Bupropion and desipramine increase dopamine transporter mRNA expression in the ventral tegmental area/substantia nigra of rat brain

The homeobox gene Otx2 is a mouse cognate of the Drosophila orthodenticle gene, which is required for development of the brain, rostral to rhombomere three. We have investigated the mechanisms involved in this neural function and specifically the requirement for Otx2 in the visceral endoderm and the neuroectoderm using chimeric analysis in mice and explant recombination assay. Analyses of chimeric embryos composed of more than 90% of Otx2-/- ES cells identified an essential function for Otx2 in the visceral endoderm for induction of the forebrain and midbrain. The chimeric studies also demonstrated that an anterior neural plate can form without expressing Otx2. However, in the absence of Otx2, expression of important regulatory genes, such as Hesx1/Rpx, Six3, Pax2, Wnt1 and En, fail to be initiated or maintained in the neural plate. Using explant-recombination assay, we could further demonstrate that Otx2 is required in the neuroectodem for expression of En. Altogether, these results demonstrate that Otx2 is first required in the visceral endoderm for the induction, and subsequently in the neuroectoderm for the specification of forebrain and midbrain territories.     

Haloperidol induces persistent down-regulation of tyrosine hydroxylase immunoreactivity in substantia nigra but not ventral tegmental area in the rat

The dopamine antagonist haloperidol can cause tardive side-effects that may persist after the drug is withdrawn. We studied the time course of changes in dopaminergic neurons of the substantia nigra and ventral tegmental area following withdrawal of haloperidol. Rats received daily intraperitoneal injections of saline or haloperidol for eight weeks and were killed at two, four or 12 weeks after the final injection. Sections of substantia nigra and ventral tegmental area were processed for tyrosine hydroxylase immunohistochemistry. Quantitative morphometric analysis was carried out blinded in order to determine the number, cell body size and topography of tyrosine hydroxylase-positive cells, and the immunoreactive area of the substantia nigra and ventral tegmental area. In haloperidol-treated rats, tyrosine hydroxylase-positive cell counts were normal in ventral tegmental area but were decreased in substantia nigra by 34% at two weeks withdrawal and by 52% at four weeks withdrawal; cell counts were almost fully recovered by 12 weeks withdrawal. Cross-sectional area of tyrosine hydroxylase immunoreactivity within the substantia nigra demonstrated a similar pattern of reduction, with full recovery by 12 weeks withdrawal. Mean cell size, by contrast, was essentially unchanged at two and four weeks withdrawal, but was significantly decreased in sub-regions of substantia nigra at 12 weeks withdrawal. These results indicate that haloperidol can produce selective changes in midbrain dopamine neurons that persist long after discontinuation of the drug. This decrease in tyrosine hydroxylase-immunoreactive cell counts may play a role in the neurobiology of the persistent tardive syndromes associated with the use of neuroleptics.     

Regulation of substantia nigra dopamine neurons

A substantial amount of research has focused on determining the factors that alter the activity of substantia nigra dopamine neurons. Much of this research has indicated that several mechanisms that regulate dopamine neuron activity have the capability to maintain the baseline activity of dopamine cells at a fairly constant rate. For example, the intrinsic membrane conductances present on dopamine neurons, which generate the spike activity of these cells, appear to maintain the activity of spontaneously active neurons and suppress the induction of activity in quiescent cells. In addition, dopamine cell activity can be regulated by afferent systems that appear to be capable of preventing dopamine neurons from displaying sustained variations in electrophysiological activity. Specifically, inputs from the striatum or from the subthalamic nucleus may each exert opposing influences on dopamine cell activity via direct vs. indirect afferent projection pathways. In addition, the dendritic release of dopamine may provide negative feedback; dopamine cell firing may increase the dendritic release of dopamine within the substantia nigra, providing a local feedback inhibition of dopamine neuron activity. Factors such as the intrinsic membrane properties, afferent input, and the dendritic release of dopamine all work together in a complex manner to regulate the activity level of dopamine neurons.     

