Differential regulation of glutamate decar☐ylase and preproenkephalin mRNA levels in the rat striatum

The mRNA levels encoding for the enzyme glutamate decar☐ylase (GAD67) and the peptide enkephalin were measured in the striatum of adult and 15 day-old rats by in situ hydridization histochemistry and radioautography after neonatal injections of 6-hydroxydopamine or after acute pharmacological blockade of dopamine receptors with haloperidol or sulpiride. In adult rats injected as neonates with 6-hydroxydopamine or treated with the D₁/D₂ dopamine receptors antagonist, haloperidol, an increase in preproenkephalin and GAD67 mRNA levels was measured in the striatum. The D₂ dopamine receptor antagonist, sulpiride, did not change the mRNA levels of either GAD67 or PPE in the striatum. In 15-day-old rats, neonatal 6-hydroxydopamine or haloperidol treatment resulted in increased preproenkephalin but unchanged GAD67 mRNA levels compared to controls. In these 15-day-old rats, however, sulpiride produced an increase in GAD67 but not preproenkephalin mRNA levels. Intrastriatal injections to adult rats of pertussis toxin which uncouples Gi/Go proteins from their receptors resulted in a dramatic increase in preproenkephalin without concomitant change in GAD67 mRNA levels. Altogether, these results show that GAD67 and preproenkephalin mRNA levels are modulated in parallel in adult but not in 15 day-old rats after 6-hydroxydopamine injections or dopaminergic blockade. In keeping with evidence of a co-localization of GAD67 and preproenkephalin mRNAs in some striatal neurons, the results indicate that these two mRNAs can be differentially regulated in the same neurons. In addition, the differential effect of haloperidol, sulpiride or pertussis toxin on GAD67 and preproenkephalin mRNA levels suggests that these two mRNAs are regulated through different dopamine receptor subtypes.     

Chronic naltrexone treatment increases expression of preproenkephalin and preprotachykinin mRNA in discrete brain regions

Long-term blockade of brain opioid receptors by the opiate antagonist naltrexone increases methionine-enkephalin content in the striatum and nucleus accumbens (Tempel et al., 1984). To determine whether these changes in peptide levels reflect increased peptide synthesis, we examined preproenkephalin mRNA content in discrete brain regions of control (placebo-treated) and chronic naltrexone-treated animals by Northern analysis. Chronic naltrexone treatment (8 d) led to an approximately 12-fold increase in the striatal content of preproenkephalin mRNA relative to that of control animals. In contrast, no statistically significant change was observed in striatal mRNA for cyclophilin (1B15) or actin. Small increases in preproenkephalin mRNA content occurred in the hippocampus (+40%) and hypothalamus (+19%). No significant changes occurred in the frontal cortex. Increases in levels of the mRNA were seen as early as 24 hr after antagonist treatment. In contrast, changes in opioid receptor density required 3-4 d to reach half-maximal up-regulation after chronic antagonist treatment. Recent evidence has suggested that substance P is regulated by opioid peptides. To determine whether substance P synthesis is altered by chronic antagonist treatment, the mRNA corresponding to the precursor for substance P was examined using a probe for exon-7 of the preprotachykinin gene. Preprotachykinin mRNA content in the striatum was increased 6-fold after chronic antagonist treatment relative to that of control animals. Substance P content was increased 3-fold after chronic antagonist treatment. These data suggest that chronic blockade of brain opioid receptors leads to the increased synthesis of both enkephalin and substance P in the striatum and that these changes are relatively specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation by dopaminergic neurotransmission of dopamine D2 mRNA and receptor levels in the striatum and nucleus accumbens of the rat.

