Faculty Bibliography

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.

Release of neuroactive substances by exocytosis from dendrites is surprisingly widespread and is not confined to a particular class of transmitters: it occurs in multiple brain regions, and includes a range of neuropeptides, classical neurotransmitters, and signaling molecules, such as nitric oxide, carbon monoxide, ATP, and arachidonic acid. This review is focused on hypothalamic neuroendocrine cells that release vasopressin and oxytocin and midbrain neurons that release dopamine. For these two model systems, the stimuli, mechanisms, and physiological functions of dendritic release have been explored in greater detail than is yet available for other neurons and neuroactive substances. (c) 2017 American Physiological Society. Compr Physiol 7:235-252, 2017.

The study of transmitter interactions in reward and motor pathways in the brain, including the striatum, requires methodology to detect stimulus-driven neurotransmitter release events. Such methods exist for dopamine, and have contributed to the understanding of local and behavioral factors that regulate dopamine release. However, factors that regulate release of another key transmitter in these pathways, acetylcholine (ACh), are unresolved, in part because of limited temporal and spatial resolution of current detection methods. We have optimized a voltammetric method for detection of local stimulus-evoked ACh release using enzyme-coated carbon-fiber microelectrodes and fast-scan cyclic voltammetry. These electrodes are based on the detection of H2O2 generated by the actions of acetylcholine esterase and choline oxidase, and reliably respond to ACh in a concentration-dependent manner. Methods for enzyme coating were optimized for mechanical stability that allowed for their use in ex vivo brain slices. We report here the first quantitative assessment of extracellular ACh concentration after local electrical stimulation in dorsal striatum in slices from control mice. The selective detection of ACh under these conditions was confirmed by showing that the response detected in the control slices was absent in slices from mice bred to lack ACh synthesis in the forebrain. These electrodes represent a new tool to study ACh and ACh-dopamine interactions with micrometer spatial resolution.

Voltammetry is an electrochemical technique that capitalises on the ability of some substances to become oxidised or reduced. A variety of voltammetric methods have been developed for the detection of biogenic amines such as dopamine, noradrenaline and serotonin in the brain. Each method differs in selectivity for the transmitter of interest and in temporal resolution. Of these, fast-scan cyclic voltammetry (FCV) at carbon fibre electrodes has been used extensively for monitoring the evoked or spontaneous release of biogenic amines in various brain regions with temporal and spatial resolutions that capture extrasynaptic transmission. Studies in rodent and non-rodent brain slices containing either monoamine cell bodies or axonal projections enable the dynamics of neurotransmitter release and its regulation by monoamine transporters, autoreceptors and local neuromodulators to be examined. Furthermore, rapid detection of release in freely moving animals can reveal the role of biogenic amines in motivated behaviour

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.

Historically, brain neurochemicals have been broadly classified as energetic or informational. However, increasing evidence implicates metabolic substrates and byproducts as signalling agents, which blurs the boundary between energy and information, and suggests the introduction of a new category for 'translational' substances that convey changes in energy state to information. One intriguing example is hydrogen peroxide (H2 O2 ), which is a small, readily diffusible molecule. Produced during mitochondrial respiration, this reactive oxygen species, can mediate dynamic regulation of neuronal activity and transmitter release by activating inhibitory ATP-sensitive K(+) (KATP ) channels, as well as a class of excitatory non-selective cation channels, TRPM2. Studies using ex vivo guinea pig brain slices have revealed that activity-generated H2 O2 can act via KATP channels to inhibit dopamine release in dorsal striatum and dopamine neuron activity in the substantia nigra pars compacta. In sharp contrast, endogenously generated H2 O2 enhances the excitability of GABAergic projection neurons in the dorsal striatum and substantia nigra pars reticulata by activating TRPM2 channels. These studies suggest that the balance of excitation vs. inhibition produced in a given cell by metabolically generated H2 O2 will be dictated by the relative abundance of H2 O2 -sensitive ion channel targets that receive this translational signal.

Dopamine (DA) is a key transmitter in motor, reward and cogitative pathways, with DA dysfunction implicated in disorders including Parkinson's disease and addiction. Located in midbrain, DA neurons of the substantia nigra pars compacta project via the medial forebrain bundle to the dorsal striatum (caudate putamen), and DA neurons in the adjacent ventral tegmental area project to the ventral striatum (nucleus accumbens) and prefrontal cortex. In addition to classical vesicular release from axons, midbrain DA neurons exhibit DA release from their cell bodies and dendrites. Somatodendritic DA release leads to activation of D2 DA autoreceptors on DA neurons that inhibit their firing via G-protein-coupled inwardly rectifying K+ channels. This helps determine patterns of DA signalling at distant axonal release sites. Somatodendritically released DA also acts via volume transmission to extrasynaptic receptors that modulate local transmitter release and neuronal activity in the midbrain. Thus, somatodendritic release is a pivotal intrinsic feature of DA neurons that must be well defined in order to fully understand the physiology and pathophysiology of DA pathways. Here, we review recent mechanistic aspects of somatodendritic DA release, with particular emphasis on the Ca2+ dependence of release and the potential role of exocytotic proteins.

Tobacco products influence striatal dopamine (DA) release primarily through the actions of nicotine, an agonist of nicotinic acetylcholine receptors (nAChR). Gutkha is a smokeless tobacco product that includes the habit-forming areca nut, and other plant-based constituents contain muscarinic acetylcholine receptor (mAChR) agonists and other cholinergic agents, as well as nicotine. The net influence of the cholinergic agents in gutkha on striatal DA release is therefore difficult to predict. This study investigated the influence of gutkha extract on evoked DA release in mouse striatal slices using fast-scan cyclic voltammetry. The potency of a given concentration of nicotine in the gutkha extract was found to be significantly lower than that of a comparable concentration of nicotine alone. Atropine, a mAChR antagonist, increased the potency of gutkha-associated nicotine; however, other experiments suggested that this was mediated in part by effects of atropine directly at nAChRs. Overall, these results suggest that the unique constituents of gutkha work together to oppose the influence of gutkha-associated nicotine on evoked striatal DA release.

Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (gamma-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.

Brain dopamine pathways serve wide-ranging functions including the control of movement, reward, cognition, learning, and mood. Consequently, dysfunction of dopamine transmission has been implicated in clinical conditions such as Parkinson's disease, schizophrenia, addiction, and depression. Establishing factors that regulate dopamine release can provide novel insights into dopaminergic communication under normal conditions, as well as in animal models of disease in the brain. Here we describe methods for the study of somatodendritic and axonal dopamine release in brain slice preparations. Topics covered include preparation and calibration of carbon-fiber microelectrodes for use with fast-scan cyclic voltammetry, preparation of midbrain and forebrain slices, and procedures of eliciting and recording electrically evoked dopamine release from in vitro brain slices.