Specific involvement of neurotensin type 1 receptor in the neurotensin-mediated in vivo dopamine efflux using knock-out mice

Abstract Neurotensin is a tridecapeptide neurotransmitter known to be involved in psychiatric disorders, various physiological processes and several different neurobiological mechanisms, including modulation of accumbal dopamine release. Two neurotensin extracellular binding sites, namely NT1- and NT2-receptor (NT1R and NT2R), have been cloned from the rat brain. These receptors are distinguishable by their different in vitro pharmacological properties but the available pharmacological tools have weak in vivo potency and specificity. The use of genetically engineered knock-out mice has provided a powerful alternative to the classical pharmacological approach to investigate their respective roles. In this study, using in vivo differential pulse amperometry, we show that, in wild-type mice, neurotensin application into the ventral tegmental area dose-dependently evokes dopamine efflux in the nucleus accumbens. This neurotensin-mediated efflux is dramatically decreased in mice lacking NT1R while it is unaffected in NT2R-deleted mice. This finding indicates that a large part of the dopamine efflux evoked by neurotensin in the nucleus accumbens of wild-type mice is mediated via NT1R present in the ventral tegmental area.     

Aphagia in the rat following microinjection of neurotensin into the ventral tegmental area

Neurotensin was microinjected into the lateral cerebral ventricle and the ventral tegmental area of rats which had been deprived of food for 18 hours. Both routes of administration resulted in a significant reduction of food intake compared to vehicle control injections. Additionally, the dose of neurotensin required to produce aphagia following ventral tegmental injection was substantially less than the dose required by the ventricular route. The results are discussed in relation to a possible site and mode of action for this neuropeptide.     

Cerebral Metabolic Effects of Monoamine Oxidase Inhibition in Normal and 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Acutely Treated Monkeys

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces dopaminergic cell death in the substantia nigra pars compacta (SNpc) and clinical parkinsonism in humans and experimental animals. Pretreatment with monoamine oxidase inhibitors prevents this cell death and associated parkinsonism by blocking the oxidation of MPTP to a toxic intermediate. The 2-deoxyglucose method was used to study the acute effects of MPTP in the monkey brain and the effects of monoamine oxidase inhibition on local cerebral glucose utilization in both normal and MPTP-treated monkeys. MPTP administration alone caused a major increase in glucose utilization in the SNpc and smaller increases in some subnuclei within the ventral tegmental area in which eventual dopaminergic cell loss also occurs. Pretreatment with pargyline abolished these metabolic increases, a finding suggesting both that the oxidized product of MPTP generates the metabolic increases and that the increased glucose consumption may contribute to cell toxicity. On the other hand, in most cortical, thalamic, striatal, brainstem, and cerebellar areas MPTP alone caused reductions in glucose utilization, and pargyline failed to prevent these effects. Pargyline alone depressed metabolism in the locus coeruleus and a few other monoaminergic structures.     

Long-term desipramine treatment attenuates clonidine-induced suppression of ventral tegmental self-stimulation

Long-term administration of the tricyclic antidepressant desipramine did not change the rate of self-stimulation responding in the A10 (ventral tegmental) area but significantly attenuated the suppressive effect of the selective alpha 2-adrenergic agonist clonidine on this behavior. These findings demonstrate an involvement of noradrenergic mechanisms in the regulation of ventral tegmental self-stimulation and further suggest that adaptive changes in inhibitory presynaptic noradrenergic receptors may be involved in desipramine's antidepressant effects.     

Haloperidol but Not Clozapine Increases Neurotensin Receptor mRNA Levels in Rat Substantia Nigra

Abstract: We examined the effects of chronic (2 weeks) treatment with a typical neuroleptic, haloperidol (1 mg/kg, s.c.), and an atypical neuroleptic, clozapine (20 mg/kg, s.c.), on neurotensin receptor (NTR) mRNA levels by in situ hybridization histochemistry. Quantitative OD analysis showed haloperidol-induced NTR mRNA levels in the substantia nigra/ventral tegmental area (SN/ VTA) 110% over control levels (significant difference from the control, p < 0.05). In contrast, the same analysis applied to the sections from clozapine-treated animals showed no significant change in NTR mRNA levels compared with matched control sections ( p > 0.05). Thus, chronic treatment with haloperidol but not clozapine resulted in elevated levels of NTR mRNA within the SN/VTA. These results suggest that the high incidence of extrapyramidal side effects of typical neuroleptics could result from changes in NTR expression in the SN/VTA.     

