Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging.

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.

Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis.

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock-out mice (p53-/-) were analyzed by two-dimensional gel electrophoresis (2-DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18-week-old non-stress-induced mice were analyzed. The morphology of brains from p53-/- mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2-D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific, whereas 12 of 1008 spots from lung tissue were p53-/- specific. N-terminal amino acid sequence was determined for p53-/- specific proteins. In all brain tissues from p53-/- mice, a newly identified mouse mitochondrial NADH-ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain-type creatine kinase B chain and tubulin beta-5 N-terminal fragment were increased in the p53-/- cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53-/- mice. The alterations in protein expression identified in this study may imply a p53-related brain function. This is the first proteomic analysis on the p53-/- mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.

Levodopa induces AP-1 and CREB DNA-binding activities in the rat striatum.

In order to elucidate the effect of levodopa and bromocriptine on the DNA-binding activities of transcription factors, AP-1 and CREB DNA-binding activities were investigated using gel-shift assay. Intraperitoneal administration of 100 mg/kg levodopa with 50 mg/kg benserazide in rats increased both AP-1 and CREB DNA-binding activities in the dorsolateral aspect of the striatum. The major proteins composing the increased AP-1 were JunB and JunD. Bromocriptine at doses of 2.5 and 5.0 mg/kg, however, did not increase these binding activities. Present results suggest that levodopa but not bromocriptine induces these transcription-regulating proteins in the striatum with normal dopaminergic functioning.

Neurotransmitter interactions in the striatum and hypothalamus of mice after single and repeated ethanol treatment.

In single treatment study, ethanol was administered intraperitoneally to ICR mice (about 34 g) in the amounts of 1.0, 2.0, 3.0 or 4.0 g/kg body weight. The 3,4-dihydroxyphenylacetic acid (DOPAC) + homovanillic acid (HVA) concentration in the striatum was elevated with 3.0 and 4.0 g/kg of ethanol. In the hypothalamus, the DOPAC, HVA and 5-hydroxyindoleacetic acid concentrations were increased after injection of 3.0 and 4.0 g/kg of ethanol. Furthermore, the acetylcholine (ACh) and gamma-aminobutyric acid (GABA) concentrations were also increased following the injection of 1.0, 2.0, 3.0 and 4.0 g/kg. To study the effects of repeated administration, mice were injected intraperitoneally with 1.0 or 2.0 g/kg of ethanol once daily for 7 days. The DOPAC + HVA level in the striatum was elevated after injection of 1.0 and 2.0 g/kg of ethanol. The GABA and ACh concentrations in the hypothalamus were decreased after repeated injections of ethanol. These results suggest that ethanol significantly alters the utilization of dopamine, ACh and GABA in the hypothalamus. This may partially explain why ethanol has such profound effects on emotional behavior and mood.

Effects of Kamikihito, a traditional Chinese medicine, on neurotransmitter receptor binding in the aged rat brain determined by in vitro autoradiography (2): changes in GABAA and benzodiazepine receptor binding.

We investigated the effects of the long-term administration of Kamikihito (KKT) on the specific binding of [3H]muscimol and [3H]flunitrazepam in the brains of young and aged rats using in vitro quantitative autoradiography. Specific [3H]muscimol binding in aged rats was decreased in all brain regions examined compared with that in young rats, whereas [3H]flunitrazepam binding did not change in any of the brain regions. Scatchard analysis revealed that the maximal number of [3H]muscimol binding sites in the cortex and thalamus was significantly decreased in aged rats compared with young rats, while its affinity remained unchanged. Long-term administration of KKT in young rats had no effect on either [3H]muscimol or [3H]flunitrazepam binding. In contrast, the same treatment in aged rats produced a significant increase in [3H]flunitrazepam binding to the cortex, caudate/putamen and accumbens, and it tended to decrease the [3H]muscimol binding. These results suggest that the selective reduction of specific [3H]muscimol binding in the brain may be responsible, at least in part, for anxiety-related behavior in aged rats. Furthermore, it appears that the significant increase in specific [3H]flunitrazepam binding produced in the brains of aged rats by the long-term administration of KKT may be responsible for the anxiolytic effects of this agent.

Effects of kamikihito, a traditional Chinese medicine, on neurotransmitter receptor binding in the aged rat brain determined by in vitro autoradiography: changes in dopamine D1 and serotonin 5-HT2A receptor binding.

Using in vitro autoradiography, we investigated the effects of Kamikihito (KKT), a traditional Chinese medicine, on specific [3H]SCH23390 binding to dopamine D1 receptors and [3H]ketanserine binding to serotonin 5-HT2A receptors in the rat brain. Specific binding of both compounds was affected by aging. Long-term administration of KKT resulted in decreases in [3H]SCH23390 binding to the cortex and hippocampus in aged rats, and in decreases in [3H]ketanserine binding to the caudate/putamen in young rats. These results suggest that the changes in dopamine Di and serotonin 5-HT2A receptor binding may be involved in the central effects of KKT.

