Search

The mTOR pathway is an essential regulator of cell growth and metabolism. Midbrain dopamine neurons are particularly sensitive to mTOR signaling status as activation or inhibition of mTOR alters their morphology and physiology. mTOR exists in two distinct multiprotein complexes termed mTORC1 and mTORC2. How each of these complexes affect dopamine neuron properties, and whether they have similar or distinct functions is unknown. Here, we investigated this in mice with dopamine neuron-specific deletion of Rptor or Rictor, which encode obligatory components of mTORC1 or mTORC2, respectively. We find that inhibition of mTORC1 strongly and broadly impacts dopamine neuron structure and function causing somatodendritic and axonal hypotrophy, increased intrinsic excitability, decreased dopamine production, and impaired dopamine release. In contrast, inhibition of mTORC2 has more subtle effects, with selective alterations to the output of ventral tegmental area dopamine neurons. Disruption of both mTOR complexes leads to pronounced deficits in dopamine release demonstrating the importance of balanced mTORC1 and mTORC2 signaling for dopaminergic function.

Ca2+ entry into nigrostriatal dopamine (DA) neurons and axons via L‐type voltage‐gated Ca2+ channels (LTCCs) contributes, respectively, to pacemaker activity and DA release and has long been thought to contribute to vulnerability to degeneration in Parkinson's disease. LTCC function is greater in DA axons and neurons from substantia nigra pars compacta than from ventral tegmental area, but this is not explained by channel expression level. We tested the hypothesis that LTCC control of DA release is governed rather by local mechanisms, focussing on candidate biological factors known to operate differently between types of DA neurons and/or be associated with their differing vulnerability to parkinsonism, including biological sex, α‐synuclein, DA transporters (DATs) and calbindin‐D28k (Calb1). We detected evoked DA release ex vivo in mouse striatal slices using fast‐scan cyclic voltammetry and assessed LTCC support of DA release by detecting the inhibition of DA release by the LTCC inhibitors isradipine or CP8. Using genetic knockouts or pharmacological manipulations, we identified that striatal LTCC support of DA release depended on multiple intersecting factors, in a regionally and sexually divergent manner. LTCC function was promoted by factors associated with Parkinsonian risk, including male sex, α‐synuclein, DAT and a dorsolateral co‐ordinate, but limited by factors associated with protection, that is, female sex, glucocerebrosidase activity, Calb1 and ventromedial co‐ordinate. Together, these data show that LTCC function in DA axons and isradipine effect are locally governed and suggest they vary in a manner that in turn might impact on, or reflect, the cellular stress that leads to parkinsonian degeneration. [ABSTRACT FROM AUTHOR]

Striatal adenosine A1 receptor (A1R) activation can inhibit dopamine release. A1Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A1Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A1R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A1R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor nitrobenzylthioinosine reduced dopamine release and promoted A1R-mediated inhibition, and, conversely, virally mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A1R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor [GPCR-activation based (GRAB)-Ado], we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mm) promoted A1R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function. [ABSTRACT FROM AUTHOR]

Nigrostriatal dopamine (DA) is critical to action selection and learning. Axonal DA release is locally influenced by striatal neurotransmitters. Striatal neurons are principally GABAergic projection neurons and interneurons, and a small minority of other neurons are cholinergic interneurons (Chls). Chls strongly gate striatal DA release via nicotinic receptors (nAChRs) identified on DA axons. Striatal GABA is thought to modulate DA, but GABA receptors have not been documented conclusively on DA axons. However, Chls express GABA receptors and are therefore candidates for potential mediators of GABA regulation of DA. We addressed whether striatal GABA and its receptors can modulate DA release directly, independently from Chi regulation, by detecting DA in striatal slices from male mice using fast-scan cyclic voltammetry in the absence of nAChR activation. DA release evoked by single electrical pulses in the presence of the nAChR antagonist dihydro-j3-erythroidine was reduced by GABA or agonists of GABAA or GABAB receptors, with effects prevented by selective GABA receptor antagonists. GABA agonists slightly modified the frequency sensitivity of DA release during short stimulus trains. GABA agonists also suppressed DA release evoked by optogenetic stimulation of DA axons. Furthermore, antagonists of GABAA and GABAB receptors together, or GABAb receptors alone, significantly enhanced DA release evoked by either optogenetic or electrical stimuli. These results indicate that striatal GABA can inhibit DA release through GABAa and GABAb receptors and that these actions are not mediated by cholinergic circuits. Furthermore, these data reveal that there is a tonic inhibition of DA release by striatal GABA operating through predominantly GABAB receptors. [ABSTRACT FROM AUTHOR]