The effect of dopamine depletion or pharmacological blockade of dopamine receptors on striatal and accumbens dopamine D2 mRNA and receptor levels was assessed by in situ hybridization histochemistry and receptor autoradiography. The time course of pharmacological blockade with haloperidol demonstrates a complex mode of regulation of dopamine D2 mRNA and receptor levels. By day 8 of haloperidol treatment, D2 mRNA and receptor levels were decreased (up to 20%) in the medial and anterior aspects of the caudate-putamen (mCPU and aCPU) and the nucleus accumbens (NAc). However, by day 21 of haloperidol treatment, D2 mRNA and receptor were increased relative to vehicle-injected controls. Likewise, unilateral dopamine depletion due to 6-hydroxydopamine (6-OHDA) lesions of mesencephalic dopaminergic neurons resulted in decreased levels of D2 receptor mRNA by day 8 post-lesion in the ipsilateral mCPU, aCPU and the NAc. However, at days 14 or 21 post-lesion, there was a reversal of the effect with increases of up to 22% in all brain regions ipsilateral to the lesion. Although no decreases in receptor level were observed at day 8, significant increases in receptor level in all three brain regions were detected at days 14 and 21 post-lesion. The results demonstrate that midbrain dopaminergic innervation exerts tonic effects on the levels of dopamine D2 receptor and mRNA in the caudate-putamen and the nucleus accumbens of the rat. Changes in receptor level are frequently accompanied by comparable changes in mRNA level, indicating a mass action relationship between receptor level and receptor biosynthesis in these forebrain regions in the rat.     

Isolation stress increases tyrosine hydroxylase mRNA in the locus coeruleus and midbrain and decreases proenkephalin mRNA in the striatum and nucleus accumbens.

Isolation of adult animals represents a form of psychological stress from which the animals cannot escape. In order to assess the effect of this stressor on neurochemical substrates in the brain, we assessed behavior and measured tyrosine hydroxylase and proenkephalin mRNA levels in selected brain areas by in situ hybridization histochemistry. Tyrosine hydroxylase (TH) mRNA levels in the locus coeruleus (LC) were significantly and progressively increased by 18, 42 and 68% after 7, 14 or 28 days of isolation, respectively. TH mRNA in the midbrain was transiently increased by isolation. Levels were significantly elevated by 34 and 48% above group-housed controls in the ventral tegmentum and the substantia nigra, respectively, after 14 days of isolation. In the forebrain, proenkephalin (PE) mRNA levels were found to be transiently decreased by 29% in the anterior and medial aspects of the caudate-putamen and the nucleus accumbens after 7 or 14 days of isolation stress, but the levels returned toward control levels after 28 days of isolation. Behavioral tests indicate that isolated animals progressively became more aggressive with duration of stress and showed a small but significant decrease in locomotor activity. The results demonstrate that a physically noninvasive stressor such as isolation of adult male rats can produce significant alterations in brain neurochemistry. The neurochemical responses observed may represent a brain mechanism designed to help the organism adapt to or protect from the deleterious effects of chronic psychological stress.     

Chronic cocaine increases neurotensin gene expression in the shell of the nucleus accumbens and in discrete regions of the striatum

The effects of chronic cocaine administration on neurotensin (NT) mRNA expression were investigated in the rat brain using in situ hybridization. Adult Wistar rats were injected daily with cocaine (15 mg/kg i.p.) or saline for 10 days. One hour after the last injection, the brains were removed and coronal sections of the nucleus accumbens and striatum processed for in situ hybridization using a ³⁵S-labeled NT mRNA oligonucleotide probe. Repeated administration of cocaine induced a specific increase in the expression of NT mRNA in the shell of the nucleus accumbens whereas no changes were observed in the core compartment. In addition, cocaine enhanced the expression of the NT gene in neurons confined to the posterior dorsomedial striatum, but did not alter this same region in the anterior striatum. A strong increase in NT mRNA expression was also observed in rats treated with cocaine in the ventrolateral region of the striatum, the fundus striati. No modifications were seen in the dorsolateral or ventromedial striatum, the lateral septum, or the olfactory tubercle. These findings demonstrate that cocaine affects NT mRNA expression in discrete populations of neurons confined to the shell of the nucleus accumbens and dorsomedial and ventrolateral striatum of the rat. The shell of the nucleus accumbens is a limbic area considered the locus of the reinforcing and locomotor activating properties of cocaine while the dorsal striatum is implicated in the regulation of motor output, and appears to be involved in the stereotypies induced by cocaine. The specific increases in NT gene expression induced by chronic cocaine suggest that these changes could be physiologically relevant for the behavioral effects of psychostimulant drugs.     

