Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (V_max) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (B_max) and affinity (K_d) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.     

Age-dependent changes of nucleic acid labeling in different rat brain regions

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-¹⁴C]thymidine incorporation into DNA of cerebral cortex and cerebelllum during aging was observed.The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging.In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A)+ RNA and poly(A)- RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.     

Maternal Undernutrition During Lactation: Effect on Amino Acids in Brain Regions of Offspring

Sprague-Dawley dams were fed either a protein-calorie deficient or control diet from day 5 to day 21 after parturition. The concentrations of seven amino acids (aspartate, glutamate, gamma-aminobutyric acid, glycine, glutamine, serine, and taurine) were determined in brain regions from 17-day-old undernourished offspring and from 35-day-old rehabilitated rats. The brain regions examined were the cortex, cerebellum, corpus striatum, hippocampus, hypothalamus, brainstem, and midbrain. At 17 days of age, taurine was the amino acid with the highest concentration, whereas at 35 days glutamate had the highest concentration. This change was due to the fact that the concentration of taurine decreased significantly in all brain regions between 17 and 35 days, whereas the concentration of glutamate remained high or increased somewhat in all brain regions except the hypothalamus and brainstem. When the age-matched offspring of control and undernourished rats were compared, several interesting and significant differences were found. The concentrations of glutamate and aspartate were significantly lower (decreased 16-34%) in the cerebellum, brainstem, cortex, and midbrain in 17-day-old undernourished rats. The aspartate level was also significantly decreased in the corpus striatum and hypothalamus in 17-day-old offspring. However, the deficiencies of aspartate and glutamate were transient and reversible. In contrast, the concentration of taurine was increased in the hypothalamus (31%) and hippocampus (12-33%) at both 17 and 35 days of age and in the midbrain (17%) at 17 days. Other transient abnormalities in amino acid levels were found in undernourished offspring. The results of these experiments suggest that undernutrition during lactation causes delayed CNS development, which is manifested in altered concentrations of the neurotransmitters aspartate, glutamate, and taurine.     

Age and Brain Structural Related Effects of Glutaric and 3-Hydroxyglutaric Acids on Glutamate Binding to Plasma Membranes During Rat Brain Development

(1) In the present study we determined the effects of glutaric (GA, 0.01–1 mM) and 3-hydroxyglutaric (3-OHGA, 1.0–100 μM) acids, the major metabolites accumulating in glutaric acidemia type I (GA I), on Na⁺-independent and Na⁺-dependent [³H]glutamate binding to synaptic plasma membranes from cerebral cortex and striatum of rats aged 7, 15 and 60 days. (2) GA selectively inhibited Na⁺-independent [³H]glutamate binding (binding to receptors) in cerebral cortex and striatum of rats aged 7 and 15 days, but not aged 60 days. In contrast, GA did not alter Na⁺-dependent glutamate binding (binding to transporters) to synaptic membranes from brain structures of rats at all studied ages. Furthermore, experiments using the glutamatergic antagonist CNQX indicated that GA probably binds to non-NMDA receptors. In addition, GA markedly inhibited [³H]kainate binding to synaptic plasma membranes in cerebral cortex of 15-day-old rats, indicating that this effect was probably directed towards kainate receptors. On the other hand, experiments performed with 3-OHGA revealed that this organic acid did not change Na⁺-independent [³H]glutamate binding to synaptic membranes from cerebral cortex and striatum of rats from all ages, but inhibited Na⁺-dependent [³H]glutamate binding to membranes in striatum of 7-day-old rats, but not in striatum of 15- and 60-day-old rats and in cerebral cortex of rats from all studied ages. We also provided some evidence that 3-OHGA competes with the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting a possible interaction of 3-OHGA with glutamate transporters on synaptic membranes. (3) These results indicate that glutamate binding to receptors and transporters can be inhibited by GA and 3-OHGA in cerebral cortex and striatum in a developmentally regulated manner. It is postulated that a disturbance of glutamatergic neurotransmission caused by the major metabolites accumulating in GA I at early development may possibly explain, at least in part, the window of vulnerability of striatum and cerebral cortex to injury in patients affected by this disorder.     

