Yolanda Mateo

Selective activation of cholinergic interneurons enhances accumbal phasic dopamine release: setting the tone for reward processing

Dopamine plays a critical role in motor control, addiction, and reward-seeking behaviors, and its release dynamics have traditionally been linked to changes in midbrain dopamine neuron activity. Here, we report that selective endogenous cholinergic activation achieved via in vitro optogenetic stimulation of nucleus accumbens, a terminal field of dopaminergic neurons, elicits real-time dopamine release. This mechanism occurs via direct actions on dopamine terminals, does not require changes in neuron firing within the midbrain, and is dependent on glutamatergic receptor activity. More importantly, we demonstrate that in vivo selective activation of cholinergic interneurons is sufficient to elicit dopamine release in the nucleus accumbens. Therefore, the control of accumbal extracellular dopamine levels by endogenous cholinergic activity results from a complex convergence of neurotransmitter/neuromodulator systems that may ultimately synergize to drive motivated behavior.

Cocaine supersensitivity and enhanced motivation for reward in mice lacking dopamine D2 autoreceptors.

Dopamine (DA) D2 receptors expressed in DA neurons (D2 autoreceptors) exert a negative feedback regulation that reduces DA neuron firing, DA synthesis and DA release. As D2 receptors are mostly expressed in postsynaptic neurons, pharmacological and genetic approaches have been unable to definitively address the in vivo contribution of D2 autoreceptors to DA-mediated behaviors. We found that midbrain DA neurons from mice deficient in D2 autoreceptors (Drd2loxP/loxP; Dat+/IRES−cre, referred to as autoDrd2KO mice) lacked DA-mediated somatodendritic synaptic responses and inhibition of DA release. AutoDrd2KO mice displayed elevated DA synthesis and release, hyperlocomotion and supersensitivity to the psychomotor effects of cocaine. The mice also exhibited increased place preference for cocaine and enhanced motivation for food reward. Our results highlight the importance of D2 autoreceptors in the regulation of DA neurotransmission and demonstrate that D2 autoreceptors are important for normal motor function, food-seeking behavior, and sensitivity to the locomotor and rewarding properties of cocaine.

Reduced dopamine terminal function and insensitivity to cocaine following cocaine binge self-administration and deprivation

Despite large numbers of studies describing neuroadaptations caused by chronic cocaine exposure, there remains considerable uncertainty as to whether alterations in dopamine (DA) neurotransmission are responsible for progression into an addicted state. High-intake, 24-h access cocaine self-administration (SA, 10 days) followed by an extended (7 days), but not 1 day deprivation period produces an increased motivation to SA cocaine as measured by a progressive ratio protocol. Following binge cocaine SA and deprivation, the status of DA terminals in the nucleus accumbens (NAc) was investigated using microdialysis in freely moving rats and voltammetry in brain slices. At 1 and 7 days following binge cocaine SA, baseline extracellular DA concentrations in the NAc core were decreased by 40 and 55% of control levels, in the 1 and 7 day deprivation groups, respectively. Acute cocaine (1.5 mg/kg, i.v.) administration increased extracellular DA (350%) in the NAc core of naïve animals but failed to significantly increase DA at 1 or 7 days following binge cocaine SA. The shell of the NAc showed a similar lack of effect of cocaine. Analysis of DA terminals in brain slices showed that cocaine was markedly less effective in inhibiting DA uptake at 1 and 7 days of cocaine deprivation (max. effect 40% of control). Electrically stimulated DA release was decreased at 1 day and further decreased at 7 days of deprivation (67 and 49% of control, respectively). The rate of DA uptake was increased (150% of control) following binge SA, irrespective of deprivation period. Finally, presynaptic autoreceptors were subsensitive at both time points, as measured by the ability of quinpirole, a D2-like DA receptor agonist, to inhibit DA release. Thus, the NAc was hypodopaminergic and DA terminals were less sensitive to cocaine following binge cocaine SA and deprivation.

Conditional expression of Parkinson's disease-related mutant α-synuclein in the midbrain dopaminergic neurons causes progressive neurodegeneration and degradation of transcription factor nuclear receptor related 1.

α-Synuclein (α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinsons disease (PD). However, only a few studies on α-syn have been performed in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We also systematically examined the subcellular abnormalities that appeared in the mDA neurons of mutant mice and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and the impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that overexpression of α-syn promoted a proteasome-dependent degradation of nuclear receptor-related 1 protein (Nurr1), whereas inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.