N-methyl-D-aspartate receptor antagonism in the ventral tegmental area diminishes the systemic nicotine-induced dopamine release in the nucleus accumbens

Systemic nicotine enhances burst firing of dopamine neurons in the ventral tegmental area and dopamine release in the nucleus accumbens, mainly via stimulation of nicotinic acetylcholine receptors in the ventral tegmental area. Given that both the neuronal activity of mesolimbic dopamine neurons and terminal dopamine release are regulated by excitatory amino acid inputs to the ventral tegmental area and that nicotine facilitates glutamatergic transmission in brain, we investigated the putative role of ionotropic glutamate receptors within the ventral tegmental area for the effects of nicotine on dopamine release in the nucleus accumbens using microdialysis, with one probe implanted in the ventral tegmental area for drug application and another in the ipsilateral nucleus accumbens for measuring dopamine, in awake rats. Systemic nicotine (0.5 mg/kg, s.c.) and infusion of nicotine (1.0 mM) into the ventral tegmental area increased dopamine output in the nucleus accumbens. Intrategmental infusion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (0.1 mM) or N-methyl-D-aspartate (0.3 mM) increased accumbal dopamine release; these effects were antagonized by concomitant infusion of a selective antagonist at N-methyl-D-aspartate receptors, 2-amino-5-phosphonopentanoic acid (0.3 mM), and non-N-methyl-D-aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (0.3 mM), respectively. Infusion of either antagonist (0.3 or 1.0 mM) into the ventral tegmental area did not affect basal dopamine levels, whereas infusion of 2-amino-5-phosphonopentanoic acid, but not 6-cyano-7-nitroquinoxaline-2,3-dione, starting 40 min before nicotine injection dose-dependently attenuated the nicotine-induced increase in accumbal dopamine release. Concurrent intrategmental infusion of 2-amino-5-phosphonopentanoic acid and nicotine decreased nicotine-induced dopamine release in the nucleus accumbens. These results indicate that the stimulatory action of nicotine on the mesolimbic dopamine system is to a considerable extent mediated via stimulation of N-methyl-D-aspartate receptors within the ventral tegmental area.     

Activity of presumed dopamine neurons in the ventral tegmental area during heroin self-administration

To assess the pattern of mesocorticolimbic dopamine (DA) activity associated with drug-seeking and drug-taking behavior, we monitored the firing rate of presumed DA neurons in the ventral tegmental area of trained rats during i.v. heroin self-administration (SA). Relative to a slow and irregular basal activity, the first SA of each session was preceded by a phasic increase and followed by a more persistent increase in discharge rate that peaked approximately 15-20 min later at the time of the second SA. All subsequent SAs were associated with a biphasic neuronal change: a transient decrease followed by a gradual increase that peaked just before the next SA. Our results support mesocorticolimbic DA activation in heroin-seeking behavior but suggest a transient inhibition of DA activity correlated with heroin reward.     

Systemic morphine-induced Fos protein in the rat striatum and nucleus accumbens is regulated by mu opioid receptors in the substantia nigra and ventral tegmental area

