freund’s adjuvant (inflammatory group: n = 8) was applied to the disc. Seven days after the second operation, T13–L5 dorsal root ganglions were processed for double staining of CGRP and IB4. Results. Of the Fluoro-Gold-labeled neurons, 50.1 ± 4.6% (mean ± SEM) were positive for CGRP and 0.7 ± 0.6% positive for IB4 in the control group, while 65.6 ± 4.7% were positive for CGRP and 1.0 ± 1.0% positive for IB4 in the inflammatory group. The percentage of CGRP-ir neurons was significantly higher than that of IB4-binding neurons in both groups (P < 0.001, each). The percentage of CGRP-ir neurons in the inflammatory group was significantly higher than in the control group (P < 0.05). Conclusions. We found that most small neurons innervating the disc were CGRP-ir. Furthermore, disc inflammation caused an increase in CGRP-ir neurons but not IB4-binding neurons, suggesting that CGRP-ir, nerve growth factor-dependent neurons are more responsible for discogenic pain.

Characterization of Microtubule-Associated Protein 2 from Mouse Brain and Its Localization in the Cerebellar Cortex

Abstract: Microtubule‐associated protein (MAP) 2 was purified from the microtubule fraction of mouse brain by heat treatment and BioGel A‐5m gel nitration. The purified preparation showed a single protein band on sodium dodecyl sul‐fate‐polyacrylamide gel electrophoresis using both a gradient gel (3.75–12.5%) and a low‐percentage gel (5%), a finding indicating that MAP2B was absent under the conditions used. Amino acid analysis revealed that mouse MAP2 was an acidic protein with an isoelectric point (pI 4.5) and amino acid composition similar to close of porcine brain MAP2. Im‐munoblot analysis indicated that the antigens that reacted with MAP2 antiserum were present in large quantities in mouse brain. However, we also found a weak reaction in various tissues other than brain, and the major antigens involved were recognized to be common molecular species with the same molecular mass, 162 and 170 kilodaltons. Using antiserum against mouse brain MAP2, the developmental localization patterns of MAP2 in the mouse cerebellar cortex were studied by immunohistochemistry. MAP2 was mainly localized in the neuronal cells throughout development, with the expression in Purkinje cell dendrites being especially remarkable in the growth of arborization from postnatal day 3 to day 20. At the mature stage, the reaction was strong in the dendritic tree but very weak in the proximal dendrites and cell bodies.

Mode of blockade by MK-801 of N-methyl-D-aspartate-induced increase in intracellular Ca2+ in cultured mouse hippocampal neurons.

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+]i) 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 degrees C, whereas it reduced the subsequent response to NMDA significantly at 37 degrees 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+.

Characterization of binding sites for spider toxin, [3H]NSTX-3, in the rat brain

A group of spider toxins (JSTX, NSTX, argiopin, argiotoxin etc.) share a basic common structure and have been reported to block strongly quisqualate- and kainate-sensitive glutamate responses in vertebrate and invertebrate nervous systems. They are presumed to be potent antagonists of both quisqualate and kainate receptors and may serve as useful tools for characterizing these receptors. We report here the synthesis of tritium-labeled NSTX-3 and the characterization of its binding sites in the rat brain. We found that high- and low-affinity binding sites exist in the cerebellum (Kd = 7.75 and 202 nM, Bmax = 0.37 and 5.54 pmol/mg protein, respectively). Synthetic NSTX analogs strongly inhibited [3H]NSTX-3 binding in the cerebellum (IC50 = 10(-7)-10(-6) M), whereas competitive agonists of glutamate receptors (AMPA, quisqualate, NMDA, kainate, glutamate and aspartate) exhibited weak or no inhibitory effects.

Neuronal cell death followed by the expression of immediate early genes in the rat olfactory system

The expression of IEGs is often observed prior to the neuronal degeneration and a close relation between the induction of apoptosis in neurons and the immediate early genes (IEGs) , such as c-Fos and c-Jun, has been supposed. In the present study, we investigated the expression of IEGs in the rat olfactory system which involves the olfactory bulb and piriform cortex using immunohistochemical technique following intraperitoneal kainate injection. Kainate is a potent excitotoxin that is known to cause the neuronal cell death in the hippocampus. amygdala, piriform cortex, olfactory bulb, etc. Transient expression (2 hr to 12 hr postinjection) of c-Fos and Egr-1 was observed in the granular layer of the olfactory bulb and the piril‘orm cortex, whereas persistent expression (2 hr to 7 days) of FosB, Fral and Fra2 was found in the same portions. Apoptotic cells were also observed in these areas.