Habenular modulation of dynorphinergic systems in rat ventral mesencephalon

Bilateral electrolytic lesion of the striatonigral pathways (which convey massive afferents to the substantia nigra) caused a marked lowering of alpha-neo-endorphin (alpha-Neo) and dynorphin A(1-8) [Dyn A(1-8)] levels in the substantia nigra without affecting the alpha-Neo content in the ventral tegmental area. Moreover, unilateral infusion of the axon sparing neurotoxin ibotenate into the striatum, but not into the substantia nigra, decrease these two opioid peptides in the substantia nigra on the side ipsilateral to the lesion, failing to modify the alpha-Neo levels in the ventral tegmental area. Bilateral electrolytic lesion of the habenula augmented alpha-Neo content in the substantia nigra and ventral tegmental area at 8-30 days postlesion without affecting the nigral Dyn A(1-8). These results add further support to the view that alpha-Neo- and Dyn A(1-8)-containing neurons projecting to the substantia nigra originate in the striatum and descend through striatonigral pathways. The present data provide evidence that the habenula may participate in the regulation of the activity of alpha-Neo-immunoreactive neurons in the substantia nigra and ventral tegmental area.     

Release of dopamine in the ventral tegmental area of rat

In this preliminary report we showed that 3,4-dihydroxyphenylacetic acid (DOPAC), the major metabolite of dopamine (DA), is present in the ventral tegmental area. This finding indicates that in the ventral tegmental area, which contains the cell bodies of dopaminergic neurons of the mesocortical and mesolimbic DA systems, DA may be released by a mechanism similar to that operating in the nerve endings. However, haloperidol, which increases DOPAC levels in the substantia nigra, failed to do so in the ventral tegmental area. The results support the contention that DA neurons in the ventral tegmental area have distinctive features from nigral DA neurones.     

Modulation of cerebral acetylcholine metabolism by the dorsal diencephalic conduction system in the rat

We investigated the effects of interruption of the impulse flow in the habenulopeduncular pathways by local infusion of tetrodotoxin on the acetylcholine and choline content in selected dopamine rich regions in the forebrain and midbrain in rats. The tetrodotoxin infusion caused a marked increase in acetylcholine content in the medial frontal cortex, striatum and ventral tegmental area+interpeduncular nucleus, but not in the limbic area or the substantia nigra, whereas choline content was reduced only in both the striatum and ventral tegmental area+interpeduncular nucleus. There was an increase in 3,4-dihydroxyphenylacetic acid content in the striatum after the manipulation. These findings suggest that the dorsal diencephalic conduction system may be involved in the integration of the activity of cholinergic neurons in the forebrain and midbrain regions and striatal dopanine neurons may play a role in the modulation of cholinergic neurons.     

Receptors in the Ventral Tegmental Area Mediating Nicotine-Induced Dopamine Release in the Nucleus Accumbens

Nicotine or cocaine, when administered intravenously, induces an increase of extracellular dopamine in the nucleus accumbens. The nicotine-mediated increase was shown to occur at least in part through increase of the activity of dopamine neurons in the ventral tegmental area. As part of our continuing studies of the mechanisms of nicotine effects in the brain, in particular, effects on reward and cognitive mechanisms, in the present study we examined the role of various receptors in the ventral tegmental area in nicotine and cocaine reward. We assayed inhibition of the increase of dopamine in the nucleus accumbens induced by intravenous nicotine or cocaine administration by antagonists administered into the ventral tegmental area. Nicotine-induced increase of accumbal dopamine release was inhibited by intrategmental nicotinic (mecamylamine), muscarinic (atropine), dopaminergic (D₁: SCH 23390, D₂: eticlopride), and NMDA glutamatergic (MK 801) and GABA_B (saclofen) antagonists, but not by AMPA-kainate (CNQX, GYKI-52466) antagonists under our experimental circumstances. The intravenous cocaine-induced increase of dopamine in the nucleus accumbens was inhibited by muscarinic (atropine), dopamine 2 (eticlopride), and GABA_B (saclofen) antagonists but not by antagonists to nicotinic (mecamylamine), dopamine D₁ (SCH 23390), glutamate (MK 801), or AMPA-kainate (CNQX, GYKI-52466) receptors. Antagonists administered in the ventral tegmental area in the present study had somewhat different effects when they were previously administered intravenously. When administered intravenously atropine did not inhibit cocaine effects. The inhibition by atropine may be indirect, since this compound, when administered intrategmentally, decreased basal dopamine levels in the accumbens. The findings indicate that a number of receptors in the ventral tegmental area mediate nicotine-induced dopamine changes in the nucleus accumbens, a major component of the nicotine reward mechanism. Some, but not all, of these receptors in the ventral tegmental area also seem to participate in the reward mechanism of cocaine. The importance of local receptors in the ventral tegmental area was further indicated by the increase in accumbal dopamine levels after intrategmental administration of nicotine or also cocaine.     

