Cloning of the cDNA encoding neural adhesion molecule F3 from bovine brain.

We cloned a bovine cDNA encoding the neural adhesion molecule F3 and analyzed its nucleotide sequence. The coding region consisted of 3054 bp encoding 1018 amino acid (aa) residues. The M(r) calculated from the deduced aa sequence was 113,383. Bovine F3 had 93, 94 and 77% aa identity with the mouse, human and chicken homologs, respectively. Bovine F3, similar to those of chicken and human, was devoid of two aa residues (Ile-Thr) in the sixth immunoglobulin type C2-like domain, as compared with the mouse homolog. Parts of bovine F3 protein were overproduced in Escherichia coli. The antibodies raised against the recombinant proteins in rabbits reacted specifically with F3. F3 protein was detected in cerebellum, cerebrum and spinal cord in Western blot analysis.

Endothelin-1 increases the levels of mRNA and protein of muscarinic acetylcholine receptors and c-fos mRNA in cerebellar granule cells.

Endothelin-1 (ET-1) induced a time- and dose-dependent increase in the levels of mRNA of m2- and m3-muscarinic acetylcholine receptors (mAChRs) in cultured cerebellar granule cells. The levels of immunoprecipitable m3-mAChR protein and total mAChR binding sites were also increased by ET-1 treatment. The up-regulation of m2- and m3-mAChR was blocked by phorbol ester pretreatment to inhibit ET-1-stimulated phosphoinositide hydrolysis and was preceded by an increase in c-fos mRNA levels. Treatments that prevented ET-1-induced c-fos mRNA increase also abolished the subsequent m2- and m3-mAChR mRNA up-regulation, suggesting that c-Fos protein is involved in the ET-1-induced mAChR expression.

Changes in cholecystokinin mRNA expression in methamphetamine-induced behavioral sensitization.

Neuropeptide cholecystokinin (CCK) mRNA was measured in the rat brain subchronically treated with methamphetamine (MAP). Male Wistar rats were injected daily with MAP (3 or 6 mg/kg, i.p., once a day) or saline for 14 days. Progressive reinforcement was observed in MAP-induced stereotyped behaviors. After 7 or 14 days of discontinuation, the rats were decapitated and the brains were prepared for Northern blot analysis using 32P-labeled cDNA probes. Northern blot analysis revealed that the levels of CCK mRNA in the frontal cortex and the hippocampus of 3 or 6 mg/kg MAP-treated rats were significantly decreased, compared to the saline-treated controls. These findings indicate that the alteration in CCK mRNA levels in the frontal cortex and the hippocampus of MAP-treated rats persisted for at least 2 weeks and might be involved in the expression of MAP-induced long-lasting behavioral sensitization (reverse tolerance).

Internalization and down-regulation of muscarinic acetylcholine receptors in cerebellar granule cells of tenascin-gene deficient mice.

The expression of tenascin-C on oligodendrocytes parallels the migration of granule cells in the developing cerebellum, indicating a role for tenascin-C as a guide for granule neurons to find their proper locations. In this study, cultured cerebellar granule neurons from tenascin-C-knockout mice were used to examine the role of tenascin-C in agonist-induced muscarinic acetylcholine receptor down-regulation. Exposure of granule cells from wild-type or tenascin-C-negative mice to the muscarinic acetylcholine receptor agonist carbachol (1 mM) resulted in normal sequestration of cell-surface muscarinic acetylcholine receptors as assessed by [3H]N-methylscopolamine binding; however, down-regulation of total muscarinic acetylcholine receptors, measured with [3H]quinuclidinyl benzilate, was inhibited in granule cells from tenascin-C-negative mice. Remarkably, incubation of the tenascin-C-negative cells with the microtubule stabilizer taxol (10 microM) restored down-regulation of total muscarinic acetylcholine receptors to normal levels. We speculate that agonist-induced down-regulation of muscarinic acetylcholine receptors is functionally associated with tenascin-C-regulated microtubule structures in the developing cerebellum.

Long-term biphasic effects of lithium treatment on phospholipase C-coupled M3-muscarinic acetylcholine receptors in cultured cerebellar granule cells.

