Faculty Bibliography

Diet influences dopamine transmission in motor- and reward-related basal ganglia circuitry. In part, this reflects diet-dependent regulation of circulating and brain insulin levels. Activation of striatal insulin receptors amplifies axonal dopamine release in brain slices, and regulates food preference in vivo. The effect of insulin on dopamine release is indirect, and requires striatal cholinergic interneurons that express insulin receptors. However, insulin also increases dopamine uptake by promoting dopamine transporter (DAT) surface expression, which counteracts enhanced dopamine release. Here we determined the functional consequences of acute insulin exposure and chronic diet-induced changes in insulin on DAT activity after evoked dopamine release in striatal slices from adult ad-libitum fed (AL) rats and mice, and food-restricted (FR) or high-fat/high-sugar obesogenic (OB) diet rats. Uptake kinetics were assessed by fitting evoked dopamine transients to the Michaelis-Menten equation and extracting C_peak and V_max . Insulin (30 nM) increased both parameters in the caudate putamen and nucleus accumbens core of AL rats in an insulin receptor- and PI3-kinase-dependent manner. A pure effect of insulin on uptake was unmasked using mice lacking striatal acetylcholine, in which increased V_max caused a decrease in C_peak . Diet also influenced V_max , which was lower in FR versus AL. The effects of insulin on C_peak and V_max were amplified by FR but blunted by OB, consistent with opposite consequences of these diets on insulin levels and insulin receptor sensitivity. Overall, these data reveal acute and chronic effects of insulin and diet on dopamine release and uptake that will influence brain reward pathways.

Fast-scan cyclic voltammetry (FCV) is an established method to monitor increases in extracellular dopamine (DA) concentration ([DA]o) in the striatum, which is densely innervated by DA axons. Ex vivo brain slice preparations provide an opportunity to identify endogenous modulators of DA release. For these experiments, local electrical stimulation is often used to elicit release of DA, as well as other transmitters, in the striatal microcircuitry; changes in evoked increases in [DA]o after application of a pharmacological agent (e.g., a receptor antagonist) indicate a regulatory role for the transmitter system interrogated. Optogenetic methods that allow specific stimulation of DA axons provide a complementary, bottom-up approach for elucidating factors that regulate DA release. To this end, we have characterized DA release evoked by local electrical and optical stimulation in striatal slices from mice that genetically express a variant of channelrhodopsin-2 (ChR2). Evoked increases in [DA]o in the dorsal and ventral striatum (dStr and vStr) were examined in a cross of a Cre-dependent ChR2 line ("Ai32" mice) with a DAT::Cre mouse line. In dStr, repeated optical pulse-train stimulation at the same recording site resulted in rundown of evoked [DA]o using heterozygous mice, which contrasted with the stability seen with electrical stimulation. Similar rundown was seen in the presence of a nicotinic acetylcholine receptor (nAChR) antagonist, implicating the absence of concurrent nAChR activation in DA release instability in slices. Rundown with optical stimulation in dStr could be circumvented by recording from a population of sites, each stimulated only once. Same-site rundown was less pronounced with single-pulse stimulation, and a stable baseline could be attained. In vStr, stable optically evoked increases in [DA]o at single sites could be achieved using heterozygous mice, although with relatively low peak [DA]o. Low release could be overcome by using mice with a second copy of the Ai32 allele, which doubled ChR2 expression. The characteristics reported here should help future practitioners decide which Ai32;DAT::Cre genotype and recording protocol is optimal for the striatal subregion to be examined.

Insulin activates insulin receptors (InsRs) in the hypothalamus to signal satiety after a meal. However, the rising incidence of obesity, which results in chronically elevated insulin levels, implies that insulin may also act in brain centres that regulate motivation and reward. We report here that insulin can amplify action potential-dependent dopamine (DA) release in the nucleus accumbens (NAc) and caudate-putamen through an indirect mechanism that involves striatal cholinergic interneurons that express InsRs. Furthermore, two different chronic diet manipulations in rats, food restriction (FR) and an obesogenic (OB) diet, oppositely alter the sensitivity of striatal DA release to insulin, with enhanced responsiveness in FR, but loss of responsiveness in OB. Behavioural studies show that intact insulin levels in the NAc shell are necessary for acquisition of preference for the flavour of a paired glucose solution. Together, these data imply that striatal insulin signalling enhances DA release to influence food choices.

Dopamine (DA) transmission is governed by processes that regulate release from axonal boutons in the forebrain and the somatodendritic compartment in midbrain, and by clearance by the DA transporter, diffusion, and extracellular metabolism. We review how axonal DA release is regulated by neuronal activity and by autoreceptors and heteroreceptors, and address how quantal release events are regulated in size and frequency. In brain regions densely innervated by DA axons, DA clearance is due predominantly to uptake by the DA transporter, whereas in cortex, midbrain, and other regions with relatively sparse DA inputs, the norepinephrine transporter and diffusion are involved. We discuss the role of DA uptake in restricting the sphere of influence of DA and in temporal accumulation of extracellular DA levels upon successive action potentials. The tonic discharge activity of DA neurons may be translated into a tonic extracellular DA level, whereas their bursting activity can generate discrete extracellular DA transients.