Age related changes in weight and the concentrations of zinc and copper in the brain of the adult rat

Two groups of adult male rats aged 15 weeks and 49 weeks, 15 rats in each group, were analysed for the concentrations of the trace elements zinc (Zn) and copper (Cu) in serum, liver, kidney, and five parts of the brain (cortex, corpus striatum, hippocampus, midbrain + medulla, and cerebellum). All organs increased in weight from 15 weeks to 49 weeks. In all parts of the brain, except for corpus striatum, there was a significant increase of the weights. The dry weight (% of wet) increased in all parts of the brain. In serum, the Zn and Cu concentrations increased from 15 weeks to 49 weeks. In the liver, both concentrations decreased and in the kidney the concentrations increased with increasing age. The Zn concentrations increased in cortex and corpus striatum and decreased in cerebellum and hippocampus. The Cu levels increased in all parts of the brain with the largest changes in corpus striatum. For rats aged 49 weeks, a significant correlation was found between the Cu concentrations of corpus striatum or midbrain + medulla and the fluid consumption. The findings of the present study reveal a dynamic age-related pattern of changes in the concentrations of Zn and Cu in different organs of the adult rat. This stresses the need of age-matching as an important control in experiment studies.     

Functional striatal hypodopaminergic activity in mice lacking adenosine A(2A) receptors.

Adenosine and caffeine modulate locomotor activity and striatal gene expression, partially through the activation and blockade of striatal A(2A) receptors, respectively. The elucidation of the roles of these receptors benefits from the construction of A(2A) receptor-deficient mice (A(2A)-R(-/-)). These mice presented alterations in locomotor behaviour and striatal expression of genes studied so far, which are unexpected regarding the specific expression of A(2A) receptor by striatopallidal neurones. To clarify the functions of A(2A) receptors in the striatum and to identify the mechanisms leading to these unexpected modifications, we studied the basal expression of immediate early and constitutive genes as well as dopamine and glutamate neurotransmission in the striatum. Basal zif268 and arc mRNAs expression was reduced in mutant mice by 60-80%, not only in the striatum but also widespread in the cerebral cortex and hippocampus. Striatal expression of substance P and enkephalin mRNAs was reduced by about 50% and 30%, respectively, whereas the expression of GAD67 and GAD65 mRNAs was slightly increased and unaltered, respectively. In vivo microdialysis in the striatum revealed a 45% decrease in the extracellular dopamine concentration and three-fold increase in extracellular glutamate concentration. This was associated with an up-regulation of D(1) and D(2) dopamine receptors expression but not with changes in ionotropic glutamate receptors. The levels of tyrosine hydroxylase and of striatal and cortical glial glutamate transporters as well as adenosine A(1) receptors expression were indistinguishable between A(2A)-R(-/-) and wild-type mice. Altogether these results pointed out that the lack of A(2A) receptors leads to a functional hypodopaminergic state and demonstrated that A(2A) receptors are necessary to maintain a basal level in immediate early and constitutive genes expression in the striatum and cerebral cortex, possibly via their control of dopamine pathways.     

Iron Deposition after Transient Forebrain Ischemia in Rat Brain

In this study, we investigated the iron deposition in the cerebral cortex, hippocampus CA1 area and corpus striatum pars dorsolateralis in a rat model of cerebral ischemia. Forebrain ischemia was induced by four-vessel occlusion for 20 min. Using iron histochemistry, regional changes were examined from 1 to 8 weeks of postischemic recirculation. Neuronal death was demonstrated in pyramidal cells of the hippocampal CA1 area and in the dorsolateral part of the corpus striatum, which are known as areas most vulnerable to ischemia. Iron deposition in hippocampal CA1 area was coupled to delayed pyramidal cell death. Perl's reaction with DAB intensification revealed of the 1 week iron deposits in the CA1 area, which gradually increased and formed clusters by 8 weeks. In the corpus striatum, strong iron staining was observed in injured cellular layer pars dorsolateralis 1 week after recirculation. Granular iron was deposited in the cytoplasm of pyramidal cells in layers III and V of the frontal cortex after 2 weeks of recirculation. In contrast to the hippocampus and striatum, the cerebral cortex did not develop severe neuronal cell death and atrophy immediately after the ischemic insult, which suggest that the neuronal cell death in the cerebral cortex occurs extremely late.     

