Toshikatsu Nakashima

Nicotine-induced sensitization to ambulatory stimulant effect produced by daily administration into the ventral tegmental area and the nucleus accumbens in rats

Bilateral injections of nicotine (30 micrograms/side) into the ventral tegmental area (VTA) and the nucleus accumbens (NACC) increased the ambulatory activity in rats. Moreover, daily injections of nicotine (10, 20 and 30 micrograms/side) into the VTA and the NACC for 6 successive days produced sensitization to the ambulatory stimulant effect of nicotine. Sensitization produced by daily injections of nicotine (20 micrograms/side) into both the sites was maintained for withdrawal periods of 10 days. Mecamylamine (2 mg/kg, i.p.), SCH23390 (0.05 mg/kg, i.p.) and spiperone (0.1 mg/kg, i.p.) antagonized nicotine-induced sensitization to the ambulatory stimulant nicotine-induced sensitization to the ambulatory stimulant effect produced by daily injections into the VTA. These results suggest that nicotine-induced sensitization to the ambulatory stimulant effect involves the stimulation of the mesolimbic dopaminergic pathway through the nicotinic acetylcholine receptor (nAChR) in the VTA and the NACC.

Cyclooxygenase-2 expression in Schwann cells and macrophages in the sciatic nerve after single spinal nerve injury in rats

Recent evidence suggested that cyclooxygenase-2 (COX-2) expression in the peripheral nerve early after nerve injury might be involved in the development of neuropathic pain. Although previous investigators have demonstrated that COX-2 is expressed in peripheral nerve at a late phase (2-4 weeks) after nerve injury, COX-2 up-regulation at an early phase after nerve injury has not been determined. Using immunohistochemistry, we observed biphasic increases of COX-2 expression after L5 single spinal nerve injury. The first increment of COX-2 positive cells was noted 1 day after nerve injury and these cells co-expressed the Schwann cell marker S-100. A second increment was noted after 7-14 days and these cells co-expressed the macrophage marker ED-1. These results suggested that the cellular sources of COX-2 expression might be different between the early and late phases after nerve injury.

Methamphetamine-induced neurotoxicity in BALB/c, DBA/2N and C57BL/6N mice

Repeated administration of methamphetamine (METH; 2 and 4 mg/kg, s.c. four times every 2 h) caused hyperthermia and a dose-dependent depletion of striatal dopamine levels 3 days after the METH-treatment in both BALB/cAnNCrj (BALB) and DBA/2NCrj (DBA) mice, but these responses were lower in C57BL/6NCrj (C57BL) mice. An acute decrease of striatal dopamine levels 30 min after the last injection of METH (4 mg/kg) was observed in both BALB and DBA mice, while an increase in dopamine was observed in C57BL mice. Striatal 3-methoxytyramine levels were drastically increased in both DBA and C57BL mice after this same treatment. Moreover, pretreatment with the superoxide dismutase inhibitor, diethyldithiocarbamate (200 mg/kg, i.p.) exacerbated the METH (4 mg/kg)-induced striatal dopamine-depletion in BALB mice. In addition, pretreatment with an inhibitor of poly(ADP-ribose) polymerase, benzamide (160 mg/kg, s.c.), significantly attenuated the METH (4 mg/kg)-induced striatal dopamine depletion in both BALB and DBA mice. These results suggest that both BALB and DBA mice possess a higher sensitivity to the METH-induced striatal dopaminergic neurotoxicity compared to C57BL mice. In addition, the striatal dopaminergic neurons of BALB mice may be more vulnerable to METH-induced oxidative stress as compared to that in C57BL mice.

Immunocytochemical localization of nicotinic acetylcholine receptor in rat cerebral cortex.

Localization of nicotinic acetylcholine receptor (nAChR) alpha 4 subunits was investigated in rat cerebral cortex using a monoclonal antibody against alpha 4 subunits. The antibody depleted more than 70% of the [3H]methylcarbamylcholine choline binding activity of the solubilized membrane fraction. By light microscopy alpha 4-like immunoreactivity (alpha 4-LI) was found through layers II to VI. The immunostaining was the most prominent in cell bodies and apical dendrites of pyramidal cells in layer V. By electron microscopy most immunoreaction products were observed in the rough endoplasmic reticulum, cytoplasmic matrix and synaptic membranes. Alpha 4-LI was detected in the postsynaptic membranes of neuronal cell bodies and apical dendrites. These findings suggest that alpha 4-containing subtypes serve as one possibly the postsynaptic nAChR in rat cerebral cortex.

The presence of corticotropin-releasing factor-like immunoreactive synaptic vesicles in axon terminals with nicotinic acetylcholine receptor-like immunoreactivity in the median eminence of the rat

Whether or not corticotropin-releasing factor (CRF) containing synaptic vesicles are located in axon terminals with nicotinic acetylcholine receptor (nAChR) in the median eminence (ME) of the rat was examined by electron microscopic double-labeling immunocytochemistry combining the pre-embedding avidin-biotin-peroxidase complex (ABC) method for nAChR with the post-embedding immunogold staining method for CRF. nAChR-like immunoreactivity (nAChR-LI) was found in the cell membranes of the axon terminals in the ME. CRF-like immunoreactivity (CRF-LI) was found in dense granular vesicles (about 100 nm in diameter) in the axon terminals. Double-labeling method revealed that some of nAChR-LI axon terminals were found to contain CRF-LI dense granular vesicles. The results indicate that nicotine may act on nAChR in axon terminals to release CRF.