Effects of kamikihito, a traditional Chinese medicine, on neurotransmitter receptor binding in the aged rat brain determined by in vitro autoradiography (1): Changes in the [3H]QNB binding.

Using in vitro autoradiography, we investigated the effects of Kamikihito (KKT), a traditional Chinese medicine, on the specific binding of [3H]quinuclidinyl benzilate (QNB) and [3H]N-(1-[2-thienyl]cyclohexyl)-3,4-piperidine (TCP) in the rat brain. The Bmax but not the Kd values for [3H]QNB binding to the caudate/putamen and accumbens in aged rats were lower than those in young rats. The [3H]TCP binding was also decreased in aged rats compared with that in young rats. Long-term administration of KKT modulated the [3H]QNB binding in young but not aged rats.

[Animal models of aggression and serotonin receptor subtypes].

Aggressive behavior is an intrinsic behavior of most animals. A number of animal models of aggression have been used to evaluate the effectiveness of anxiolytics and antidepressants in the preclinical trials. These models interested investigators in search of the mechanism of presentation of aggression. Over the past decade, serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes have been identified. Various serotonergic agents have been tested, and some of them successfully reduced aggressive behavior in animal models of aggression. These include 5-HT1A receptor agonists. In the present review, after a description of various animal models of aggression is given, authors overview the role of serotonergic mechanism in the CNS and discuss its relation to aggressive behaviors.

Changes in muscarinic cholinergic, PCP, GABAA, D1, and 5-HT2A receptor binding, but not in benzodiazepine receptor binding in the brains of aged rats.

We used in vitro quantitative autoradiography to investigate changes in neurotransmitter receptor binding, including muscarinic cholinergic, PCP, GABAA, benzodiazepine, D1 and 5-HT2A receptor, in the brains of aged rats, compared with such binding in young rats. Scatchard analysis revealed that the maximal number of binding sites for [3H]quinuclidinyl benzilate (QNB) in the caudate/putamen and accumbens was significantly decreased in aged rats compared with young rats, while its affinity remained unchanged. The specific binding of [3H]N-(1-[2-thienyl]cyclohexyl)3,4-piperidine (TCP) for the ion channels coupled with N-methyl-D-aspartate receptors in the caudate/putamen and hippocampus was significantly decreased in aged rats compared with young rats. The [3H]muscimol binding in aged rats was decreased in all brain regions examined compared with that in young rats, whereas [3H]flunitrazepam binding was not changed in any brain regions. The [3H]SCH23390 binding for dopamine D1 receptors was significantly increased in the parietal cortex, but decreased in the caudate/putamen and accumbens of aged rats compared with that in young rats. The [3H]ketanserin binding for 5-HT2A receptors in the cortex and accumbens was significantly decreased in aged rats compared with young rats. These results suggest that uneven changes in receptors for various neurotransmitters throughout the brain may be responsible for the decline of brain function in aged rats.

The effect of acute and repeated ethanol administration on monoamines and their metabolites in brain regions of rats.

Concentrations of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were determined in eleven brain regions of rats following acute and repeated ethanol administration: (a) an intraperitoneal (i.p.) injection of 1, 2, 3 or 4g ethanol/kg body weight and (b) i.p. injection of 1 or 2g ethanol/kg body weight for seven consecutive days. After acute administration, the concentrations of monoamines and their metabolites appeared to be altered in all brain regions examined except substantia nigra and dorsal amygdala, with maximal variation 2 or 3h after 3g ethanol administration. After repeated administration, the alterations following injections of 2.0g/kg were more marked than the injections of 1.0g/kg. Generally, the levels of NE, DA and 5-HT were decreased while the levels of HVA, DOPAC and 5-HIAA were increased with a few exception. The most prominent findings were seen in the striatum, nucleus accumbens and locus coeruleus. These data indicate that concentrations of monoamines and their metabolites can be determined simultaneously in discrete brain regions and that monoaminergic systems in the brain respond region-specifically to ethanol treatment.

Action of peripherally administered cholecystokinin on monoaminergic and GABAergic neurons in the rat brain.

