Dopamine neuron glutamate cotransmission: frequency-dependent modulation in the mesoventromedial projection

Mesoventromedial dopamine neurons projecting from the medial ventral tegmental area to the ventromedial shell of the nucleus accumbens play a role in attributing incentive salience to environmental stimuli that predict important events, and appear to be particularly sensitive to the effects of psychostimulant drugs. Despite the observation that these dopamine neurons make up almost the entire complement of neurons in the projection, stimulating their cell bodies evokes a fast glutamatergic response in accumbens neurons. This is apparently due to dopamine neuron glutamate cotransmission, suggested by the extensive coexpression of vesicular glutamate transporter 2 (VGLUT2) in the neurons. To examine the interplay between the dopamine and glutamate signals, we used acute quasi-horizontal brain slices made from DAT-YFP mice in which the intact mesoventromedial projection can be visualized. Under current clamp, when dopamine neurons were stimulated repeatedly, dopamine neuron glutamate transmission showed dopamine-mediated facilitation, solely at higher, burst-firing frequencies. Facilitation was diminished under voltage clamp and flipped to inhibition by intracellular Cs+ or GDPβS, indicating that it was mediated postsynaptically. Postsynaptic facilitation was D1 mediated, required activation of NMDA receptors and closure of voltage gated K+-channels. When postsynaptic facilitation was blocked, D2-mediated presynaptic inhibition became apparent. These counterbalanced pre- and postsynaptic actions determine the frequency dependence of dopamine modulation; at lower firing frequencies dopamine modulation is not apparent, while at burst firing frequency postsynaptic facilitation dominates and dopamine becomes facilitatory. Dopamine neuron glutamate cotransmission may play an important role in encoding the incentive salience value of conditioned stimuli that activate goal-directed behaviors, and may be an important subtract for enduring drug-seeking behaviors. Key words mesolimbic projection; ventral tegmental area; addiction; schizophrenia; transgenic mice; VGLUT

Ratio and time requirements on operant schedules: effort-related effects of nucleus accumbens dopamine depletions

Accumbens dopamine (DA) depletions produce deficits that are related to the ratio requirement of the operant schedule; however, it is also possible that time without reinforcement is a factor. The present study examined the effects of accumbens DA depletions in rats using variable interval (VI) schedules with additional fixed ratio (FR) requirements. Four VI schedules were used (VI 60/FR 1, VI 120/FR 1, VI 60/FR 10, VI 120/FR 10). Attachment of the additional work requirement increased response rates under control conditions. After surgery, there was no interaction between interval level (i.e. 60 vs. 120 s) and DA depletion, but there was a significant interaction between ratio requirement (i.e. 1 vs. 10) and DA depletion within the first week after surgery. DA depletions substantially impaired performance on the schedules with added FR 10 requirements, an effect that was largely dependent upon a reduction in fast responses (i.e. interresponse times less than 1.0 s). There was little effect of DA depletion on overall responding on VI 60/FR 1 and VI 120/FR 1 schedules. DA depletions also increased the tendency to take long pauses in responding (i.e. > 20.0 s), and this effect was evident across all schedules tested. Thus, accumbens DA depletions interact with work requirements and blunt the rate-enhancing effects of moderate size ratios, and also enhance the tendency to pause. Attachment of ratio requirements to interval schedules is a work-related response cost that provides a challenge to the organism, and DA in nucleus accumbens appears to be necessary for adapting to this challenge

Functional Connectome Analysis of Dopamine Neuron Glutamatergic Connections in Forebrain Regions

In the ventral tegmental area (VTA), a subpopulation of dopamine neurons express vesicular glutamate transporter 2 and make glutamatergic connections to nucleus accumbens (NAc) and olfactory tubercle (OT) neurons. However, their glutamatergic connections across the forebrain have not been explored systematically. To visualize dopamine neuron forebrain projections and to enable photostimulation of their axons independent of transmitter status, we virally transfected VTA neurons with channelrhodopsin-2 fused to enhanced yellow fluorescent protein (ChR2-EYFP) and used DATIREScre mice to restrict expression to dopamine neurons. ChR2-EYFP-expressing neurons almost invariably stained for tyrosine hydroxylase, identifying them as dopaminergic. Dopamine neuron axons visualized by ChR2-EYFP fluorescence projected most densely to the striatum, moderately to the amygdala and entorhinal cortex (ERC), sparsely to prefrontal and cingulate cortices, and rarely to the hippocampus. Guided by ChR2-EYFP fluorescence, we recorded systematically from putative principal neurons in target areas and determined the incidence and strength of glutamatergic connections by activating all dopamine neuron terminals impinging on recorded neurons with wide-field photostimulation. This revealed strong glutamatergic connections in the NAc, OT, and ERC; moderate strength connections in the central amygdala; and weak connections in the cingulate cortex. No glutamatergic connections were found in the dorsal striatum, hippocampus, basolateral amygdala, or prefrontal cortex. These results indicate that VTA dopamine neurons elicit widespread, but regionally distinct, glutamatergic signals in the forebrain and begin to define the dopamine neuron excitatory functional connectome