Dopamine D2 Receptor Overexpression Alters Behavior and Physiology in Drd2-EGFP Mice

Bacteria artificial chromosome (BAC) transgenic mice expressing the reporter protein enhanced green fluorescent protein (EGFP) under the control of the D1 and D2 dopamine receptor promoters (Drd1-EGFP and Drd2-EGFP) have been widely used to study striatal function and have contributed to our understanding of the physiological and pathological functions of the basal ganglia. These tools were produced and promptly made available to address questions in a cell-specific manner that has transformed the way we frame hypotheses in neuroscience. However, these mice have not been fully characterized until now. We found that Drd2-EGFP mice display an ∼40% increase in membrane expression of the dopamine D2 receptor (D2R) and a twofold increase in D2R mRNA levels in the striatum when compared with wild-type and Drd1-EGFP mice. D2R overexpression was accompanied by behavioral hypersensitivity to D2R-like agonists, as well as enhanced electrophysiological responses to D2R activation in midbrain dopaminergic neurons. Dopamine (DA) transients evoked by stimulation in the nucleus accumbens showed slower clearance in Drd2-EGFP mice, and cocaine actions on DA clearance were impaired in these mice. Thus, it was not surprising to find that Drd2-EGFP mice were hyperactive when exposed to a novel environment and locomotion was suppressed by acute cocaine administration. All together, this study demonstrates that Drd2-EGFP mice overexpress D2R and have altered dopaminergic signaling that fundamentally differentiates them from wild-type and Drd1-EGFP mice.

Neurochemical characterization of the release and uptake of dopamine in ventral tegmental area and serotonin in substantia nigra of the mouse.

In the present report, fast‐scan cyclic voltammetry was used to identify the monoamines that were released by electrical stimulation in mouse brain slices containing ventral tegmental area (VTA), substantia nigra (SN) ‐pars compacta (SNc) and ‐pars reticulata (SNr). We showed that voltammograms obtained in mouse VTA were consistent with detection of a catecholamine, while those in both subregions of the SN were consistent with detection of an indolamine, based on the reduction peak potentials. We used pharmacological blockade and genetic deletion of monoamine transporters to further confirm the identity of released monoamines in mouse midbrain and to assess the control of monoamines by their transporters in each brain region. Inhibition of dopamine and norepinephrine transporters by nomifensine (1 and 10 μm) decreased uptake rates in the VTA, but did not change uptake rates in either subregion of the SN. Serotonin transporter inhibition by fluoxetine (10 μm) decreased uptake rates in the SNc and SNr, but was without effect in the VTA. Selective inhibition of the norepinephrine transporter by desipramine (10 μm) had no effect in any brain region. Using dopamine transporter‐ and serotonin transporter‐knockout mice, we found decreased uptake rates in VTA and SN subregions, respectively. Peak signals recorded in each midbrain region were pulse number dependent and exhibited limited frequency dependence. Thus, dopamine is predominately detected by voltammetry in mouse VTA, while serotonin is predominately detected in mouse SNc and SNr. Furthermore, active uptake occurs in these areas and can be altered only by specific uptake inhibitors, suggesting a lack of heterologous uptake. In addition, somatodendritic dopamine release in VTA was not mediated by monoamine transporters. This work offers an initial characterization of voltammetric signals in the midbrain of the mouse and provides insight into the regulation of monoamine neurotransmission in these areas.

Role of serotonin in cocaine effects in mice with reduced dopamine transporter function

The mesolimbic dopaminergic system, especially the nucleus accumbens, has received attention for its involvement in the reinforcing and addictive properties of cocaine and other drugs of abuse. It is generally accepted that the ability of cocaine to inhibit the dopamine transporter (DAT) is directly related to its reinforcing actions. However, mice with a genetic deletion of the DAT (DAT-KO mice) still experience the rewarding effects of cocaine. These behavioral findings suggest that there is an alternate site for cocaine reinforcement. We demonstrate here that modulation of the serotonergic system in the ventral tegmental area, where the mesolimbic dopamine system originates, is a target of cocaine action. The ultimate effect of this serotonin mechanism in animal models with sustained elevations of dopamine may be a feed-forward enhancement of dopamine levels in the nucleus accumbens.