Chronic phencyclidine treatment increases dendritic spine density in prefrontal cortex and nucleus accumbens neurons

We examined whether repeated exposure to the noncompetitive NMDA receptor antagonist phencyclidine (PCP) produces enduring changes in dendritic structure in a manner similar to the stimulants cocaine and amphetamine. Adult rats were treated with i.p. injections of PCP (5 mg/kg) or saline, twice a day, for 5 consecutive days, for a total of 4 weeks. One month after the last injection, their brains were removed and processed for Golgi-Cox staining. Prior exposure to PCP increased dendritic spine density in the mPFC and NAcc core, but not in the parietal cortex. These findings, which are similar to those observed after chronic treatment with cocaine and amphetamine, raise the possibility that, despite differences in their mechanisms of action, PCP and stimulant drugs may induce some of their enduring effects via common processes.     

Microheterogeneity of dopamine transporters in rat striatum and nucleus accumbens.

Previously we have shown that the [125I]DEEP-labeled dopamine transporter from the rat nucleus accumbens has a higher apparent molecular weight than that from striatum. The present study confirms and extends these observations. Experiments with nucleus accumbens showed [125I]-DEEP to specifically bind to a protein with an apparent molecular weight of 76 kDa and with the pharmacological properties of the dopamine transporter. In exoglycosidase studies, treatment with neuraminidase, but not alpha-mannosidase, reduced the apparent molecular weight of the dopamine transporter from both the striatum and nucleus accumbens; however, a difference in the apparent molecular weight was still observed. N-Glycanase treatment, on the other hand, did reduce the apparent molecular weight of the dopamine transporters from the two regions to a similar value, approximately 56 kDa. In radioligand binding studies examining the effect of partial deglycosylation on striatal dopamine transporters, neuraminidase did not affect specific [3H]WIN 35,428 binding at 4 and 40 nM concentrations. In conclusion, the present study demonstrates that the difference in the apparent molecular weight of the dopamine transporter from these two regions is due to a difference in glycosylation and that the dopamine transporter from both regions contains similar amounts of sialic acid in their carbohydrate structure. Furthermore, the present data also indicate that the polypeptide portion of the dopamine transporter from both regions could be the same gene product.     

Fluoxetine increases the extracellular levels of serotonin in the nucleus accumbens

The effects of an IP injection of the monoamine uptake inhibitor fluoxetine on the extracellular concentration of serotonin (5-HT), dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens of awake and freely moving rats were examined using a push-pull perfusion technique. Baseline values of 5-HT, 5-HIAA, DA, DOPAC and HVA in the perfusates were approximately 0.07, 13, 0.8, 49 and 12 pmol/hr, respectively. The IP administration of 5 and 10 mg/kg fluoxetine dose-dependently elevated the amounts of 5-HT 3- and 13-fold, respectively, in the push-pull perfusate, with the maximum reached within one hour after drug administration. Moreover, 10 mg/kg fluoxetine also significantly decreased the levels of 5-HIAA in the perfusate as much as 50% within 2-3 hours. On the other hand, no significant effect of 5 or 10 mg/kg fluoxetine was observed on the contents of DA, DOPAC and HVA in the push-pull perfusates. The data indicate that fluoxetine, in accord with its role as a 5-HT uptake inhibitor, increases the physiologically active pool of 5-HT in the nucleus accumbens under in vivo conditions.     