D4 Dopamine and Metabotropic Glutamate Receptors in Cerebral Cortex and Striatum in Rat Brain

The characterization of the functional interactions between the metabotropic glutamate receptors (mGluR) and the dopaminergic (DR) receptors in the corticostriatal projections may provide a possible interpretation of synaptic events in the basal ganglia. It has been suggested that presynaptic D2-type receptor located on glutamatergic corticostriatal neurons regulates the release of glutamate. In a first approach we have studied the cellular distribution of the D4R and the mGluRs in cerebral cortex and striatum employing immunocytochemistry. D4R positive neurons were particularly numerous in medial prefrontal cortex mainly occupying layers II and III. An even distribution was found on small round-shaped neurons in the striatum. Group I mGluR1-like immunoreactivity (mGluR1-LI) was found in medial and deep layers of the cerebral cortex while group III mGluR4a labeled more superficial layers; group II mGluR2/3 signal was intense on fine fibers with a punctate appearance. In the striatum, mGluR1 and mGluR2/3 stained mainly fibers while mGluR4a labeled round shaped cell bodies. After lateral ventricular injection of colchicine, an axonal transport and firing activity blocker, D4R labeling significantly increased in cerebral cortex and decreased in the striatum. mGluR1 and mGluR4a signal decreased in cerebral cortex and only mGluR4a signal decreased in the striatum. These results support previous reports indicating a presynaptic localization of D4R in the striatum. In contrast, striatal mGluR1 appears to be a postsynaptic receptor probably synthesized in situ. Our results do not support the hypothesis of a colocalization of D4 receptor and one or more of the metabotropic glutamatergic receptors studied here.     

Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

The binding of 3H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. 3H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of 3H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (Bmax value) and apparent dissociation constant (Kd value) values of 3H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midbrain, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of 3H-naltrexone binding to membranes of hypothalamus of SHR rats was 518% higher than WKY rats but the Kd values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with 3H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats.     

RNA polymerase I and II activities in different brain regions of young, adult, and old rats

The activities of RNA polymerase I and II were assayed in nuclei isolated from different regions (cerebral cortex, cerebellum, hypothalamus, hippocampus, corpus striatum and pituitary) of brains from young (10 days), adult (6 months), and old (2 years) rats. The RNA polymerases I and II activities generally increased during maturation, i.e., from 10 days to 6 months of postnatal age and then showed a decrease from 6 months to 2 years of age in all the regions except in cerebral cortex where the RNA polymerase II activity was highest at 10 days but showed a gradual decrease through the lifespan up to 2 years.     

NMDA receptor function is enhanced in the hippocampus of aged rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [³H]CGP 39653 and [³H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [³H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 M glycine and 10 M glutamate increased [³H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [³H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [³H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [³H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.     

Dopamine D1-stimulated adenylyl cyclase activity in cerebral cortex of autopsied human brain.

Although the cerebral cortical dopamine D(1) receptor is considered to play a role in normal and abnormal brain function, little information is available on its characteristics in human brain. We compared dopamine-stimulated adenylyl cyclase (AC) activity in homogenates of cerebral cortex (frontal, temporal, parietal, occipital and cingulate cortex) of autopsied brain of neurologically normal subjects to that in striatum. Cerebral cortical AC activity was modestly and dose-dependently stimulated by dopamine (maximal 20-30%) with low microM EC50s and such stimulation was inhibited by the selective dopamine D1 receptor antagonist SCH23390. The magnitude of the maximal stimulation by dopamine was similar in autopsied and biopsied cerebral cortex. The extent of maximal stimulation was similar to that in dopamine-rich striatum (caudate, putamen and nucleus accumbens), despite much lower density of dopamine D1 receptors in cerebral cortex vs. striatum. The EC50 for dopamine stimulation in cerebral cortex (approximately 1 microM) was lower than that for caudate and putamen (approximately 3 microM). No detectable dopamine stimulation was observed in cerebellar cortex, thalamus or hippocampus. Dopamine stimulation in both cerebral cortex and striatum was independent of calcium activation. We conclude that dopamine stimulated AC can be measured in cerebral cortex of human brain allowing for the possibility that this process can be examined in human brain disorders in which dopaminergic abnormalities are suspected.     