Mutations in the PRKN gene encoding the protein parkin cause autosomal recessive juvenile parkinsonism (ARJP). Harnessing this mutation to create an early-onset Parkinson's disease mouse model would provide a unique opportunity to clarify the mechanisms involved in the neurodegenerative process and lay the groundwork for the development of neuroprotective strategies. To this end, we created a knock-in mouse carrying the homozygous PrknR275W mutation, which is the missense mutation with the highest allelic frequency in PRKN patients. We evaluated the anatomical and functional integrity of the nigrostriatal dopamine (DA) pathway, as well as motor behaviour in PrknR275W mice of both sexes. We report here that PrknR275W mice show early DA neuron dysfunction, age-dependent loss of DA neurons in the substantia nigra, decreased DA content and stimulus-evoked DA release in the striatum, and progressive motor impairment. Together, these data show that the PrknR275W mouse recapitulates key features of ARJP. Thus, these studies fill a critical need in the field by introducing a promising new Parkinson's disease model in which to study causative mechanisms of the disease and test therapeutic strategies.

Monitoring the coordinated signaling of dopamine (DA) and serotonin (5-HT) is important for advancing our understanding of the brain. However, the co-detection and robust quantification of these signals at low concentrations is yet to be demonstrated. Here, we present the quantification of DA and 5-HT using nano-graphitic (NG) sensors together with fast-scan cyclic voltammetry (FSCV) employing an engineered N-shape potential waveform. Our method yields 6% error in quantifying DA and 5-HT analytes present in in vitro mixtures at concentrations below 100 nM. This advance is due to the electrochemical properties of NG sensors which, in combination with the engineered FSCV waveform, provided distinguishable cyclic voltammograms (CVs) for DA and 5-HT. We also demonstrate the generalizability of the prediction model across different NG sensors, which arises from the consistent voltammetric fingerprints produced by our NG sensors. Curiously, the proposed engineered waveform also improves the distinguishability of DA and 5-HT CVs obtained from traditional carbon fiber (CF) microelectrodes. Nevertheless, this improved distinguishability of CVs obtained from CF is inferior to that of NG sensors, arising from differences in the electrochemical properties of the sensor materials. Our findings demonstrate the potential of NG sensors and our proposed FSCV waveform for future brain studies.

Striatal dopamine axons co-release dopamine and gamma-aminobutyric acid (GABA), using GABA provided by uptake via GABA transporter-1 (GAT1). Functions of GABA co-release are poorly understood. We asked whether co-released GABA autoinhibits dopamine release via axonal GABA type A receptors (GABA_ARs), complementing established inhibition by dopamine acting at axonal D2 autoreceptors. We show that dopamine axons express α3-GABA_AR subunits in mouse striatum. Enhanced dopamine release evoked by single-pulse optical stimulation in striatal slices with GABA_AR antagonism confirms that an endogenous GABA tone limits dopamine release. Strikingly, an additional inhibitory component is seen when multiple pulses are used to mimic phasic axonal activity, revealing the role of GABA_AR-mediated autoinhibition of dopamine release. This autoregulation is lost in conditional GAT1-knockout mice lacking GABA co-release. Given the faster kinetics of ionotropic GABA_ARs than G-protein-coupled D2 autoreceptors, our data reveal a mechanism whereby co-released GABA acts as a first responder to dampen phasic-to-tonic dopamine signaling.

Insulin crosses the blood-brain barrier to enter the brain from the periphery. In the brain, insulin has well-established actions in the hypothalamus, as well as at the level of mesolimbic dopamine neurons in the midbrain. Notably, insulin also acts in the striatum, which shows abundant expression of insulin receptors (InsRs) throughout. These receptors are found on interneurons and striatal projections neurons, as well as on glial cells and dopamine axons. A striking functional consequence of insulin elevation in the striatum is promoting an increase in stimulated dopamine release. This boosting of dopamine release involves InsRs on cholinergic interneurons, and requires activation of nicotinic acetylcholine receptors on dopamine axons. Opposing this dopamine-enhancing effect, insulin also increases dopamine uptake through the action of insulin at InsRs on dopamine axons. Insulin acts on other striatal cells as well, including striatal projection neurons and astrocytes that also influence dopaminergic transmission and striatal function. Linking these cellular findings to behavior, striatal insulin signaling is required for the development of flavor-nutrient learning, implicating insulin as a reward signal in the brain. In this review, we discuss these and other actions of insulin in the striatum, including how they are influenced by diet and other physiological states.