Quantitative analysis of the effects of lithium on the reverse tolerance and the c-Fos expression induced by methamphetamine in mice.

To elucidate the mechanism of psychostimulant-induced reverse tolerance [A. Kifune, S. Tadokoro, Modification of stereotype producing and ambulation-increasing effects following repeated administration of methamphetamine in rats, Jpn. J. Psychopharmacol. 11 (1991) 207-214 [11]; N.J. Leith, R. Kuczenski, Chronic amphetamine: tolerance and reverse tolerance reflect different behavioral actions of the dog, Pharmacol. Biochem. Behav. 15 (1981) 399-405 [13]; S. Tadokoro, H. Kuribara, Reverse tolerance to the ambulation-increasing effect of methamphetamine in mice as an animal model of amphetamine-psychosis, Psychopharmacol, Bull. 22 (1986) 757-762 [18]; S. Tadokoro, H. Kuribara, Modification of the behavioral effects of drugs after repeated administration: special reference to the reverse tolerance, Folia Pharmacologica Japonica 95 (1990) 229-238 [19]], the effects of lithium on ambulatory activity [P. Cappeliez, E. Moore, Effects of lithium on an amphetamine animal model of bipolar disorder, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 14 (1990) 347-358 [1]; M. Hirabayashi, M.K. Alam, Enhancing effect of methamphetamine on ambulatory activity produced by repeated administration on mice, Pharmacol. Biochem. Behav. 15 (1981) 925-932 [7]; M. Hirabayashi, S. Okada, S. Tadokoro, Comparison of sensitization to ambulation-increasing effects of cocaine and methamphetamine after repeated administration in mice, J. Pharm. Pharmacol. 43 (1991) 827-830 [8]; T. Miyauchi, K. Kikuchi, S. Satoh, Further studies on the potentiating effect of lithium chloride on methamphetamine-induced stereotypy in mice, Jpn. J. Pharmacol. 31 (1981) 61-68 [14]; H. Ozawa, T. Nozu, H. Aihara, F. Akiyama, M. Sasajima, Pharmacokinetics and general pharmacological actions of lithium salts administered singly or repeatedly, Folia Pharmacologica Japonica 72 (1976) 433-443 [15].] and cerebral c-Fos expression [S. Ceccatelli, M.J. Villar, M. Goldstein, T. Hokfelt, Expression of c-Fos immunoreactivity in transmitter-characterized neurons after stress, Proc. Natl. Acad. Sci. USA 86 (1989) 9569-9573 [2]; L. Giovannelli, P.J. Shiromani, G.F. Jirikoski, F.E. Bloom, Expression of c-fos protein by immunohistochemically identified oxytocin neurons in the rat hypothalamus upon osmotic stimulation, Brain Research 588 (1992) 41-48 [4]; B.T. Hope, H.E. Nye, M.B. Kelz, D.W. Self, M.J. Iadarola, Y. Nakabeppu, R.S. Duman, E.J. Nestler, Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments, Neuron 13 (1994) 1235-1244 [10]; T. Miyauchi, K. Kikuchi, S. Satoh, Further studies on the potentiating effect of lithium chloride on methamphetamine-induced stereotypy in mice, Jpn. J. Pharmacol. 31 (1981) 61-68 [14]; F.R. Sharp, S.M. Sager, K. Hicks, D. Lowenstein, K. Hisanaga, c-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress, J. Neurosci. 11 (1991) 2321-2331 [16].] were investigated in mice injected with methamphetamine (2 mg/kg, s.c., one to five times). The ambulatory activity enhanced by either acute or chronic methamphetamine injection was delayed or diminished by lithium chloride (LiCl) pretreatment [R.G. Fessler, R.D. Sturgeon, S.F. London, H.Y. Meltzer, Effects of lithium on behaviour induced by phencyclidine and amphetamine in rats. Psychopharmacology 78 (1982) 373-376 [3].]. How the Li-sensitive c-Fos expression in the dorsolateral geniculate nucleus and striatum is related to methamphetamine-induced behavioral excitation is unclear. This protocol, in combination with c-Fos expression of mouse cerebral regions, may provide a useful tool for quantitation of ambulatory activity during c-Fos expression.

Effect of growth factor on GTPase-activating protein (Ras GAP) in Chinese hamster ovary cells.