Effect of the Non-NMDA Receptor Antagonist GYKI 52466 on the Microdialysate and Tissue Concentrations of Amino Acids Following Transient Forebrain Ischaemia

Abstract: The effect of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) on ischaemia-induced changes in the microdialysate and tissue concentrations of glutamate, aspartate, and γ-aminobutyric acid (GABA) was studied in rats. Twenty minutes of four-vessel occlusion resulted in a transient increase in microdialysate levels of glutamate, aspartate, and GABA in striatum, cortex, and hippocampus. Administration of GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min intravenously starting 20 min before onset of ischaemia) inhibited ischaemia-induced increases in microdialysate glutamate and GABA in striatum without affecting the increases in hippocampus or cortex. Twenty minutes of four-vessel occlusion resulted in immediate small decreases and larger delayed (72 h) decreases in tissue levels of glutamate and aspartate. Transient increases in tissue levels of GABA were shown in all three structures at the end of the ischaemic period. At 72 h, after the ischaemic period, significantly reduced GABA levels were observed in striatum and hippocampus. GYKI 52466, given under identical conditions as above, augmented the ischaemia-induced decrease in striatal tissue levels of glutamate and aspartate, without significantly affecting the decreases in hippocampus and cortex. Twenty minutes of ischaemia resulted in a large increase in microdialysate dopamine in striatum. GYKI 52466 failed to inhibit this increase. Kainic acid (500 μM infused through the probe for 20 min) caused increases in microdialysate glutamate and aspartate in the striatum. GYKI 52466 (10 mg/ kg bolus + 10 mg/kg/60 min) completely inhibited the kainic acid-induced glutamate release. In conclusion, the action of the non-NMDA antagonist, GYKI 52466, in the striatum is different from that in the cortex and hippocampus. The inhibition by GYKI 52466 of ischaemia-induced and kainate-induced increases in microdialysate glutamate concentration in the striatum may be related to the neuroprotection provided by GYKI 52466 in this region.     

Rosmarinic acid prevents lipid peroxidation and increase in acetylcholinesterase activity in brain of streptozotocin‐induced diabetic rats

We investigated the efficacy of rosmarinic acid (RA) in preventing lipid peroxidation and increased activity of acetylcholinesterase (AChE) in the brain of streptozotocin‐induced diabetic rats. The animals were divided into six groups (n = 8): control, ethanol, RA 10 mg/kg, diabetic, diabetic/ethanol and diabetic/RA 10 mg/kg. After 21 days of treatment with RA, the cerebral structures (striatum, cortex and hippocampus) were removed for experimental assays. The results demonstrated that the treatment with RA (10 mg/kg) significantly reduced the level of lipid peroxidation in hippocampus (28%), cortex (38%) and striatum (47%) of diabetic rats when compared with the control. In addition, it was found that hyperglycaemia caused significant increased in the activity of AChE in hippocampus (58%), cortex (46%) and striatum (30%) in comparison with the control. On the other hand, the treatment with RA reversed this effect to the level of control after 3 weeks. In conclusion, the present findings showed that treatment with RA prevents the lipid peroxidation and consequently the increase in AChE activity in diabetic rats, demonstrating that this compound can modulate cholinergic neurotransmission and prevent damage oxidative in brain in the diabetic state. Thus, we can suggest that RA could be a promising compound in the complementary therapy in diabetes. Copyright © 2013 John Wiley & Sons, Ltd.     

Regional Differences in 5-Hydroxytryptamine and Catecholamine Uptake in Primary Astrocyte Cultures

The uptake of 3H-labelled 5-hydroxytryptamine (5-HT, serotonin) norepinephrine ([3H]NE), and 3,4-dihydroxyphenylethylamine ([ 3H]dopamine, [3H]DA) was studied in primary astrocyte cultures prepared from the cerebral cortex, corpus striatum, and hippocampal regions of neonatal rat brain. Na+-dependent uptake showed marked regional differences. For [3H]5-HT the magnitude of uptake was corpus striatum greater than or equal to cerebral cortex greater than hippocampus, whereas for [3H]NE the order was hippocampus greater than corpus striatum greater than cerebral cortex. For [3H]DA, only the hippocampal cultures showed significant Na+-dependent uptake. [3H]5-HT uptake was specifically inhibited by 10(-7) M fluoxetine whereas [3H]NE uptake was preferentially inhibited by 10(-7) M desipramine. These results may reflect regional brain specialization and/or different developmental patterns of high affinity uptake of serotonin and catecholamines by astrocytes in situ.     