We have studied the long-term effects of lithium on neuronal morphology and the functional expression of phospholipase C-coupled m3-muscarinic acetylcholine receptors (mAChRs) in cerebellar granule cells. There was a biphasic dose-dependent effect on cell morphology following treatment with lithium for 7 days. At low concentrations (< or = 2 mM), this drug elicited an increase in the number and thickness of connecting nerve fibers, and the size of neuronal aggregates. At high concentrations (5-10 mM), lithium induced a severe deterioration of cell morphology, which ultimately resulted in neuronal death. Carbachol-induced phosphoinositide (PI) turnover was similarly affected by lithium treatment with a significant potentiation at concentrations up to 2 mM and a marked inhibition at doses higher than 5 mM due to lithium-induced neurotoxicity. The biphasic effect on mAChR-mediated PI hydrolysis was associated with corresponding changes in the maximal extent of carbachol-induced inositol phosphate accumulation, and was accompanied by similar changes in [3H]N-methyl-scopolamine binding to mAChRs and the levels of mRNAs for m3-mAChR and c-Fos. The up-regulation of m3-mAChR mRNA induced by low concentrations of lithium was associated with a down-regulation of m2-mAChR mRNA and no change in either total RNA or beta-actin mRNA. Lithium's effects on m2- and m3-mAChR mRNAs were time-dependent, requiring a pretreatment time of > or = 3 days. The biphasic effect was also demonstrated by the binding of [3H]ouabain to Na+, K(+)-ATPase, which was shown to be a convenient method for quantifying viable neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional heterogeneity within the nucleus accumbens concerning the effects of dopaminergic agents on the content of cholecystokinin.

In the anterior nucleus accumbens of the rat, the high dose of apomorphine (5 mg/kg i.p.) increased and r-butyrolactone decreased cholecystokinin octapeptide-like immunoreactivity, but the low dose of apomorphine (0.02 mg/kg i.p.) did not induce any change. In contrast, all administrations increased immunoreactivity in the posteromedial nucleus accumbens. These results suggests that the different responses of cholecystokinin may reflect the heterogeneity in neuronal organization within the nucleus accumbens.

Differential expression of c-fos mRNA in rat prefrontal cortex, striatum, N. accumbens and lateral septum after typical and atypical antipsychotics: an in situ hybridization study.

The regional difference in the expression of c-fos mRNA induced by typical and atypical antipsychotics was determined in prefrontal cortex, striatum, N. accumbens and lateral septum in rats by in situ hybridization. Two typical antipsychotics, haloperidol (2 mg/kg) and fluphenazine (2 mg/kg), and three atypical antipsychotics, (-)sulpiride (100 mg/kg), clozapine (20 mg/kg) and OPC-14597 (40 mg/kg), were used. Brains were fixed with 4% paraformaldehyde 45 min after drug administration (i.p.). Brain sections of 30 microns-thickness were made in a cryostat and hybridized with 35S-labelled for c-fos oligonucleotide probe. These sections were apposed to X-ray films and the autoradiograms were semi-quantitatively analysed by computer-assisted densitometry. All antipsychotics used increased c-fos mRNA expression in N. accumbens shell, a region of the forebrain associated with limbic systems. On the other hand, two typical antipsychotics (haloperidol and fluphenazine) that cause a high incidence of acute motor side effects increased the expression of c-fos mRNA in the dorsolateral striatum, an extrapyramidal region primarily involved in motor control. Only clozapine induced c-fos mRNA in the medial prefrontal cortex and lateral septum. These results strongly suggest that the shell region of N. accumbens may be a common site of therapeutic action of antipsychotics.

Neurochemical analysis of the tyrosine hydroxylase expression in methamphetamine-sensitized rats.

To elucidate the expression of TH mRNA in MAP-induced behavioral sensitization, rats were daily injected with MAP (5 mg/kg i.p.) or saline for 14 days. Progressive enhancement was observed in MAP-induced stereotyped behavior. After 7 days of discontinuation of MAP treatment, the rats were decapitated and the brains were prepared for either in situ hybridization or Northern blot hybridization. In situ hybridization revealed that the signals of TH mRNA were localized to the dopaminergic perikarya of substantia nigra and ventral tegmental area in the midbrain, and Northern blot analysis showed that the levels of TH mRNA in these areas decreased by 37% compared to that in the saline-treated controls. These findings indicate that MAP-induced dopaminergic hyperactivity is not associated with enhanced expression of TH gene mRNA.

Effects of cholecystokinin-B receptor antagonist on dopamine system in tenascin mutant mice.

The aim of this study was to investigate the effect of cholecystokinin (CCK) receptor antagonist on the abnormal behavior and dopamine (DA) transmission of tenascin (TN)-gene knockout mice. Recently, we demonstrated that TN-gene deficient mice show hyperlocomotion that is related to reduced DA transmission and tyrosine hydroxylase (TH) activities in the brain. In this report, we show that the intraperitoneal administration of a CCK-B receptor antagonist, PD135158 (0.1 mg/kg), but not a CCK-A receptor antagonist, lorglumide, inhibited hyperlocomotion. Moreover, PD135158 reversed the low levels of DA turnover rate and TH activities in the striatum of TN-gene knockout mouse brain. These results suggest that CCK-B receptor is involved in the behavior of TN-gene knockout mouse through striatal DA transmission.

The reduction of preprotachykinin mRNA in the methamphetamine-induced behavioral sensitization.