Central integration of cardiovascular and drinking responses elicited by central administration of angiotensin II: divergence of regulation by the ventral tegmental area and nucleus accumbens

Previous studies had implicated the involvement of the ventral tegmental area and its dopamine projections to the nucleus accumbens in goal-directed behavior. This study investigated whether or not the GABAergic inputs to the ventral tegmental area and, in turn, dopaminergic input to the nucleus accumbens from the ventral tegmental area modify drinking and cardiovascular responses elicited by central administration of angiotensin II. Injections of 25 ng of angiotensin II into a lateral cerebral ventricle of the rat elicited water intakes averaging 7-8 mL in 15 min with latencies usually less than 3 min. Pretreatment of the nucleus accumbens with spiperone, a dopamine antagonist, or the ventral tegmental area with gamma-amino butyric acid (GABA) produced dose-dependent reductions in water intake and number of laps taken while increasing the latency to drink. The spiperone injection did not alter the pressor response. On the other hand, the GABA injections attenuated the pressor responses to central angiotensin II administration. These findings suggest that GABA input to the ventral tegmental area modifies both the cardiovascular and drinking responses elicited following central administration of angiotensin II. However, the dopamine projections to the nucleus accumbens appear to be involved only in the drinking responses elicited by central injections of angiotensin II. Divergence for the coordination of the skeletal motor behavioral component and the cardiovascular component elicited by central administration of angiotensin II must occur before the involvement of these dopamine pathways.     

Neurotensin and Neuromedin N Are Differently Metabolized in Ventral Tegmental Area and Nucleus Accumbens

Whole homogenates and membrane-bound and cytosoluble fractions prepared from rat ventral tegmental area (VTA) and nucleus accumbens were examined for their content of peptidasic activities and for their ability to metabolize neurotensin and its natural related hexapeptide neuromedin N. No qualitative differences were observed between these two brain regions concerning the presence and the subcellular distribution of a series of activities able to hydrolyze various specific fluorimetric enzymatic substrates. However, aminopeptidase B, endopeptidase 24-15, and endopeptidase 24-11 were significantly lower in the VTA than in the nucleus accumbens membrane preparations, while proline endopeptidase was detected in significantly higher amount only in the cytosolic fraction prepared from nucleus accumbens. Both neurotensin and neuromedin N were metabolized more rapidly in the nucleus accumbens than in the VTA. Furthermore, the degradation rate of neuromedin N was considerably faster than that of neurotensin whatever the cerebral area examined. Studies carried out with highly specific peptidase inhibitors revealed that endopeptidase 24-15 mainly contributed to the catabolism of neurotensin in homogenates and membrane-bound preparations of nucleus accumbens and VTA, while aminopeptidase B appeared predominantly responsible for the rapid disappearance of neuromedin N in both cerebral tissues. The possibility that the different metabolic processes of the two peptide congeners could explain their distinct pharmacological profiles observed after their microinjection in the nucleus accumbens and in the VTA is discussed.     

Orphanin FQ/Nociceptin Modulation of Mesolimbic Dopamine Transmission Determined by Microdialysis

Orphanin FQ has been reported to suppress extracellular dopamine levels in the nucleus accumbens after intracerebroventricular administration. This study sought to provide evidence for an intra-ventral tegmental site of action for this effect using a dual-probe microdialysis experimental design. Orphanin FQ was applied to the ventral tegmental area of anesthetized rats by reverse dialysis while extracellular dopamine was sampled with a second dialysis probe in the nucleus accumbens. Orphanin FQ at a probe concentration of 1 mM (but not at 0.1 mM) significantly reduced nucleus accumbens dialysate dopamine levels. The receptor-inactive analogue, des-Phe1-orphanin FQ (1 mM), produced a small but significant increase in nucleus accumbens dialysate dopamine levels. Simultaneous measurement of ventral tegmental area dialysate amino acid content revealed significant increases in both GABA and glutamate during infusion of orphanin FQ (1 mM). To determine if increased GABA overflow mediates the action of orphanin FQ on mesolimbic neurons, orphanin FQ (10 nmol) was microinjected directly into the ventral tegmental area in the presence or absence of the GABA(A) receptor antagonist, bicuculline (1 nmol). Bicuculline transiently blocked the suppressive action of orphanin FQ on accumbens dialysate dopamine levels. These data indicate that orphanin FQ decreases dopamine transmission in the nucleus accumbens by inhibiting dopamine neuronal activity in the ventral tegmental area through a mechanism that may involve an increased overflow of GABA.     