Cocaine Self-Administration Produces Pharmacodynamic Tolerance: Differential Effects on the Potency of Dopamine Transporter Blockers, Releasers, and Methylphenidate

The dopamine transporter (DAT) is the primary site of action for psychostimulant drugs such as cocaine, methylphenidate, and amphetamine. Our previous work demonstrated a reduced ability of cocaine to inhibit the DAT following high-dose cocaine self-administration (SA), corresponding to a reduced ability of cocaine to increase extracellular dopamine. However, this effect had only been demonstrated for cocaine. Thus, the current investigations sought to understand the extent to which cocaine SA (1.5 mg/kg/inf × 40 inf/day × 5 days) altered the ability of different dopamine uptake blockers and releasers to inhibit dopamine uptake, measured using fast-scan cyclic voltammetry in rat brain slices. We demonstrated that, similar to cocaine, the DAT blockers nomifensine and bupropion were less effective at inhibiting dopamine uptake following cocaine SA. The potencies of amphetamine-like dopamine releasers such as 3,4-methylenedioxymethamphetamine, methamphetamine, amphetamine, and phentermine, as well as a non-amphetamine releaser, 4-benzylpiperidine, were all unaffected. Finally, methylphenidate, which blocks dopamine uptake like cocaine while being structurally similar to amphetamine, shared characteristics of both, resembling an uptake blocker at low concentrations and a releaser at high concentrations. Combined, these experiments demonstrate that after high-dose cocaine SA, there is cross-tolerance of the DAT to other uptake blockers, but not releasers. The reduced ability of psychostimulants to inhibit dopamine uptake following cocaine SA appears to be contingent upon their functional interaction with the DAT as a pure blocker or releaser rather than their structural similarity to cocaine. Further, methylphenidates interaction with the DAT is unique and concentration-dependent.

Cocaine-Insensitive Dopamine Transporters with Intact Substrate Transport Produced by Self-Administration

BACKGROUND Psychomotor stimulant drugs such as cocaine and amphetamine activate brain dopamine (DA) neurotransmission and support self-administration in humans and laboratory animals. Cocaine amplifies DA signaling by blocking the DA transporter (DAT), and this has been described as the most important mechanism underlying cocaines reinforcing effects. Amphetamine has the added mechanism of reverse transport of intracellular DA through the DAT. METHODS We used cocaine and amphetamine self-administration under a fixed-ratio 1 schedule followed by microdialysis in freely moving rats to measure extracellular DA levels and fast scan cyclic voltammetry in brain slices to measure subsecond DA release and uptake parameters. RESULTS Following a high dose (1.5 mg/kg intravenous) cocaine self-administration paradigm (40 injections/day × 5 days), the DAT was markedly less sensitive to cocaine, as measured by microdialysis and voltammetry in the nucleus accumbens core. In contrast, the DAT substrate amphetamine retained the same efficacy at the DAT in cocaine self-administering animals, and amphetamine did not mimic cocaines effect on the DAT when self-administered. A single session of cocaine self-administration caused a significant decrease in the ability of cocaine to inhibit the DAT, a finding that may provide a neurochemical basis for rapid tolerance. The effects of cocaine returned to normal within a few weeks following cessation of self-administration. CONCLUSIONS Here, we, for the first time, demonstrate an in vivo, pharmacologically induced alteration in the sensitivity of the DAT to cocaine that is specific to cocaine, spares DAT and substrate/releaser interactions, and is independent of maximal rate of DA uptake (V(max)).

Subcortical dopaminergic deficits in a DISC1 mutant model: a study in direct reference to human molecular brain imaging

Imaging of the human brain has been an invaluable aid in understanding neuropsychopharmacology and, in particular, the role of dopamine in the striatum in mental illness. Here, we report a study in a genetic mouse model for major mental illness guided by results from human brain imaging: a systematic study using small animal positron emission tomography (PET), autoradiography, microdialysis and molecular biology in a putative dominant-negative mutant DISC1 transgenic model. This mouse model showed augmented binding of radioligands to the dopamine D2 receptor (D2R) in the striatum as well as neurochemical and behavioral changes to methamphetamine administration. Previously we reported that this model displayed deficits in the forced swim test, a representative indicator of antidepressant efficacy. By combining the results of our two studies, we propose a working hypothesis for future studies that this model might represent a mixed condition of depression and psychosis. We hope that this study will also help bridge a major gap in translational psychiatry between basic characterization of animal models and clinico-pharmacological assessment of patients mainly through PET imaging.