Differential responsiveness of metabotropic glutamate receptors coupled to phosphoinositide hydrolysis to agonists in various brain areas of the adult rat

The effects of metabotropic glutamate receptor (mGluR) agonists on inositol phosphates (IP) accumulation were investigated in slices of the cerebral cortex, hippocampus, striatum and cerebellum of adult Sprague-Dawley rats. EC₅₀ values for 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) did not differ significantly between various brain areas (range 10⁻⁵ M), quisqualate was the most potent in all the brain areas (range 10⁻⁷−10⁻⁶ M), except the cerebellum (10⁻⁵ M), ibotenate was the most potent in the striatum (range 10⁻⁶ M) and the least potent in the cerebral cortex and hippocampus (range 10⁻⁴ M). The efficacy in the four brain areas showed the following trend of ranking order for ACPD and quisqualate: hippocampus > striatum > cerebral cortex > cerebellum, and for ibotenate: hippocampus > cerebral cortex > striatum > cerebellum, although the observed differences reached the level of statistical significance only in the case of ACPD (hippocampus and striatum vs cerebellum) and ibotenate (hippocampus vs cerebellum). Co-incubation of the agonists at maximally effective concentrations in any pairwise combination resulted in no substantial additivity of IP accumulation. D,L-1-amino-3-phosphonopropionic acid (AP3) and D,L-2-amino-4-phosphonobutyric acid (AP4) at 0.5 mM concentration antagonized ACPD-induced IP accumulation by about 70 and 45%, respectively, without differences between brain areas. On the other hand, the antagonistic effects ofl-serine-o-phosphate (SOP) at 1 mM concentration were the highest in the hippocampus (75%) and the lowest in the cerebellum (25%). The comparative data indicate considerable regional receptor heterogeneity, in terms of different ratios of response to the agonists (but not antagonists, except SOP). There is a robust responsiveness of mGluRs not only in the hippocampus and cerebral cortex, but also in the striatum which exhibits the highest affinity to both quisqualate and ibotenate.     

Effect of Chronic Nicotine Treatment on Nicotinic Autoreceptor Function and N-[3H]Methylcarbamylcholine Binding Sites in the Rat Brain

It has been reported that N-methylcarbamylcholine (MCC), a nicotinic agonist, binds to central nicotinic receptors and causes an increase of acetylcholine (ACh) release from certain central cholinergic nerve terminals. The present experiments determine whether these two phenomena change in response to the chronic administration of nicotine, a procedure known to result in an increase in nicotinic binding sites. Chronic nicotine caused a brain region-specific up-regulation of [3H]MCC sites; binding increased in the frontal cortex, parietal cortex, striatum, and hippocampus, but not in the occipital cortex or cerebellum. The effect of nicotine was selective to nicotinic binding sites, because muscarinic sites, both M1 ([ 3H]pirenzepine) and M2 ([3H]ACh), were unaffected by chronic nicotine treatment. MCC increased the release of ACh from the frontal cortex and hippocampus by a calcium-dependent mechanism; MCC did not alter ACh release from striatum or occipital cortex of control animals. The MCC-induced increase in ACh release was not apparent in those animals which had been treated with nicotine. There was a partial recovery of nicotinic autoreceptor function when animals were allowed to recover (4 days) following chronic nicotine treatment, but the density of binding sites remained increased compared to control. Chronic nicotine did not change the potassium-evoked release of ACh from the frontal cortex or hippocampus, but decreased this measure from striatum. It also decreased the ACh content of the striatum, but not that of the cortex or the hippocampus; the activity of choline acetyltransferase was not altered in any of the regions tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.     

Effects of ethanol on brain monoamine content of spontaneously hypertensive rats (SHR)

Spontaneously hypertensive rats (SHR) were administered either 2.4 g/kg ethanol or an isocaloric glucose daily for 4 weeks and the levels of norepinephrine (NE), epinephrine (EP), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in different brain regions were determined. Results indicated a 3-fold increase in NE level in brain stem and hypothalamus and more than 2-fold increase in DA in corpus striatum in alcohol-treated rats as compared to controls. There was a significant increase in the level of DA in the corpus striatum but the levels in cerebral cortex, brain stem and hippocampus were decreased instead. Decreases in 5-HT levels were found in hypothalamus, brain stem, cortex and cerebellum of alcohol-treated brain as compared to untreated controls. These results indicate alterations of the biogenic amine contents in different regions of the SHR brain after chronic ethanol ingestion. Since stimulated release of biogenic amines in the SHR brain has been implicated in the regulation of blood pressure, changes due to ethanol ingestion may be a risk factor in hypertensive patients.