In an acute study, cholecystokinin octapeptide sulfate (CCK) in doses of 1, 10 or 100 micrograms/kg body weight was injected intraperitoneally into rats just prior to the dark cycle. Rats were sacrificed two hours following the CCK injection. Norepinephrine levels were elevated in the dorsal amygdala of rats injected with 10 micrograms of CCK as well as in the septum of rats injected with 1 and 10 micrograms of CCK. The dopamine level in the septum of rats injected with 1 microgram of CCK as well as the gamma-aminobutyric acid (GABA) level in the lateral hypothalamus of rats injected with 10 micrograms of CCK were also elevated. In a chronic study, CCK (1 microgram/kg body weight/h) was subcutaneously infused into rats with Alzet osmotic minipump for seven consecutive days. The daily food consumption did not change during the 7 days of CCK infusion. The dopamine turnover in the striatum accelerated and the GABA level increased. On the contrary, dopamine metabolism in the substantia nigra and locus coeruleus decreased. Furthermore, the serotonin level in the substantia nigra decreased. Norepinephrine levels decreased in the nucleus paraventricularis, the locus coeruleus and the substantia nigra. The results suggest that peripherally administered CCK may act on the monoaminergic neurons and GABAergic neurons in the brain.

Chronic methamphetamine and its withdrawal modify behavioral and neuroendocrine circadian rhythms.

Chronic methamphetamine (MA) administration via the drinking water not only induced hyperactivity, but phase delayed the rat rest-activity cycle under entrained conditions. The minimum and/or maximum of the rhythms in eating, drinking, body weight, core body temperature and plasma/urine corticosterone were delayed. The different phase shifts of peak and trough values can also be a result of modulation in the wave form of the rhythms. The fall, but not the rise, of nocturnal pineal melatonin secretion occurred 4 hours earlier in MA-treated rats than in controls: this pattern was still present 1 week after withdrawal, but no longer after 4 weeks withdrawal. Neither chronic MA nor its withdrawal had any effect on plasma thyrotropin. These patterns after chronic MA intake fall into two groups: those rhythms whose peak and/or trough are delayed and those that are not. We thus interpret chronic MA application as modulating the eating rhythm (though not directly through rhythmic MA levels in the CNS), and that this in turn changes all food-dependent rhythms. In contrast, the circadian rhythms of melatonin and thyrotropin remain independent.

Temporal distribution of [3H]-imipramine binding in rat brain regions is not changed by chronic methamphetamine.

Specific binding of [3H]-imipramine in the rat suprachiasmatic nuclei, occipital cortex and caudate putamen underwent significant and replicable changes throughout 24 hr under a light-dark cycle or under constant conditions. Daily variations were also found in the medial and dorsal raphe nuclei and the lateral hypothalamus. Methamphetamine, a psychoactive drug with marked effect on circadian rhythms in physiological and hormonal parameters and adrenergic receptors, did not have any significant effect on imipramine binding rhythms in eight discrete brain regions. Thus a drug known to reduce serotoninergic neurotransmission did not change characteristics of the modulatory binding site related to serotonin uptake.

Hypothalamic alpha 2- and beta-adrenoceptor rhythms are correlated with circadian feeding: evidence from chronic methamphetamine treatment and withdrawal.

The circadian regulation of food intake in rats is correlated with a bimodal rhythm of beta-adrenoceptor binding in the lateral hypothalamus and a unimodal rhythm of alpha 2-adrenoceptor binding in the medial hypothalamus. Chronic methamphetamine treatment provides evidence for a functional correlation: beta-adrenoceptor binding in the lateral hypothalamus is reduced at dusk, together with reduction of food intake; alpha 2-adrenoceptor binding in the medial hypothalamus is increased at dawn, together with persistent food intake. Long-term changes in these two adrenergic systems are also correlated with homeostasis of food intake: 24-h mean beta-adrenoceptor binding is reduced and alpha 2-adrenoceptor binding is increased upon methamphetamine withdrawal, when rebound feeding occurs. Corticosterone, although normally coupled to adrenergic mechanisms that regulate feeding, is phase delayed after chronic methamphetamine treatment.

Relationship of tyrosine concentration to catecholamine levels in rat brain.

Rats were fed a choline-free low protein diet for 12 or 26 weeks. In the 12-week group, the brain tyrosine concentration did not change. Dopamine levels were low in both the cerebral cortex and striatum. Norepinephrine level was low in the diencephalon. In the 26-week group, the tyrosine concentration was high in the brain. However, the dopamine and norepinephrine levels did not change in the cerebral cortex, striatum and hypothalamus. Furthermore, in another group of rats which were intraperitoneally injected with tyrosine, the brain tyrosine concentration was high, whereas the dopamine and norepinephrine levels in the hypothalamus were not significantly different from control levels.

Circadian rhythm of [3H]imipramine binding in the rat suprachiasmatic nuclei.

High affinity imipramine binding undergoes circadian variations of ca. 35% amplitude in many rat brain nuclei. The suprachiasmatic nuclei of the anterior hypothalamus (considered to be the circadian pacemaker driving many overt rhythms) has highest imipramine binding at the end of the dark and lowest at the end of the light phase. A similar circadian rhythm has previously been observed for serotonin uptake in the suprachiasmatic nuclei. In conjunction with other findings, these data indicate that serotonergic turnover in the suprachiasmatic nuclei decreases at lights on and increases at lights off.