Increased preproenkephalin mRNA and preprotachykinin mRNA in the striatum of Rolling mouse Nagoya

Although Rolling mouse Nagoya (RMN) has been considered to demonstrate cerebellar dysfunction, our previous metabolic and electrophysiological studies also revealed a dysfunction of the basal ganglia, with the presumable primary site of dysfunction being the striatum. In the present study., we investigated the neurochemical functions of the striatum. In RMN, both preproenkephalin mRNA and preprotachykinin mRNA increased significantly in the striatum, with unaltered GAD mRNA, [³H]spiperone binding, [³H]QNB binding and preprosomatostatin mRNA, thus indicating the dysfunction of striatal projection neurons. These findings support the hypothesis that the site of primary dysfunction in the basal ganglia is in the striatum of RMN.     

Chronic nicotine administration increases binding of [3H]domperidone in rat nucleus accumbens

An apparent inverse relationship between smoking and Parkinson's disease prompted an investigation of the effect of chronic nicotine administration on dopaminergic and serotonergic receptors in rat brain. Nicotine, 0.8 mg/kg, was injected once daily, five times per week, for 6 weeks. In nucleus accumbens the Kd for [3H]domperidone was increased 2-4-fold, and the Bmax was increased 1.5-2-fold. No changes were observed in the binding of [3H]domperidone in caudate-putamen or in that of [3H]ketanserin in frontal cortex. It is concluded that chronic nicotine administration may have a suppressant effect on central nervous system release of dopamine that in pre-parkinsonian persons causes an aversion to the effects of smoking.     

Adenosine A2A receptor mRNA expression is increased in rat striatum and nucleus accumbens after memantine administration

Quantitative in situ hybridization revealed a significant increase (23-43%) of A(2A) receptor mRNA levels 8-48 h after administration of memantine (25 mg/kg, i.p.) in the striatum and nucleus accumbens regions of rat brain. These results indicate an effect on adenosine A(2A) receptors at the molecular level by a NMDA receptor antagonist.     

Experimental tooth movement upregulates preproenkephalin mRNA in the rat trigeminal nucleus caudalis and oralis

Levels of preproenkephalin mRNA expression in trigeminal subnucleus complex by noxious tooth movement stimuli were examined using in situ hybridization. At 24 h, preproenkephalin mRNA expression was significantly upregulated in the ipsilateral trigeminal subnucleus caudalis (P<0.05), and in the subnucleus oralis (P<0.05). These findings suggested that enkephalinergic inhibitory systems could be activated during tooth movement, and that subnucleus oralis may be involved in modulation of the nociception, as well as the subnucleus caudalis.     

Role of the striatum and nucleus accumbens in paced copulatory behavior in the female rat

Female rats engage in a series of approach and avoidance behaviors (pacing behavior) directed at the male in order to achieve a preferred rate of intromissions that make pregnancy more likely to occur with insemination. The striatum and nucleus accumbens have been implicated in the modulation of pacing behavior. It is unclear, however, whether these areas of the brain are necessary for the display of pacing behavior. To address this question, ovariectomized female rats received either bilateral quinolinic acid lesions of the striatum or nucleus accumbens or sham surgeries. After hormone priming, rats were allowed to engage in mating behaviors in an apparatus in which they could pace the rate of the copulatory bout. There was a significant reduction in pacing efficiency after striatal lesions, in that females were less likely to leave the male's side of the chamber after a contact. Animals with lesions of the nucleus accumbens that included the shell were more likely to avoid sexual interaction altogether than animals with control lesions. Therefore, it is concluded that the striatum and nucleus accumbens modulate specific aspects of pacing behavior in the female rat.     

Effect of lead exposure on dopaminergic receptors in rat striatum and nucleus accumbens

Haloperidol- and sulpiride-displaceable [3H]spiroperidol binding and the dopamine-inhibited adenylate cyclase were measured in rats chronically exposed to lead acetate. Haloperidol-displaceable [3H]spiroperidol binding was unmodified while sulpiride-displaceable binding was increased in striatum and decreased in nucleus accumbens. In addition, the decrease of sulpiride-displaceable binding in nucleus accumbens was paralleled by a reduced ability of bromocriptine to inhibit cAMP formation in presence of the D1 receptor antagonist SCH 23390. The results support the concept that in vivo lead treatment affects dopaminergic receptors and that the binding sites labelled by [3H]spiroperidol displaced by haloperidol may be different from those which recognize sulpiride.     