Intra-accumbens injections of the adenosine A(2A) agonist CGS 21680 affect effort-related choice behavior in rats

Rationale: Nucleus accumbens dopamine (DA) participates in the modulation of instrumental behavior, including aspects of behavioral activation and effort-related choice behavior. Rats with impaired accumbens DA transmission reallocate their behavior away from food-reinforced activities that have high response requirements, and instead select less-effortful types of food-seeking behavior. Although accumbens DA is considered a critical component of the brain circuitry regulating effort-related processes, emerging evidence also implicates adenosine A2A receptors. Objective: The present work was undertaken to test the hypothesis that accumbens A2A receptor stimulation would produce effects similar to those produced by DA depletion or antagonism. Methods: Three experiments assessed the effects of the adenosine A2A agonist CGS 21680 on performance of a concurrent choice task (lever pressing for preferred food vs. intake of less preferred chow) that is known to be sensitive to DA antagonists and accumbens DA depletions. Results: Systemic injections of CGS 21680 reduced lever pressing but did not increase feeding. In contrast, bilateral infusions of the adenosine A2A receptor agonist CGS 21680 (6.0- 24.0 ng) into the nucleus accumbens decreased lever pressing for the preferred food, but substantially increased consumption of the less preferred chow. Injections of CGS 21680 into a control site dorsal to the accumbens were ineffective. Conclusions: Taken together, these results are consistent with the hypothesis that local stimulation of adenosine A2A receptors in nucleus accumbens produces behavioral effects similar to those induced by accumbens DA depletions. Accumbens adenosine A2A receptors appear to be a component of the brain circuitry regulating effort-related choice behavio

Genetic Pharmacotherapy as an Early CNS Drug Development Strategy: Testing Glutaminase Inhibition for Schizophrenia Treatment in Adult Mice

Genetic pharmacotherapy is an early drug development strategy for the identification of novel CNS targets in mouse models prior to the development of specific ligands. Here for the first time, we have implemented this strategy to address the potential therapeutic value of a glutamate-based pharmacotherapy for schizophrenia involving inhibition of the glutamate recycling enzyme phosphate-activated glutaminase. Mice constitutively heterozygous for GLS1, the gene encoding glutaminase, manifest a schizophrenia resilience phenotype, a key dimension of which is an attenuated locomotor response to propsychotic amphetamine challenge. If resilience is due to glutaminase deficiency in adulthood, then glutaminase inhibitors should have therapeutic potential. However, this has been difficult to test given the dearth of neuroactive glutaminase inhibitors. So, we used genetic pharmacotherapy to ask whether adult induction of GLS1 heterozygosity would attenuate amphetamine responsiveness. We generated conditional floxGLS1 mice and crossed them with global CAGERT2cre∕+ mice to produce GLS1 iHET mice, susceptible to tamoxifen induction of GLS1 heterozygosity. One month after tamoxifen treatment of adult GLS1 iHET mice, we found a 50% reduction in GLS1 allelic abundance and glutaminase mRNA levels in the brain. While GLS1 iHET mice showed some recombination prior to tamoxifen, there was no impact on mRNA levels. We then asked whether induction of GLS heterozygosity would attenuate the locomotor response to propsychotic amphetamine challenge. Before tamoxifen, control and GLS1 iHET mice did not differ in their response to amphetamine. One month after tamoxifen treatment, amphetamine-induced hyperlocomotion was blocked in GLS1 iHET mice. The block was largely maintained after 5 months. Thus, a genetically induced glutaminase reduction—mimicking pharmacological inhibition—strongly attenuated the response to a propsychotic challenge, suggesting that glutaminase may be a novel target for the pharmacotherapy of schizophrenia. These results demonstrate how genetic pharmacotherapy can be implemented to test a CNS target in advance of the development of specific neuroactive inhibitors. We discuss further the advantages, limitations, and feasibility of the wider application of genetic pharmacotherapy for neuropsychiatric drug development

Nucleus Accumbens Adenosine A2A Receptors Regulate Exertion of Effort by Acting on the Ventral Striatopallidal Pathway