To study the role of the preprotachykinin A (PPT) mRNA in methamphetamine (MAP)-induced behavioral sensitization, rats were daily injected within MAP (3 or 6 mg/kg, i.p., once a day) or saline for 14 days. Progressive enhancement was observed in MAP-induced stereotyped behavior. After 7 or 14 days of discontinuation of MAP treatment, the rats were decapitated and the brains were prepared for Northern blot analysis using a 32P-labeled oligonucleotide probe. Northern blot analysis revealed that the level of PPT mRNA in the striatum of 3 or 6 mg/kg MAP-treated rats was significantly decreased, compared to that in the saline-treated controls. These findings indicate that PPT mRNA expression plays an important role in MAP-induced long-lasting behavioral sensitization and dopamine hyperactivity.

Preprotachykinin A and cholecystokinin mRNAs in tenascin-gene knockout mouse brain.

Tenascin (TN), an extracellular matrix glycoprotein, exhibits various functions in the developmental stage of the mammalian brain. TN-gene deficient mice show abnormal behavior such as hyperlocomotion and poor swimming ability, and this abnormal behavior may derive from a low level of dopamine transmission in the striatum or hippocampus of the TN-gene disrupted mouse brain. We assayed preprotachykinin A (PPT) and cholecystokinin (CCK) mRNAs in the terminal fields of the dopamine neuron. The levels of PPT mRNA were significantly higher in the striatum, and the expression of CCK mRNA was markedly augmented in the hippocampus of the TN-knockout mice, compared to the wild or heterozygous mice. One possible explanation of the changes of PPT and CCK mRNA expressions is functional compensation against the low level of dopamine turnover rate in the TN-knockout mouse brain.

Regional effects of apomorphine on rat brain cholecystokinin-8 like immunoreactivity following electrolytic lesions of the ventral tegmental area.

After bilateral electrocoagulation of the ventral tegmental area (VTA) of rats, apomorphine (APO) was administered intraperitoneally to study the effect on the cholecystokinin-8 like immunoreactivity (CCK-8 IR) in the medial prefrontal cortex, the anterior and posterior part of the nucleus accumbens and the corpus striatum. In the medial prefrontal cortex and the posterior part of the nucleus accumbens, CCK-8 IR was markedly decreased by lesioning of the VTA. Then recovery of CCK-8 IR was noted in the APO treated rats following electrolytic lesioning of the VTA. This recovery corresponds to the response of CCK neurons not originating in the VTA or CCK interneurons to APO via DA receptors. In the anterior part of the nucleus accumbens and the corpus striatum, no significant decrease in CCK-8 IR was noted even following lesioning of the VTA. APO administration following the lesioning, however, resulted in a marked increase in CCK-8 IR in the anterior part of the nucleus accumbens and the corpus striatum. According to this result, in these sites, very few CCK neurons originating in the VTA exist and CCK neurons originating in sites other than the VTA or CCK interneurons react on APO via DA receptors.

Reduced mRNA expression of neuropeptide Y in the limbic system of tenascin gene disrupted mouse brain.

Tenascin-C (TN), an extracellular matrix glycoprotein which reveals both neurite outgrowth-promoting and growth-inhibiting effects, is generated in the central nervous system. A previous study reported that TN-gene null mutant mice display hyperlocomotion and do not easily habituate to unfamiliar environments. Additionally, these mice display poor appetite, abnormal circadian rhythm and low pregnancy rate. The present study demonstrated that neuropeptide Y (NPY) mRNA expression is reduced in the limbic area of the TN gene-deficient mouse brain as compared to wild-type mice. NPY has been shown to affect emotion, circadian rhythm and food intake, and the present results suggest that the some behavioural abnormalities exhibited by TN-mutant mice may be in part due to the low level of expression of NPY mRNA in the limbic system.

The effects of central administration of neuropeptide Y on behavior of tenascin-gene knockout mice.

Tenascin-C (TN), an extracellular matrix glycoprotein, plays a pivotal role in the regulation of neuronal pattern formation. TN-gene deficient mice produced by homologous recombination show hyperlocomotive activities. We have recently demonstrated that neuropeptide Y (NPY) mRNA expression is reduced in the limbic area of the TN-gene knockout mouse brain, compared to that in wild-type mice. We now report the effect of NPY on the behavior of TN mutant mice. Intracerebroventricular injection of NPY transiently decreased the hyperlocomotion of TN mutant mice. Concurrently, mice given NPY showed anxiolytic behavioral change as assessed by an exploratory model. The results presented here suggest that the hyperlocomotion of TN mutant mice is partially derived from severe tensity.

Pre-synaptic dopaminergic control mechanisms for CCK-8 like immunoreactivity in the rat medial frontal cortex.

