Faculty Bibliography

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.

Here we review evidence that the reactive oxygen species, hydrogen peroxide (H(2)O(2)), meets the criteria for classification as a neuromodulator through its effects on striatal dopamine (DA) release. This evidence was obtained using fast-scan cyclic voltammetry to detect evoked DA release in striatal slices, along with whole-cell and fluorescence imaging to monitor cellular activity and H(2)O(2) generation in striatal medium spiny neurons (MSNs). The data show that (1) exogenous H(2)O(2) suppresses DA release in dorsal striatum and nucleus accumbens shell and the same effect is seen with elevation of endogenous H(2)O(2) levels; (2) H(2)O(2) is generated downstream from glutamatergic AMPA receptor activation in MSNs, but not DA axons; (3) generation of modulatory H(2)O(2) is activity dependent; (4) H(2)O(2) generated in MSNs diffuses to DA axons to cause transient DA release suppression by activating ATP-sensitive K(+) (K(ATP)) channels on DA axons; and (5) the amplitude of H(2)O(2)-dependent inhibition of DA release is attenuated by enzymatic degradation of H(2)O(2), but the subsecond time course is determined by H(2)O(2) diffusion rate and/or K(ATP)-channel kinetics. In the dorsal striatum, neuromodulatory H(2)O(2) is an intermediate in the regulation of DA release by the classical neurotransmitters glutamate and GABA, as well as other neuromodulators, including cannabinoids. However, modulatory actions of H(2)O(2) occur in other regions and cell types, as well, consistent with the widespread expression of K(ATP) and other H(2)O(2)-sensitive channels throughout the CNS.

Dopamine transmission is critical for exploratory motor behaviour. A key regulator is acetylcholine; forebrain acetylcholine regulates striatal dopamine release, whereas brainstem cholinergic inputs regulate the transition of dopamine neurons from tonic to burst firing modes. How these sources of cholinergic activity combine to control dopamine efflux and exploratory motor behaviour is unclear. Here we show that mice lacking total forebrain acetylcholine exhibit enhanced frequency-dependent striatal dopamine release and are hyperactive in a novel environment, whereas mice lacking rostral brainstem acetylcholine are hypoactive. Exploratory motor behaviour is normalized by the removal of both cholinergic sources. Involvement of dopamine in the exploratory motor phenotypes observed in these mutants is indicated by their altered sensitivity to the dopamine D2 receptor antagonist raclopride. These results support a model in which forebrain and brainstem cholinergic systems act in tandem to regulate striatal dopamine signalling for proper control of motor activity.

Dopamine (DA) is a key transmitter in the basal ganglia, yet DA transmission does not conform to several aspects of the classic synaptic doctrine. Axonal DA release occurs through vesicular exocytosis and is action potential- and Ca(2+) -dependent. However, in addition to axonal release, DA neurons in midbrain exhibit somatodendritic release by an incompletely understood, but apparently exocytotic, mechanism. Even in striatum, axonal release sites are controversial, with evidence for DA varicosities that lack postsynaptic specialization, and largely extrasynaptic DA receptors and transporters. Moreover, DA release is often assumed to reflect a global response to a population of activities in midbrain DA neurons, whether tonic or phasic, with precise timing and specificity of action governed by other basal ganglia circuits. This view has been reinforced by anatomical evidence showing dense axonal DA arbors throughout striatum, and a lattice network formed by DA axons and glutamatergic input from cortex and thalamus. Nonetheless, localized DA transients are seen in vivo using voltammetric methods with high spatial and temporal resolution. Mechanistic studies using similar methods in vitro have revealed local regulation of DA release by other transmitters and modulators, as well as by proteins known to be disrupted in Parkinson's disease and other movement disorders. Notably, the actions of most other striatal transmitters on DA release also do not conform to the synaptic doctrine, with the absence of direct synaptic contacts for glutamate, GABA, and acetylcholine (ACh) on striatal DA axons. Overall, the findings reviewed here indicate that DA signaling in the basal ganglia is sculpted by cooperation between the timing and pattern of DA input and those of local regulatory factors. This article is part of a Special Issue entitled: Function and Dysfunction of the Basal Ganglia