Developmental change of the expression of cell-cycle regulatory proteins in embryonal mouse retinae

Cyclin-dependent kinases (CDKs), cyclins and CDK inhibitors (CDKIs) are involved in the regulation of cell-cycle progression. However, developmental change of the expression of these proteins are not fully known in the developing and differentiating tissues. We investigated the alteration of the expression of CDKs, cyclins and CDKIs in the retinae of mouse embryos using in situ hybridization and immunohistochemistry. CDC2, CDK4, cyclin A and cyclin B 1 mRNAs were highly expressed in the outer layer of embryonal mouse retinae, where the active cell proliferation occurred. PCNA was also highly expressed in the outer layer. Immunoreactivity of CDC2, CDK4 and PCNA was also intense in the same portions. On the contrary, the expression of p2 1 Waf 1 is higher in the inner layer of the embryonal mouse retinae.

Relationship between the neuronal cell death and the expression of the cell-cycle regulatory proteins

After cerebellar granule neurons from 1 l-day-old rats were cultured under the high potassium concentration (26 mM) medium for 4 days, cells were changed to the low concentration of potassium (5 mM) medium, the most cells were going to die. This low K’ -induced cell death is thought to be apoptosis because DNA fragmentation was observed and cell death was blocked by Actinomycin D, RNA synthesis inhibitor. We cultured cerebellar neurons under high K’ medium for 4 days. and RNA was obtained from 3 hours after changing to the low K’ medium, then the RNA was subtracted from RNA which was not induced cell death, then we cloned the subtracted RNA. Positive clones were screened by RNase protection assay. We found that one of the clones increased the RNA expression after changing to the low K’ medium 3 hours later. Northern hybridization results said that mRNA of this clone was approximately 4-5 kbp and was expressed especially in neurons.

1227 Neuronal cell death and expression of cell-cycle regulatory factors in the rat hippocampus following the systemic kainate administration

HIDETOSHI INO, TANEMICHI CHIBA The delayed neuronal cell death (apoptosis) is induced in the rat hippocampus, amygdala, etc. by intraperitoneal injection of kainate, a potent excitotoxin. When the apoptosis of hippocampal pyramidal cells was induced with kainate, expression of the cell-cycle regulating protein kinases, CDC2, CDK2 and CDK4, and the activating factor of the DNA polymerase, PCNA were immunohistochemically observed in the hippocampus. A part of the cells expressing these proteins were identified as microglia, which suggests that the proliferation of microglia has simultaneously occurred with the apoptotic cell death of pyramidal cells. In the normal condition, the expression of these cell-cycle related proteins was not observed in the central nervous system except for in the subventricular zone.

1214 Cell cycle regulatory proteins in neurons of the rat adrenal gland

A quantitative analysis of movements of mouse fetuses at a high temporal and spatial resolution has been di t’i‘icult in experiments in viva. This is, at least partly, due to the presence of movements of the mother animal that causes changes of the gross position and orientation of fetuses. The irregular gross movements of fetal body wiously obscure small genuine fetal movements and make their measurements meaningless. The gross movements of fetuses, however, can be minimized in in vitro system, where fetuses are isolated from their mother. In the present study, in vitro transplacental perfusion method of mouse fetuses (Suzue ,1990, 1994) was used for maintaining live mouse fetuses in vitro and a high resolution quantitative analysis of fetal movements was attempted. A new position marking system of fetal body parts (more than 10 points at a time), a recording of Images by a high quality CCD camera and acquisition by a computer of the images at frame-by-frame basis allowed automatic and accurate measurements of movements of multiple body parts. In the present system, gross movements of fetal body could be reduced to less than 0.5% of the total body length. This method, allowing objective descriptions and precise distinction of patterns of fetal movements, therefore, appears to provide excellent opportunity for detailed studies of the motor functions of mammalian fetuses and their development.