GTPase-activating proteins (GAPs) stimulate the hydrolysis of GTP bound to small G-proteins and regulate the signal transduction pathway. Changes in the expression of p21-Ras p120-GAP induced by growth factor treatment were examined in cultured Chinese hamster ovary (CHO) and human choriocarcinoma (BeWo) cells. Expression of p120-GAP and GAP activity were measured. Fetal bovine serum induced a significant increased level of GAP in CHO cells, but did not increase GAP in BeWo cells. The results suggest that growth factors affect Ras GAP expression in CHO cells, while they do not in other cells such as BeWo cells.

Lithium inhibits the reverse tolerance and the c-Fos expression induced by methamphetamine in mice.

To elucidate the mechanism of psychostimulant-induced reverse tolerance, the effects of lithium on ambulatory activity and cerebral c-Fos protein expression were investigated in mice injected with methamphetamine (2 mg/kg, s.c., 1-5 times). The ambulatory activity enhanced by either acute or chronic methamphetamine injection was delayed or diminished by LiCl pretreatment (170 mg/kg, s.c., 1 h before methamphetamine). The c-Fos expression in the dorsal lateral geniculate nucleus and in the striatum was significantly increased by acute but not chronic injection of methamphetamine, and the increases were significantly suppressed by LiCl pretreatment. Although how the Li-sensitive c-Fos expressions in the dorsolateral geniculate nucleus and striatum are related to methamphetamine-induced behavioral excitation is unclear, these results suggest that lithium at least functionally interferes with the formation of the state of reverse tolerance to methamphetamine in the mouse.

Localization of GTPase-activating protein-(GAP) like immunoreactivity in mouse cerebral regions.

GTPase-activating protein is known to regulate the conversion between ras-GTP and ras-GDP. We studied the basal expression of GTPase-activating protein-like immunoreactivity in mouse cerebral regions using a polyclonal anti-GTPase-activating protein antibody. Cells with GTPase-activating protein-like immunoreactivity were distributed in frontal cortical layers IV and V, and in the parietal cortex, piriform cortex, amygdaloid area, septum, lateral thalamus, and hypothalamus. The GTPase-activating protein-like immunoreactivity was also observed in fiber-like structures in the caudate putamen, stria terminalis, internal capsule, and medial forebrain bundle, and around CA2 pyramidal cells in Ammon's horn. These results imply that GTPase-activating protein is constitutively expressed in mouse brain regions and may have physiological functions in specific neuronal pathways in the brain.

Expression of Ras GTPase-activating protein (GAP) in human normal chorionic villi and hydatidiform mole.

Ras GTPase-activating protein (GAP), an important downregulator of Ras activity, has previously been shown to be abundant in human placenta. The expression of p120 and p100 isoforms of GAP in human normal chorionic villi (n=5) and hydatidiform mole (n=5) was investigated to clarify the involvement of Ras GAP in the growth of chorionic villi in the first trimester of pregnancy. Immunoblot analysis revealed that both p120- and p100-GAP isoforms were remarkably less expressed in mole villi than in normal chorionic villi. The expression of p100-GAP significantly reduced in comparison with that of pl20-GAP in mole villi. Northern blot analysis showed that the amount of GAP mRNA reduced in hydatidiform mole less than one-third of that in normal chorionic villi. The GAP activity, measured by the effect of tissue extract on the hydrolysis of Ras-bound GTP, was significantly lower in hydatidiform mole than in normal chorionic villi. These results suggest that Ras GAP may play an important role in the normal growth and differentiation of human chorionic villi in the first trimester.

c-fos antisense blocks methamphetamine-induced ambulatory activity reversibly.

To investigate the involvement of immediate early genes (IEGs) in the ambulatory activity induced by a low dose of methamphetamine (MAP) in mice, we injected an antisense phosphorothioate oligodeoxynucleotide (ODN) to c-fos mRNA stereotactically into the striatum of ddY male mice. MAP (2 mg kg-1)-induced ambulatory activity was significantly suppressed when the mouse was pretreated by the ODN (2 mM, 1 microliter), and this was accompanied by a decrease in c-Fos-like protein immunoreactivity. Repetitive intermittent administration of MAP at intervals of 3 days confirmed that the suppressive effect was transient and reproducible. These results suggest that c-Fos is at least partly the key molecule regulating the psychomotor stimulation by MAP in mouse brain.