To characterize how systemic morphine induces Fos protein in dorsomedial striatum and nucleus accumbens (NAc), we examined the role of receptors in striatum, substantia nigra (SN), and ventral tegmental area (VTA). Morphine injected into medial SN or into VTA of awake rats induced Fos in neurons in ipsilateral dorsomedial striatum and NAc. Morphine injected into lateral SN induced Fos in dorsolateral striatum and globus pallidus. The morphine infusions produced contralateral turning that was most prominent after lateral SN injections. Intranigral injections of [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), a mu opioid receptor agonist, and of bicuculline, a GABAA receptor antagonist, induced Fos in ipsilateral striatum. Fos induction in dorsomedial striatum produced by systemic administration of morphine was blocked by (1) SN and VTA injections of the mu1 opioid antagonist naloxonazine and (2) striatal injections of either MK 801, an NMDA glutamate receptor antagonist, or SCH 23390, a D1 dopamine receptor antagonist. Fos induction in dorsomedial striatum and NAc after systemic administration of morphine seems to be mediated by dopamine neurons in medial SN and VTA that project to medial striatum and NAc, respectively. Systemic morphine is proposed to act on mu opioid receptors located on GABAergic interneurons in medial SN and VTA. Inhibition of these GABA interneurons disinhibits medial SN and VTA dopamine neurons, producing dopamine release in medial striatum and NAc. This activates D1 dopamine receptors and coupled with the coactivation of NMDA receptors possibly from cortical glutamate input induces Fos in striatal and NAc neurons. The modulation of target gene expression by Fos could influence addictive behavioral responses to opiates.     

Projections of the ventral tegmental area of the midbrain, the substantia nigra, and the amygdaloid body in different parts of the putamen in the dog

INTRODUCTION: We investigated the accuracy of endorectal coil Magnetic Resonance Imaging (MRI) and Fast Spin Echo (FSE) technique in staging prostate cancer. MATERIAL AND METHODS: MRI was performed in 70 patients with biopsy proved prostatic cancer. A total of 33 patients subsequently underwent radical prostatectomy. T2-weighted FSE sequences (TR 3400-4100, TE 120, Echo train length 13) were acquired in all patients. Axial, sagittal and coronal 4-5 mm images were obtained with 13-14 cm field of view and 256 x 256 matrix. Additional T1-weighted spin echo images were acquired in 9 patients. Lesion staging on MR images was performed according to the American Urological System. MR data were compared with the pathologic findings of whole-mount sections of the surgical specimens. RESULTS: Overall accuracy for endorectal coil MR imaging was 60%; ten cases were underestimated and 3 cases were overestimated. The sensitivity and the specificity of endorectal coil MRI in diagnosing capsular penetration were 77% and 81%, respectively. Seminal vesicle invasion was detected with 87% sensitivity and 96% specificity. CONCLUSIONS: Endorectal coil MRI provides a more accurate preoperative local staging.     

A subset of ventral tegmental area neurons is inhibited by dopamine, 5-hydroxytryptamine and opioids

Neurons originating in the ventral tegmental area are thought to play a key role in the formation of addictive behaviors, particularly in response to drugs such as cocaine and opioids. In this study we identified different populations of ventral tegmental area neurons by the pharmacology of their evoked synaptic potentials and their response to dopamine, 5-hydroxytryptamine and opioids. Intracellular recordings were made from ventral tegmental area neurons in horizontal slices of guinea-pig brain and electrical stimulation was used to evoke synaptic potentials. The majority of cells (61.3%) hyperpolarized in response to dopamine, depolarized to 5-hydroxytryptamine, failed to respond to [Met]5enkephalin and exhibited a slow GABAB-mediated inhibitory postsynaptic potential. A smaller proportion of cells (11.3%) hyperpolarized in response to [Met]5enkephalin, depolarized to 5-hydroxytryptamine, failed to respond to dopamine and did not exhibit a slow inhibitory postsynaptic potential. These two groups of cells corresponded to previously described "principal" and "secondary" cells, respectively. A further group of cells (27.4%) was identified that like the principal cells, hyperpolarized to dopamine. However, these "tertiary cells" also hyperpolarized to both 5-hydroxytryptamine and [Met]5enkephalin and exhibited a slow, cocaine-sensitive 5-hydroxytryptamine(1A)-mediated inhibitory postsynaptic potential. When principal and tertiary cells were investigated immunohistochemically, 82% of the principal cells were positive for tyrosine hydroxylase compared with only 29% of the tertiary cells. The 5-hydroxytryptamine innervation of both these cell types was investigated and a similar density of putative contacts was observed near the somata and dendrites in both groups. This latter finding suggests that the existence of a 5-hydroxytryptamine-mediated inhibitory postsynaptic potential in the tertiary cells may be determined by the selective expression of 5-hydroxytryptamine receptors, rather than the distribution or density of the 5-hydroxytryptamine innervation. We conclude that tertiary cells are a distinct subset of ventral tegmental area neurons where cocaine and mu-opioids both mediate inhibition.     