Changes in local cerebral glucose utilization associated with Parkinson's syndrome induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the primate

The quantitative 2-[14C]deoxyglucose autoradiographic method was used to map the pattern of alterations in local cerebral glucose utilization associated with the Parkinsonian syndrome induced by the administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to rhesus monkeys. Monkeys treated with the neurotoxin exhibited both behavioral symptoms (e.g. akinesia, rigidity, flexed posture, and eyelid closure) and neuropathological changes (degeneration of the cells of the substantia nigra pars compacta) that closely paralleled those in human Parkinson's disease. Glucose utilization was significantly reduced in the pars compacta of the substantia nigra and in the subthalamus, and increased in the external segment of the globus pallidus. Outside the basal ganglia reductions in glucose utilization were limited to the mediodorsal nucleus of the thalamus, frontal eye fields, and ventral tegmental area. The results of these studies indicate that the profound functional and behavioral deficits in MPTP-induced Parkinson's syndrome are the consequences of highly selective functional changes in a few cerebral structures, mainly within the basal ganglia.     

Neurochemical changes following kainic acid lesions of the nucleus accumbens: implications for a GABAergic accumbal-ventral tegmental pathway.

Unilateral injection of kainic acid into the nucleus accumbens of the rat produced moderate depletions of the GABA synthesising enzyme glutamic acid decarboxylase in the accumbens and ventral tegmental area, but failed to alter these parameters in the striatum or substantia nigra. Similar injections into the striatum produced opposite effects to those seen following accumbens injections. These results are consistent with a GABA-ergic accumbal-ventral tegmental pathway analogous to the well defined striatonigral pathway. However, alternative interpretations, possibly in terms of a non-GABAergic accumbal-ventral tegmental pathway modulating GABA interneurons intrinsic to the tegmentum, must be considered.     

Reciprocal opioid-opioid interactions between the ventral tegmental area and nucleus accumbens regions in mediating mu agonist-induced feeding in rats

Feeding elicited by the mu-selective agonist, [D-Ala2, M-Phe4, Gly-ol5]-encephalin administered into the nucleus accumbens is blocked by accumbal pre-treatment with mu, delta1, delta2 and kappa, but not mu1 opioid antagonists. Correspondingly, mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by ventral tegmental area pre-treatment with mu and kappa, but not delta opioid antagonists. A bi-directional opioid-opioid feeding interaction has been firmly established such that mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by accumbal naltrexone, and that accumbal mu-agonist-induced feeding is blocked by naltrexone pre-treatment in the ventral tegmental area. To determine which opioid receptor subtypes mediate the regional bi-directional opioid-opioid feeding interactions between these two sites, the present study examined the dose-dependent ability of either general (naltrexone), mu (beta-funaltrexamine), kappa (nor-binaltorphamine) or delta (naltrindole) opioid antagonists administered into one site to block mu-agonist-induced feeding elicited from the other site. General, mu and kappa, but not delta opioid receptor antagonist pre-treatment in the ventral tegmental area dose-dependently reduced mu-agonist-induced feeding elicited from the nucleus accumbens. General, mu and delta, and to a lesser degree kappa, opioid receptor antagonist pre-treatment in the nucleus accumbens dose-dependently reduced mu-agonist-induced feeding elicited from the ventral tegmental area. Thus, multiple, but different opioid receptor subtypes are involved in mediating opioid-opioid feeding interactions between the nucleus accumbens and ventral tegmental area regions.     