In order to study the control mechanism of cholecystokinin (CCK) contents of the rat brain mediated by pre-synaptic receptors in dopamine (DA) neurons, R(+) and S(-) compounds of 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP), which are selective pre-synaptic dopaminergic agents, were administered in rats at low and high doses. CCK-8-like immunoreactivity (CCK-8 LI) was measured in the medial frontal cortex. In another experiment, a neurotoxin, N-methyl-D-aspartic acid (NMDA) was used to degenerate efferent CCK neurons and CCK interneurons of the medial frontal cortex, followed by an intraperitoneal administration of apomorphine hydrochloride (APO) to study the effect on CCK-8 LI via the pre-synaptic DA receptor. According to the results of these experiments, CCK-8 LI was increased in the medial frontal cortex in response to the stimulation of pre-synaptic DA receptor, suggesting a control of CCK-8 release, at least in part, by the pre-synaptic DA receptor.

Apomorphine affects cholecystokinin content via preferentially D1 or D2 dopamine receptor according to the regions of the rat brain.

Cholecystokinin octapeptide-like immunoreactivity (CCK-8IR) was measured in several regions of the rat brain after the intraperitoneal administration of apomorphine, SKF-38393 (D1 agonist), LY-171555 (D2 agonist). In the medial prefrontal cortex and striatum, apomorphine and SKF 3839 decreased CCK-8IR. In the anterior and posterior nucleus accumbens, on the other hand, the inhibitory effect of apomorphine was mimicked by LY-171555. These results suggest that apomorphine affects CCK-8IR via either the D1 dopamine (DA)-receptor or D2 DA-receptor according to the brain region.

Dopaminergic agents affected neuronal transmission of cholecystokinin in the rat brain.

In order to study the interaction between cholecystokinin (CCK) and dopamine (DA), we prepared an anti-CCK-8 antibody with low cross reactivity, and observed effects of administered various dopaminergic agents on CCK-immunoreactivity (CCK-IR) in discrete brain regions of rats. CCK-8 IR (boiling water extraction) and CCK-33 IR (acetic acid extraction) were also measured in the same sample. A single administration of haloperidol decreased the CCK-8 IR in the corpus striatum and that of racemic sulpiride significantly decreased the CCK-8 IR in the frontal cortex and the limbic system. In contrast, a single administration of apomorphine or methamphetamine increased the CCK-8 IR in the same regions. These findings suggest that an acute response of the CCK system to administered dopaminergic agents may be due to a change in the rate of release of CCK-8, but not to a change in its synthesis in areas in which DA neurons originating in the midbrain innervate. After chronic administration of racemic sulpiride or methaphetamine, CCK-8 IR in various brain regions exhibited a tendency close to that of control.

The chronic administration of dopamine antagonists and methamphetamine changed the [3H]-cholecystokinin-8 binding sites in the rat frontal cortex.

The specific cholecystokinin (CCK) binding in the slide-mounted tissue sections of the rat frontal cortex was measured with [3H]-CCK-8. The binding was saturable, reversible, high in affinity, and inhibited by caerulein. The chronic administration of dopamine (DA) antagonists and methamphetamine (MAP) showed a tendency to increase the [3H]-CCK-8 binding sites (Bmax) in the frontal cortex. Long-term treatments with DA antagonists led to the depletion of CCK-8 and may have caused an observed proliferation of CCK-8 receptors in the frontal cortex.

Upregulation of brain somatostatin and neuropeptide Y following lidocaine-induced kindling in the rat.

Male Sprague-Dawley rats received a daily injection of 60 mg/kg of lidocaine (> 30 days). Twenty percent of rats developed convulsions (kindled rats) and remaining rats did not show convulsions (non-kindled rats). The level of immunoreactive somatostatin (IR-SRIF) in kindled rats was significantly increased in amygdala than that in non-kindled rats and control rats. Immunoreactive neuropeptide Y (IR-NPY) contents in kindled rats were significantly increased in amygdala, hippocampus, cortex and striatum compared to non-kindled and control rats. The expression of SRIF mRNA in kindled rats produced a significant increase in amygdala, while NPY mRNA in kindled rats showed an elevated expression in both amygdala and hippocampus. These results coincide with the previous findings with the elevated expression of SRIF and NPY mRNA in electrically and pharmacologically kindled models, suggesting the important role of these peptides in the kindling phenomenon.

Paradoxical behavioral response to apomorphine in tenascin-gene knockout mouse.

Tenascin is a large extracellular matrix glycoprotein which is highly expressed in the developing nervous system. To examine the role of tenascin in vivo, we have produced mice in which the tenascin-gene is inactivated. These animals did not easily habituate to unfamiliar circumstances and displayed hyperlocomotion. A dopamine receptor agonist, apomorphine, reduced this hyperlocomotion dose dependently, but this phenomenon was not due to the appearance of apomorphine-induced stereotypic behavior, suggesting that tenascin-gene mutant mice have a paradoxical behavioral response to apomorphine compared to wild-type mice.