Immunogold localization of the dopamine transporter: an ultrastructural study of the rat ventral tegmental area

The dopamine transporter (DAT) plays an important role in the plasmalemmal reuptake of dopamine and, thus, in the termination of normal dopaminergic neurotransmission. DAT is also a major binding site for cocaine and other stimulants, the psychoactive effects of which are associated primarily with the inhibition of dopamine reuptake within mesocorticolimbic dopaminergic neurons. We used electron microscopy with an anti-peptide antiserum directed against the N-terminal domain of DAT to determine the subcellular localization of this transporter in the rat ventral tegmental area (VTA), the region that contains the cell bodies and dendrites of these dopaminergic neurons. We show that in the VTA, almost 95% of the DAT immunogold-labeled profiles are neuronal perikarya and dendrites, and the remainder are unmyelinated axons. Within perikarya and large proximal dendrites, almost all of the DAT immunogold particles are associated with intracellular membranes, including saccules of Golgi and cytoplasmic tubulovesicles. In contrast, within medium- to small-diameter dendrites and unmyelinated axons, most of the DAT gold particles are located on plasma membranes. In dually labeled tissue, peroxidase reaction product for the catecholamine-synthesizing enzyme tyrosine hydroxylase is present in DAT-immunoreactive profiles. These findings suggest that intermediate and distal dendrites are both the primary sites of dopamine reuptake and the principal targets of cocaine and related psychostimulants within dopaminergic neurons in the VTA.     

Excitotoxic lesions of the prefrontal cortex reduce dopamine D1-like receptors in the ventral tegmental area

Ventral mesencephalic dopamine D1-like receptors were quantified in brains of male rats ten days after unilateral microinjections of ibotenic acid (2 or 10 microg/microl) or its vehicle into the medial prefrontal cortex. The density of dopamine D1-like receptors was reduced by more than 40% in the ipsilateral ventral tegmental area (both doses) and by 15% (low dose) and 44% (high dose) in the contralateral side; no significant reduction was observed in the substantia nigra. These results suggest that a significant number of ventral tegmental D1-like receptors are localized on afferent terminals from the medial prefrontal cortex.     

Metabotropic glutamate receptor-mediated inhibition and excitation of substantia nigra dopamine neurons

Microiontophoretic drug application and extracellular recording techniques were used to evaluate the effects of the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate(1S,3R-ACPD) on dopamine (DA) neurons in the substantia nigra zona compacta (SNZC) of chloral hydrate-anesthetized rats. 1S,3R-ACPD had a biphasic effect on the firing rate of DA cells, initially decreasing, then increasing the firing rate. 1S,3R-ACPD also increased the burst-firing activity of DA neurons. Application of the ionotropic receptor (iGluR) agonists (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) or N-methyl-D-aspartate (NMDA) increased the firing rates of neurons which had responded to 1S,3R-ACPD, indicating that mGluRs and iGluRs reside on the same neurons. The initial inhibitory period was not antagonized by systemic haloperidol or iontophoretic bicuculline, indicating a lack of DA or gamma-amino-n-butyric acid (GABA) involvement in this effect. Combined application of the AMPA antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), and the NMDA antagonist, (I)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphoric acid (CPP), at currents which antagonized AMPA and NMDA, did not antagonize either the inhibitory or excitatory effects of 1S,3R-ACPD. Application of the metabotropic antagonist (S)-4-carboxy-phenylglycine antagonized both the inhibitory and excitatory effects of 1S,3R-ACPD. These results indicate that mGluRs may play a role in the modulation of dopaminergic activity in the SNZC.     