Phencyclidine-induced expression of c-Fos-like immunoreactivity in mouse brain regions.

For the purpose of studying a role of immediate early genes in psychotomimetic-induced behavioral excitation, we experimentally enhanced the locomotor activity of mice by acute administration of phencyclidine and examined the expression and localization of the c-Fos-like and c-Jun-like immunoreactivities in brain regions. A single injection of phencyclidine (5.0 mg/kg, i.p.) significantly increased not only the locomotor activity but also the expression of c-Fos-like immunoreactivity in several brain regions, particularly in the parietal cortex, hippocampal dentate gyrus, piriform cortex and hypothalamus. Interestingly, the c-Fos-like immunoreactivity in the parietal cortex continued to increase for 1 week after the phencyclidine injection. These results indicate that phencyclidine, even injected only once, can induce the persistent expression of c-Fos or c-Fos-related protein(s) in the mouse brain, and also suggest the possibility that such a c-Fos expression may underlie the behavioral and/or psychotomimetic effects of phencyclidine.

[The influence of the sympathetic nervous system on brown adipose tissue: an experimental and histopathologic study on aging].

Brown adipose tissue (BAT) is known to be a principal energy source of non-shivering thermogenesis and related diet-induced thermogenesis. These regulate body temperature and body weight and are controlled by the dissipation of excessive dietary caloric intake. We carried out histopathologic, immunohistochemical and biochemical studies of BAT in rats in relation to aging changes. Four groups of Donryu strain male rats (5 each of 1 month, 2 months, 4 months and 20 months of age) were used. They had been given commercial chow and tap water ad libitum and were kept in an air-conditioned room. Body weight (BW), interscapular BAT weight (IBATW) and g IBATW/g BW of rats were measured. Nor-adrenalin (NA), and dopamine-beta-hydroxylase (DBH) of IBAT were determined. To evaluate the catecholaminergic effects of BAT, morphometric quantitation of BAT was carried out based on the cytoplasmic locularity of fat globules in the BAT cells. Distribution of DBH in BAT was assessed immunohistochemically by the avidin biotin peroxidase complex method. With the use of statistical analysis of variance procedure, there were highly significant decreases in the ratio of g IBATW/g BW (p less than 0.0001) and in the concentrations of NA (p less than 0.0001) and DBH (p less than 0.01) between young (weaning at 1 month old) rats and adult (aged from 4 to 20 months) rats. In the morphometric measurement, by the statistical analysis system (SAS) Spearman correlation coefficient method, there was a significant increase of Type 5 cell (monocular brown adipose tissue cell) in 4 month and 20 month rats, compared to 1 month and 2 month rats (p less than 0.05). Immunohistochemical study of BAT showed localization of DBH in perivascular mesenchymal cells which corresponded with the morphologic distribution of catecholamine as reported by Lever. The results suggest that in the processes of aging in the rat there are reductions in the ratio of g IBATW/g BW, NA and the activity in DBH.

Modulatory action of prostaglandin D2 on the release of 3H-norepinephrine from rat cerebellar slices.

The modulatory action of prostaglandin D2 (PGD2) on the high-K+-induced release of 3H-norepinephrine (3H-NE) from rat cerebellar slices was investigated in relation to the presynaptic feedback mechanisms for the NE release. PGD2 (10(-7)-10(5) M) dose-dependently suppressed the release of 3H-NE. The 3H-NE release was also dose-dependently decreased by 10(-10)-5 X 10(-9) M clonidine and increased by 10(-9)-10(-6) M yohimbine, and there was an antagonism between clonidine and yohimbine, indicating the presence of an alpha 2-adrenoceptor-mediated negative feedback mechanism in the rat cerebellum. The inhibitory action of clonidine was not additive to that of PGD2, while there appeared to be an additiveness between the effects of PGD2 and yohimbine. The 3H-NE release was increased by I-isoproterenol and decreased by I-propranolol, but only at concentrations higher than 10(-6) M. PGD2 nearly abolished the actions of these beta-adrenergic agents, and the 3H-NE release remained at a level similar to that induced by 10(-5) M PGD2 alone. Based on these results, it was tentatively suggested that PGD2 inhibits the 3H-NE release by a mechanism independent of adrenoceptor-mediated feedback mechanisms.

Inhibitory effect on 3H-diazepam binding and potentiating action on GABA of ethyl loflazepate, a new minor tranquilizer.