Olfactory bulbectomy increases prepro-enkephalin mRNA levels in the ventral striatum in rats

The effects of bilateral olfactory bulbectomy (OBX) on prepro-enkephalin, thyrotropin-releasing hormone, and D-2 receptor mRNA levels in the ventral striatum were examined by in situ hybridization histochemistry. Pre- pro-enkephalin mRNA levels were significantly increased in the olfactory tubercle (OT), but not in the nucleus accumbens, 14 days following bilateral OBX. Levels of D-2 receptor mRNA were also increased in the OT, though to a lesser degree. Prepro-thyrotropin-releasing hormone mRNA was unaffected by OBX. A separate experiment revealed no effect of OBX on enkephalin gene expression 7 days following surgery but a comparable elevation in pre- pro-enkephalin mRNA 14 and 28 days post-surgery. The findings are consistent with previously-reported effects of dopamine lesions on striatal gene expression, suggesting that the observed effects may be mediated by deafferentation-induced alterations in dopaminergic transmission in the OT. Altered dopaminergic function in the OT may be particularly relevant to the 'anhedonia' that has been associated with the olfactory bulbectomized rat model of depression.     

Blockade of glutamate reuptake in the rat nucleus accumbens increases locomotor activity

The effect on locomotor activity of blocking glutamate reuptake in the nucleus accumbens (NAcc) was investigated in the rat. Bilateral intracranial microinjections into the NAcc of the selective glutamate reuptake blocker, l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), were made in the freely moving rat and locomotor activity subsequently measured for 2 h. Different groups of rats injected with one of three doses of PDC (0.5, 5 or 10 nmole/0.5 microl/side) showed significant dose-dependent increases in both horizontal and vertical locomotor activity relative to control rats that received injections of the saline vehicle. These findings indicate that glutamate in the NAcc plays an important role in the production of locomotor behaviors.     

Mesencephalic dopamine neurons interfacing the shell of nucleus accumbens and the dorsolateral striatum in the rat

Parallel corticostriatonigral circuits have been proposed that separately process motor, cognitive, and emotional‐motivational information. Functional integration requires that interactions exist between neurons participating in these circuits. This makes it imperative to study the complex anatomical substrate underlying corticostriatonigral circuits. It has previously been proposed that dopaminergic neurons in the ventral mesencephalon may play a role in this circuit interaction. Therefore, we studied in rats convergence of basal ganglia circuits by depositing an anterograde neuroanatomical tracer into the ventral striatum together with a retrograde fluorescent tracer ipsilaterally in the dorsolateral striatum. In the mesencephalon, using confocal microscopy, we looked for possible appositions of anterogradely labeled fibers and retrogradely labeled neurons, “enhancing” the latter via intracellular injection of Lucifer Yellow. Tyrosine hydroxylase (TH) immunofluorescence served to identify dopaminergic neurons. In neurophysiological experiments, we combined orthodromic stimulation in the medial ventral striatum with recording from ventral mesencephalic neurons characterized by antidromic stimulation from the dorsal striatum. We observed terminal fields of anterogradely labeled fibers that overlap populations of retrogradely labeled nigrostriatal cell bodies in the substantia nigra pars compacta and lateral ventral tegmental area (VTA), with numerous close appositions between boutons of anterogradely labeled fibers and nigrostriatal, TH‐immunopositive neurons. Neurophysiological stimulation in the medial ventral striatum caused inhibition of dopaminergic nigrostriatal neurons projecting to the ventrolateral striatal territory. Responding nigrostriatal neurons were located in the medial substantia nigra and adjacent VTA. Our results strongly suggest a functional link between ventromedial, emotional‐motivational striatum, and the sensorimotor dorsal striatum via dopaminergic nigrostriatal neurons.