Electrophysiological characterization of GABAergic neurons in the ventral tegmental area

GABAergic neurons in the ventral tegmental area (VTA) play a primary role in local inhibition of mesocorticolimbic dopamine (DA) neurons but are not physiologically or anatomically well characterized. We used in vivo extracellular and intracellular recordings in the rat VTA to identify a homogeneous population of neurons that were distinguished from DA neurons by their rapid-firing, nonbursting activity (19.1 +/- 1.4 Hz), short-duration action potentials (310 +/- 10 microseconds), EPSP-dependent spontaneous spikes, and lack of spike accommodation to depolarizing current pulses. These non-DA neurons were activated both antidromically and orthodromically by stimulation of the internal capsule (IC; conduction velocity, 2.4 +/- 0.2 m/sec; refractory period, 0.6 +/- 0.1 msec) and were inhibited by stimulation of the nucleus accumbens septi (NAcc). Their firing rate was moderately reduced, and their IC-driven activity was suppressed by microelectrophoretic application or systemic administration of NMDA receptor antagonists. VTA non-DA neurons were recorded intracellularly and showed relatively depolarized resting membrane potentials (-61.9 +/- 1.8 mV) and small action potentials (68.3 +/- 2.1 mV). They were injected with neurobiotin and shown by light microscopic immunocytochemistry to be multipolar cells and by electron microscopy to contain GABA but not the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Neurobiotin-filled dendrites containing GABA received asymmetric excitatory-type synapses from unlabeled terminals and symmetric synapses from terminals that also contained GABA. These findings indicate that VTA non-DA neurons are GABAergic, project to the cortex, and are controlled, in part, by a physiologically relevant NMDA receptor-mediated input from cortical structures and by GABAergic inhibition.     

Striatal extracellular dopamine levels in rats with haloperidol-induced depolarization block of substantia nigra dopamine neurons

Correlations between substantia nigra (SN) dopamine (DA) cell activity and striatal extracellular DA were examined using simultaneous extracellular single-unit recordings and in vivo microdialysis performed in drug-naive rats and in rats treated repeatedly with haloperidol (HAL). Intact rats treated with HAL for 21-28 d exhibited significantly fewer active DA cells, indicating the presence of depolarization block (DB) in these cells. However, in rats that received surgical implantation of the microdialysis probe followed by a 24 hr recovery period, HAL-induced DA cell DB was reversed, as evidenced by a number of active DA neurons that was significantly higher than that in HAL-treated intact rats and similar to that of drug-naive rats. In contrast, using a modified probe implantation procedure that did not reverse SN DA neuron DB, we found striatal DA efflux to be significantly lower than in controls and significantly correlated with the reduction in DA neuron spike activity. Furthermore, although basal striatal DA efflux was independent of SN DA cell burst-firing activity in control rats, these variables were significantly correlated in rats with HAL-induced DA cell DB. Therefore, HAL-induced DB of SN DA neurons is disrupted by implantation of a microdialysis probe into the striatum using standard procedures. However, a modified microdialysis method that allowed reinstatement of DA neuron DB revealed that the HAL-induced inactivation of SN DA neurons was associated with significantly lower extracellular DA levels in the striatum. Moreover, the residual extracellular DA maintained in the presence of DB may, in part, depend on the burst-firing pattern of the noninactivated DA neurons in the SN.     

Behavioral depression during cocaine withdrawal is associated with decreased spontaneous activity of ventral tegmental area dopamine neurons.