A new benzodiazepine compound, ethyl loflazepate (ethyl-7-chloro-2,3-dihydro-5-(2-fluorophenyl)-2-oxo-1H,1,4- benzodiazepine-3-carboxylate; CM6912) was studied using in vitro experimental systems for its displacement activity on 3H-diazepam binding to the synaptosomal membrane fraction of rat cerebrum and potentiating action on GABA. CM6912 inhibited the specific binding of 3H-diazepam by 25%, 75% and 90% at concentrations of 0.01 microM, 0.1 microM and 1 microM, respectively, while its metabolites CM6913 and CM7116, at 0.1 microM, completely inhibited the binding. Concentrations for 50% inhibition (IC50) were 25 nM for CM6912, 3.2 nM for CM6913 and 1.4 nM for CM7116. These results suggest that the metabolite CM7116 is stronger than its parent compound in displacing the 3H-diazepam binding, and they also suggest that the long-lasting anti-anxietic action of CM6912 might be due to the in vivo formation of CM7116. CM6912, CM7116 and diazepam potentiated the suppressive action of GABA on spontaneous spikes of Purkinje cells in guinea pig cerebellar slices in a dose-dependent manner. Concentrations for 50% suppression (IC50) were 96.0 microM for GABA alone, 75.0 microM for GABA plus diazepam (5 microM), 78.9 microM for GABA plus CM6912 (5 microM) and 60.8 microM for GABA plus CM7116 (5 microM). These findings suggest that CM6912 and CM7116 may potentiate the postsynaptic inhibitory action of GABA in a manner similar to and probably more strongly than diazepam.

Studies on the muscle relaxation effects of ethyl loflazepate (CM6912) and evaluation as an anti-anxiety drug.

A new anti-anxiety drug, CM6912 (ethyl loflazepate, ethyl 7-chloro-2,3-dihydro-5-(2-fluorophenyl)-2-oxo-1H-1,4-benzodiazepine-3- carboxylate), was investigated for its effects on the alpha- and gamma-motor systems and on the cooperative muscular motions and for its interactions with other CNS drugs. The results obtained are as follows: Muscular discharges (EMG) induced by decerebrate rigidity were unaffected by 10 mg/kg (p.o.) of CM6912, but the amplitudes of the EMG were reduced by 50% for 3 hr by 30 mg/kg of CM6912 at 30 min after administration. Diazepam (10 mg/kg) also decreased the amplitudes of the EMG even at 5 min after administration, indicating that diazepam had a stronger than CM6912. Both monosynaptic spinal reflex (MSR) and polysynaptic spinal reflex (PSR) were unaffected by CM6912 (100 mg/kg). Dorsal root reflex potential was slightly enhanced by CM6912 (100 mg/kg), but not at a dose of 30 mg/kg. Diazepam (10 mg/kg) did not decrease MSR, but slightly reduced PSR. Dorsal root reflex potential was almost doubled by diazepam. The frequency of spontaneous discharges of Gla spindle afferent fiber of the extensor muscle of the hindlimb of anesthetized cats was unchanged by 10 mg/kg CM6912, but was suppressed by diazepam at the same dose while at a dose of 30 mg/kg, it was reduced mildly by CM6912, and markedly by diazepam. ED50 values for the antagonistic action on bemegride-induced convulsions were 0.30 mg/kg for CM6912 and 0.49 mg/kg for diazepam at 1 hr after administration, and they were 0.30 mg/kg and 0.67 mg/kg for CM6912 and diazepam, respectively, at 4 hr. The potentiating action of CM6912 on chlorprothixene-induced anesthesia was far weaker than that of diazepam. The suppressive potency of CM6912 on the adaptability to the rotarod was about half that of diazepam, and the muscle relaxant action of CM6912, examined by the inclined board test and the hanging test, was found to be similar to that of diazepam. These results suggest that CM6912 is less potent than diazepam in reducing muscular tone and in inducing sleep, while it has a stronger and longer-lasting anti-anxietic activity than diazepam.

Modulatory action of taurine on the release of GABA in cerebellar slices of the guinea pig.

For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [3H] gamma-aminobutyric acid ([3H]GABA) and L-[3H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [3H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of L-[3H]glutamate was little affected by taurine. The release of [3H]GABA, was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [3H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [3H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca2+ into cerebellar tissues was tentatively suggested.