Striatal adenosine A 1 receptor (A 1 R) activation can inhibit dopamine release. A 1 Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A 1 Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A 1 R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A 1 R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor nitrobenzylthioinosine reduced dopamine release and promoted A 1 R-mediated inhibition, and, conversely, virally mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A 1 R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor [GPCR-activation based (GRAB)-Ado], we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mm) promoted A 1 R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function. SIGNIFICANCE STATEMENT Dopamine axons in the mammalian striatum are emerging as strategic sites where neuromodulators can powerfully influence dopamine output in health and disease. We found that ambient levels of the neuromodulator adenosine tonically inhibit dopamine release in nucleus accumbens core via adenosine A 1 receptors (A 1 Rs), to a variable level that promotes the contrast in dopamine signals released by different frequencies of activity. We reveal that the equilibrative nucleoside transporter 1 (ENT1) on astrocytes limits this tonic inhibition, and that ethanol promotes it by diminishing adenosine uptake via ENT1. These findings support the hypotheses that A 1 Rs on dopamine axons inhibit dopamine release and, furthermore, that astrocytes perform important roles in setting the level of striatal dopamine output, in health and disease., (Copyright © 2022 the authors.)

Nigrostriatal dopamine (DA) is critical to action selection and learning. Axonal DA release is locally influenced by striatal neurotransmitters. Striatal neurons are principally GABAergic projection neurons and interneurons, and a small minority of other neurons are cholinergic interneurons (ChIs). ChIs strongly gate striatal DA release via nicotinic receptors (nAChRs) identified on DA axons. Striatal GABA is thought to modulate DA, but GABA receptors have not been documented conclusively on DA axons. However, ChIs express GABA receptors and are therefore candidates for potential mediators of GABA regulation of DA. We addressed whether striatal GABA and its receptors can modulate DA release directly, independently from ChI regulation, by detecting DA in striatal slices from male mice using fast-scan cyclic voltammetry in the absence of nAChR activation. DA release evoked by single electrical pulses in the presence of the nAChR antagonist dihydro-β-erythroidine was reduced by GABA or agonists of GABA A or GABA B receptors, with effects prevented by selective GABA receptor antagonists. GABA agonists slightly modified the frequency sensitivity of DA release during short stimulus trains. GABA agonists also suppressed DA release evoked by optogenetic stimulation of DA axons. Furthermore, antagonists of GABA A and GABA B receptors together, or GABA B receptors alone, significantly enhanced DA release evoked by either optogenetic or electrical stimuli. These results indicate that striatal GABA can inhibit DA release through GABA A and GABA B receptors and that these actions are not mediated by cholinergic circuits. Furthermore, these data reveal that there is a tonic inhibition of DA release by striatal GABA operating through predominantly GABA B receptors. SIGNIFICANCE STATEMENT The principal inhibitory transmitter in the mammalian striatum, GABA, is thought to modulate striatal dopamine (DA) release, but definitive evidence for GABA receptors on DA axons is lacking. Striatal cholinergic interneurons regulate DA release via axonal nicotinic receptors (nAChRs) and also express GABA receptors, but they have not been eliminated as potentially critical mediators of DA regulation by GABA. Here, we found that GABA A and GABA B receptors inhibit DA release without requiring cholinergic interneurons. Furthermore, ambient levels of GABA inhibited DA release predominantly through GABA B receptors. These findings provide further support for direct inhibition of DA release by GABA receptors and reveal that striatal GABA operates a tonic inhibition on DA output that could critically influence striatal output., (Copyright © 2019 the authors 0270-6474/19/391058-08$15.00/0.)

Optogenetics is a technology that is growing rapidly in neuroscience, establishing itself as a fundamental investigative tool. As this tool is increasingly utilized across the neuroscience community and is one of the primary research techniques being presented at neuroscience conferences and in journals, we believe that it is important that this technology is introduced into the undergraduate neuroscience research laboratory. While there has been a significant body of work concentrated to deploy optogenetics in invertebrate model organisms, little to no work has focused on brining this technology to mammalian model organisms in undergraduate neuroscience laboratories. The establishment of in vivo optogenetics could provide for high-impact independent research projects for upper-level undergraduate students. Here we review the considerations for establishing in vivo optogenetics with the use of rodents in an undergraduate laboratory setting and provide some cost-saving guidelines to assist in making optogenetic technologies financially accessible. We discuss opsin selection, cell-specific opsin expression strategies, species selection, experimental design, selection of light delivery systems, and the construction of implantable optical fibers for the application of in vivo optogenetics in rodents.