Methamphetamine-induced striatal dopamine neurotoxicity and cyclooxygenase-2 protein expression in BALB/c mice

The expression of cyclooxygenase-2 (COX-2) and striatal dopamine (DA) depletion in BALB/cAnNcrj (BALB/c) mice following a neurotoxic dose of methamphetamine (METH) was investigated. METH-treatment (4 mg/kg x 4, 2 h intervals, s.c.) induced a significant hyperthermia and a persistent depletion of striatal DA levels 72 h after the treatment. COX-2, a marker of the cytotoxic effect of inflammation and oxidative stress and thiobarbituric acid (TBA) were significantly induced in the striatum 72 h after the METH-treatment, but not in the hippocampus. These results suggest that COX-2 may participate in METH-induced neurotoxicity in striatum.

Methamphetamine-induced striatal dopamine release, behavior changes and neurotoxicity in BALB/c mice

The behaviors associated with the neurotoxic effects of methamphetamine were evaluated in BALB/c mice. Hyperthermia and behavioral observations were measured 60 min after each subcutaneous injection of methamphetamine (4×4 or 8 mg/kg) or saline, each given 2 h apart. The behavioral observations included stereotyped behaviors, incidence of hemorrhage in breast, salivation and self‐injurious behavior (SIB). Repeated administration of methamphetamine produced these behavioral changes and hyperthermia, but resulted in hypothermia by the final injection (8 mg/kg). In addition, the methamphetamine treatment induced a long‐lasting dopamine depletion of similar magnitude in the 4 and 8 mg/kg‐treated animals. In a time course study striatal monoamine levels were measured 60 min after each injection of these doses. The first and second injections of methamphetamine (8 mg/kg) produced a drastic increase in striatal 3‐methoxytyramine; this failed to occur after the third or fourth injection of the same dose. In contrast, 4 mg/kg of methamphetamine also produced an increase in 3‐methoxytyramine after the second and third injections of the drug and, in this case, these were maintained for the duration of the treatment. Striatal 3,4‐dihydroxyphenylacetic acid levels also drastically decreased following both doses of methamphetamine, suggesting inhibition of monoamine oxidase in striatum. Moreover, a single injection of methamphetamine increased striatal 2,3‐dihydroxybenzoic acid formation.

Immunocytochemical localization of nicotinic acetylcholine receptor in rat hypothalamus

Immunocytochemical localization of neuronal nicotinic acetylcholine receptor (nAChR) was examined in rat hypothalamus. Monoclonal antibody against alpha 4 ACh-binding subunits of nAChR was used in the avidin-biotin-peroxidase complex (ABC) immunocytochemical method at both the light and electron microscopic levels. By light microscopy nAChR-like immunoreactivity was found in many neuronal cell bodies and their fibers in the paraventricular nucleus (PVN) and in many axons and axon terminals in the median eminence (ME). The immunoreactivity of nAChR was the most intense in the ME. By electron microscopy immunoreaction products occurred on the rough endoplasmic reticulum, nuclear envelope, cytoplasmic matrices and postsynaptic densities of synaptic junctions in some neurons in the parvocellular part of the PVN. In the external layer of the ME, nAChR-like immunoreactivity was found over the entire plasma membranes of many axon terminals. Involvement of nAChRs in the release of neurotransmitters and neuropeptides both in the PVN and the ME is discussed.

Phosphorylation of rat brain nicotinic acetylcholine receptor by cAMP-dependent protein kinase in vitro

The participation of protein kinases in phosphorylation of nicotinic acetylcholine receptor (nAChR) in electric organ and muscle has been precisely investigated in vitro and in vivo whereas phosphorylation of neuronal nAChR is not yet fully characterized. Here, we first report the in vitro phosphorylation of brain nAChR. nAChR purified from rat brains was phosphorylated in vitro by cAMP-dependent protein kinase (PKA), immunoprecipitated with monoclonal antibody against the receptor, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. PKA specifically phosphorylated nAChR on the alpha 4 subunits, and H8, an inhibitor of PKA, inhibited completely the phosphorylation. Under the conditions used, a maximal stoichiometry of the phosphorylation by PKA was near to 1 mol of phosphate/mol of the alpha 4 subunits. The 32P-labeled subunits were digested with S. aureas V8 protease followed by SDS-PAGE autoradiography and the resultant phosphopeptide maps revealed three distinct phosphopeptide bands, one major band and two minor bands. Phosphoamino acid analysis of the 32P-labeled alpha 4 subunits showed that serine residues were exclusively phosphorylated. Based on these results, participation of PKA in the regulation of neuronal nAChR is discussed.

Occurrence of a new polyamine, canavalmine, in the sword bean Canavalia gladiata

Abstract A new tetraamine was detected in the seed of sword bean Canavalia gladiata and named canavalmine. The chemical structure was determined to be NH2 (CH2) 4NH(CH2) 3NH(CH2) 4NH2 (1,13-diamino-5,9-diazatridecane) based on gas chromatography-mass spectrometry after derivatization of polyamines with pentafluoropropionic anhydride. The proof of identity was established by comparison of infrared and 1H-NMR spectra of the tetraamine isolated from sword bean with those of a synthetic compound.