Withdrawal from an escalating-dose, bingelike regimen of cocaine administration in rats produced significantly depressed levels of locomotor activity during the nocturnal portion of the day-night cycle. This effect was observed during the first 48 hrs of testing. Extracellular single-unit recordings of ventral tegmental area (VTA) dopamine (DA) neurons revealed no differences between saline- and cocaine-treated rats with respect to basal firing rates. However, significantly fewer spontaneously active VTA DA neurons were encountered in rats withdrawn from binge cocaine. As with the nocturnal hypoactivity, this effect was observed only during the first 48 hrs of withdrawal. These findings suggest that short-term DA neuron dysfunction during cocaine withdrawal temporally corresponds to behavioral disruptions that are similar to those described in human addicts. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Central mediation of naloxone-induced anorexia in the ventral tegmental area.

Central naloxone injections were used to show that endogenous opioids in the ventral tegmental area (VTA) regulate consumption of palatable foods. Peripheral injections of naloxone were more effective in reducing the consumption of a sweet solution in normally fed rats than in animals maintained at 85% of their free-feeding body weight. A dose of 10 μg/side naloxone injected into the VTA reduced consumption in normally fed rats, whereas a dose of 25 μg/side did the same in food-restricted animals. The inactive isomer, (+) naloxone, did not reduce consumption; the effect has anatomical specificity. Naloxone effectively decreases the eating of palatable foods, but not eating for survival. This has important implications for the use of opioid antagonists in weight-loss programs. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The pharmacology of mesocortical dopamine neurons: a dual-probe microdialysis study in the ventral tegmental area and prefrontal cortex of the rat brain

The development of receptor function at corticothalamic synapses during the first 20 days of postnatal development is described. Whole cell excitatory postsynaptic currents (EPSCs) were evoked in relay neurons of the ventral posterior nucleus (VP) by stimulation of corticothalamic fibers in in vitro slices of mouse brain from postnatal day 1 (P1). During P1-P12, excitatory postsynaptic conductances showed strong voltage dependence at peak current and at 100 ms after the stimulus and were almost completely antagonized by -2-amino-5-phosphonopentoic acid (APV), indicating that N-methyl--aspartate (NMDA) receptor-mediated currents dominate corticothalamic EPSCs at this time. After P12, in 42% of cells, excitatory postsynaptic conductances showed no voltage-dependence at peak current but still showed voltage-dependence 100-ms poststimulus. This voltage-dependent conductance was antagonized by APV. The nonvoltage-dependent component was APV resistant, showed fast decay, and was antagonized by the nonNMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In the remaining 58% of cells after P12, excitatory postsynaptic conductances showed moderate voltage dependence at peak conductance and strong voltage dependence 100 ms after the stimulus. Analysis of EPSCs before and after APV showed a significant increase in the relative contribution of the non-NMDA conductance after the second postnatal week. From P1 to P16, there was a significant decrease in the time constant of decay of the NMDA EPSC but no change in the voltage dependence of the NMDA response. After P8, slow EPSPs, 1.5-30 s in duration and mediated by metabotropic glutamate receptors (mGluRs), could be evoked by high-frequency stimulation of corticothalamic fibers in the presence of APV and CNQX. Similar slow depolarizations could be evoked by local application of the mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) but from P0. Both conductances were blocked by the mGluR antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine. Hence functional mGluR receptors are present on VP cells from birth, but their synaptic activation at corticothalamic synapses can only be detected after P8. In voltage clamp, the extrapolated reversal potential of the t-ACPD current, with potassium gluconate-based internal solution, was +12 +/- 10 (SE) mV, and the measured reversal potential with cesium gluconate-based internal solution was 1.5 +/- 9.9 mV, suggesting that the mGluR-mediated depolarization was mediated by a nonselective cation current. Replacement of NaCl in the external solution caused the reversal potential of the current to shift to -18 +/- 2 mV, indicating that Na+ is a charge carrier in the current. The current amplitude was not reduced by application of Cs+, Ba2+, and Cd2+, indicating that the t-ACPD current was distinct from the hyperpolarization-activated cation current (IH) and distinct from certain other previously characterized mGluR-activated, nonselective cation conductances.