海洛因成瘾模型建立及中脑腹侧被盖区Bax的表达

目的探索海洛因对中脑腹侧被盖区细胞Bax表达的影响。方法肌肉注射海洛因,建立成瘾大鼠模型,用免疫组化方法检测中脑腹侧被盖区细胞Bax的表达。结果连续给大鼠注射海洛因7d后,大鼠出现明显的戒断症状;中脑腹侧被盖区细胞Bax表达阳性细胞比对照组明显增多,与对照组相比差异有显著性（P〈0.01）。结论海洛因具有诱导Bax基因表达、损伤脑组织细胞的作用。     

Acute and Repeated Systemic Amphetamine Administration: Effects on Extracellular Glutamate, Aspartate, and Serine Levels in Rat Ventral Tegmental Area and Nucleus Accumbens

Abstract: Recent work indicates an important role for excitatory amino acids in behavioral sensitization to amphetamine. We therefore examined, using in vivo microdialysis in awake rats, the effects of amphetamine on efflux of glutamate, aspartate, and serine in the ventral tegmental area and nucleus accumbens, brain regions important for the initiation and expression of amphetamine sensitization, respectively. Water-pretreated and amphetamine-pretreated rats were compared to determine if sensitization altered such effects. In both brain regions, Ca²⁺-dependent efflux of glutamate accounted for ∼20% of basal glutamate efflux. A challenge injection of water or 2.5 mg/kg of amphetamine did not significantly alter glutamate, aspartate, or serine efflux in the ventral tegmental area or nucleus accumbens of water- or amphetamine-pretreated rats. However, 5 mg/kg of amphetamine produced a gradual increase in glutamate efflux in both regions that did not reverse, was observed in both water- and amphetamine-pretreated rats, and was prevented by haloperidol. Although increased glutamate efflux occurred with too great a delay to mediate acute behavioral responses to amphetamine, it is possible that repeated augmentation of glutamate efflux during repeated amphetamine administration results in compensatory changes in levels of excitatory amino acid receptors in the ventral tegmental area and nucleus accumbens that contribute to development or expression of amphetamine sensitization.     

Leptin action via neurotensin neurons controls orexin, the mesolimbic dopamine system and energy balance

Leptin acts on leptin receptor (LepRb)-expressing neurons throughout the brain, but the roles for many populations of LepRb neurons in modulating energy balance and behavior remain unclear. We found that the majority of LepRb neurons in the lateral hypothalamic area (LHA) contain neurotensin (Nts). To investigate the physiologic role for leptin action via these LepRb(Nts) neurons, we generated mice null for LepRb specifically in Nts neurons (Nts-LepRbKO mice). Nts-LepRbKO mice demonstrate early-onset obesity, modestly increased feeding, and decreased locomotor activity. Furthermore, consistent with the connection of LepRb(Nts) neurons with local orexin (OX) neurons and the ventral tegmental area (VTA), Nts-LepRbKO mice exhibit altered regulation of OX neurons and the mesolimbic DA system. Thus, LHA LepRb(Nts) neurons mediate physiologic leptin action on OX neurons and the mesolimbic DA system, and contribute importantly to the control of energy balance.     

Short- and Long-Term Alterations of Gene Expression in Limbic Structures by Repeated Electroconvulsive-Induced Seizures

Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.     

Different Interactions of Prolyl Oligopeptidase and Neurotensin in Dopaminergic Function of the Rat Nigrostriatal and Mesolimbic Pathways

Prolyl oligopeptidase (PREP) is an intracellular enzyme digesting small proline-containing peptides. Since PREP resides the same brain areas as neurotensin in the nigrostriatal and mesolimbic dopaminergic pathways, we were interested to study if there is an intracellular interaction between them. A colocalization of PREP with neurotensin and neurotensin receptor 1 (NTS1) in the rat striatum, nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) was studied with immunofluorescence. From the same brain areas, the levels of dopamine and its metabolites were measured 1 h after the injection of saline, NTS1 ligands (JMV-449; 5 μg) or antagonist (SR142948; 5 μg) to the rat striatum or NAcc. We also studied whether an intraperitoneal injection of a PREP inhibitor (KYP-2047; 5 mg/kg) affects the levels of dopamine and its metabolites alone or modifies the effects of the NTS1 ligands. PREP was highly colocalized with neurotensin and NTS1 in the VTA, and with NTS1 in the SN. Colocalization was moderate or low in other brain areas. When injected to the striatum, JMV-449 had a tendency to increase dopamine (p = 0.052) and metabolite levels in the striatum and SN, whereas SR142948 did not. After the injection to the NAcc, JMV-449 but not SR142948, increased dopamine levels in the VTA and dopamine metabolite levels in the NAcc and VTA. KYP-2047 decreased the dopamine levels in the striatum, but increased dopamine metabolite levels in the NAcc and VTA. Our results suggest a novel role for PREP in the modulation of dopaminergic transmission, which may be different in nigrostriatal and mesolimbic pathways.