Goal-directed actions are sensitive to work-related response costs, and dopamine in nucleus accumbens is thought to modulate the exertion of effort in motivated behavior. Dopamine-rich striatal areas such as nucleus accumbens also contain high numbers of adenosine A2A receptors, and, for that reason, the behavioral and neurochemical effects of the adenosine A2A receptor agonist CGS 21680 [2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine] were investigated. Stimulation of accumbens adenosine A2A receptors disrupted performance of an instrumental task with high work demands (i.e., an interval lever-pressing schedule with a ratio requirement attached) but had little effect on a task with a lower work requirement. Immunohistochemical studies revealed that accumbens neurons that project to the ventral pallidum showed adenosine A2A receptors immunoreactivity. Moreover, activation of accumbens A2A receptors by local injections of CGS 21680 increased extracellular GABA levels in the ventral pallidum. Combined contralateral injections of CGS 21680 into the accumbens and the GABAA agonist muscimol into ventral pallidum (i.e., “disconnection” methods) also impaired response output, indicating that these structures are part of a common neural circuitry regulating the exertion of effort. Thus, accumbens adenosine A2A receptors appear to regulate behavioral activation and effort-related processes by modulating the activity of the ventral striatopallidal pathway. Research on the effort-related functions of these forebrain systems may lead to a greater understanding of pathological features of motivation, such as psychomotor slowing, anergia, and fatigue in depression

Dopamine/adenosine interactions related to locomotion and tremor in animal models: Possible relevance to parkinsonism

Abstract Adenosine A 2A antagonists can exert antiparkinsonian effects in animal models. Recent experiments studied the ability of MSX-3 (an adenosine A 2A antagonist) to reverse the locomotor suppression and tremor produced by dopamine antagonists in rats. MSX-3 reversed haloperidolinduced suppression of locomotion, and reduced the tremulous jaw movements induced by haloperidol, pimozide, and reserpine. Infusions of MSX-3 into the nucleus accumbens core increased locomotion in haloperidol-treated rats, but there were no effects of infusions into the accumbens shell or ventrolateral neostriatum. In contrast, MSX-3 injected into the ventrolateral neostriatum reduced pimozide-induced tremulous jaw movements. Dopamine/adenosine interactions in different striatal subregions are involved in distinct aspects of motor function

Axo-glial interactions between midbrain dopamine neurons and oligodendrocyte lineage cells in the anterior corpus callosum

Oligodendrocyte progenitor cells (OPCs) receive synaptic innervation from glutamatergic and GABAergic axons and can be dynamically regulated by neural activity, resulting in activity-dependent changes in patterns of axon myelination. However, it remains unclear to what extent other types of neurons may innervate OPCs. Here, we provide evidence implicating midbrain dopamine neurons in the innervation of oligodendrocyte lineage cells in the anterior corpus callosum and nearby white matter tracts of male and female adult mice. Dopaminergic axon terminals were identified in the corpus callosum of DAT-Cre mice after injection of an eYFP reporter virus into the midbrain. Furthermore, fast-scan cyclic voltammetry revealed monoaminergic transients in the anterior corpus callosum, consistent with the anatomical findings. Using RNAscope, we further demonstrate that ~ 40% of Olig2 + /Pdfgra + cells and ~ 20% of Olig2 + /Pdgfra- cells in the anterior corpus callosum express Drd1 and Drd2 transcripts. These results suggest that oligodendrocyte lineage cells may respond to dopamine released from midbrain dopamine axons, which could affect myelination. Together, this work broadens our understanding of neuron-glia interactions with important implications for myelin plasticity by identifying midbrain dopamine axons as a potential regulator of corpus callosal oligodendrocyte lineage cells

Forebrain neurochemical interactions regulating effort-related processes

An emerging body of evidence indicates that striatal DA systems interact with adenosine A2A receptors, and these interactions may regulate the behavioral functions of the nucleus accumbens. The following experiments were designed to characterize the effects of the adenosine A2A agonist CGS 21680 on effort-related processes known to be modulated by the nucleus accumbens. The first set of experiments (exp. 1-3) showed that of systemic or intracranial injections into the nucleus accumbens of CGS 21680 interfere with an effort-related choice procedure know as the concurrent FR5/feeding procedure. The second set of experiments (exp.4-6), the same neurochemical manipulations produced suppressant effects on operant responding that interacted with the work requirement of the task. The last experiments included anatomical, neurochemical and behavioral studies that investigated the functional relation between adenosine A2A receptors in nucleus accumbens and GABA transmission in the ventral pallidum (VP). Using immunohistochemistry and track-tracing methods it was shown that ventral striatopallidal neurons expressed A 2A receptor immunoreactivity (exp. 7). Stimulation of these receptors increased extracellular levels of GABA in the ventral pallidum, as measured my microdialysis (exp. 8). Simultaneous intracranial unilateral injections of CGS 21680 into nucleus accumbens and contralateral injections of muscimol into the VP, produced a synergistic effect that dramatically suppressed operant responding in a schedule with a high ratio requirement (exp.9). ^ In conclusion, these experiments showed that stimulation of adenosine A2A receptors alters the exertion of effort in a manner that is similar to the effects of interference with DA transmission. These studies implicated the role of adenosine A2A receptor mechanisms in activational aspects of motivation. Further research into the function of these receptors in forebrain systems may lead to a greater understanding of both normal and pathological features of motivation, promoting the development of novel treatments for effort-related disorders in humans, such as psychomotor slowing in depression.