Faculty Bibliography

J. Neurochem. (2011) 118, 714-720. ABSTRACT: Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1) -receptor activation in axonal DA release regulation in dorsal striatum using a D(1) -receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of approximately 65% in 5 muM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 muM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3) H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 muM. Binding studies with [(3) H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 muM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT

J. Neurochem. (2011) 118, 721-736. ABSTRACT: ATP-sensitive K(+) (K(ATP) ) channels are composed of pore-forming subunits, typically Kir6.2 in neurons, and regulatory sulfonylurea receptor subunits. In dorsal striatum, activity-dependent H(2) O(2) produced from glutamate receptor activation inhibits dopamine release via K(ATP) channels. Sources of modulatory H(2) O(2) include striatal medium spiny neurons, but not dopaminergic axons. Using fast-scan cyclic voltammetry in guinea-pig striatal slices and immunohistochemistry, we determined the time window for H(2) O(2) /K(ATP) -channel-mediated inhibition and assessed whether modulatory K(ATP) channels are on dopaminergic axons. Comparison of paired-pulse suppression of dopamine release in the absence and presence of glibenclamide, a K(ATP) -channel blocker, or mercaptosuccinate, a glutathione peroxidase inhibitor that enhances endogenous H(2) O(2) levels, revealed a time window for inhibition of 500-1000 ms after stimulation. Immunohistochemistry demonstrated localization of Kir6.2 K(ATP) -channel subunits on dopaminergic axons. Consistent with the presence of functional K(ATP) channels on dopaminergic axons, K(ATP) -channel openers, diazoxide and cromakalim, suppressed single-pulse evoked dopamine release. Although cholinergic interneurons that tonically regulate dopamine release also express K(ATP) channels, diazoxide did not induce the enhanced frequency responsiveness of dopamine release seen with nicotinic-receptor blockade. Together, these studies reveal subsecond regulation of striatal dopamine release by endogenous H(2) O(2) acting at K(ATP) channels on dopaminergic axons, including a role in paired-pulse suppression

Midbrain dopamine (DA) neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) exhibit somatodendritic release of DA. Previous studies indicate a difference between the Ca(2+) dependence of somatodendritic DA release in the SNc and that of axonal DA release in dorsal striatum. Here, we evaluated the Ca(2+) dependence of DA release in the VTA and nucleus accumbens (NAc) shell for comparison with that in the SNc and dorsal striatum. Release of DA was elicited by single-pulse stimulation in guinea-pig brain slices and monitored with subsecond resolution using carbon-fiber microelectrodes and fast-scan cyclic voltammetry. In dorsal striatum and NAc, DA release was not detectable at extracellular Ca(2+) concentrations ([Ca(2+)](o)) below 1 mM; however, a progressive increase in evoked extracellular DA concentration ([DA](o)) was seen with [Ca(2+)](o) >/= 1.5 mM. By contrast, in SNc and VTA, robust increases in [DA](o) could be elicited in 0.25 mM [Ca(2+)](o) that were approximately 60% of those seen in 1.5 mM [Ca(2+)](o). In SNc, a plateau in single-pulse evoked [DA](o) was seen at [Ca(2+)](o) >/= 1.5 mM, mirroring the release plateau reported previously for pulse-train stimulation in SNc. In VTA, however, evoked [DA](o) increased progressively throughout the range of [Ca(2+)](o) tested (up to 3.0 mM). These functional data are consistent with the microanatomy of the VTA, which includes DA axon collaterals as well as DA somata and dendrites. Differences between axonal and somatodendritic release data were quantified using Hill analysis, which showed that the Ca(2+) dependence of axonal DA release is low affinity with high Ca(2+) cooperativity, whereas somatodendritic release is high affinity with low cooperativity. Moreover, this analysis revealed the dual nature of DA release in the VTA, with both somatodendritic and axonal contributions

Dystonia is a neurological disorder characterized by involuntary movements. We examined striatal dopamine (DA) function in hyperactive transgenic (Tg) mice generated as a model of dystonia. Evoked extracellular DA concentration was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry in striatal slices from non-Tg mice, Tg mice with a positive motor phenotype, and phenotype-negative Tg littermates. Peak single-pulse evoked extracellular DA concentration was significantly lower in phenotype-positive mice than in non-Tg or phenotype-negative mice, but indistinguishable between non-Tg and phenotype-negative mice. Phenotype-positive mice also had higher functional D2 DA autoreceptor sensitivity than non-Tg mice, which would be consistent with lower extracellular DA concentration in vivo. Multiple-pulse (phasic) stimulation (five pulses, 10-100 Hz) revealed an enhanced frequency dependence of evoked DA release in phenotype-positive versus non-Tg or phenotype-negative mice, which was exacerbated when extracellular Ca(2+) concentration was lowered. Enhanced sensitivity to phasic stimulation in phenotype-positive mice was reminiscent of the pattern seen with antagonism of nicotinic acetylcholine receptors. Consistent with a role for altered cholinergic regulation, the difference in phasic responsiveness among groups was lost when nicotinic receptors were blocked by mecamylamine. Together, these data implicate compromised DA release regulation, possibly from cholinergic dysfunction, in the motor symptoms of this dystonia model

Recent evidence suggests the intriguing possibility that midbrain dopaminergic (DAergic) neurons may use fast glutamatergic transmission to communicate with their postsynaptic targets. Because of technical limitations, direct demonstration of the existence of this signaling mechanism has been limited to experiments using cell culture preparations that often alter neuronal function including neurotransmitter phenotype. Consequently, it remains uncertain whether glutamatergic signaling between DAergic neurons and their postsynaptic targets exists under physiological conditions. Here, using an optogenetic approach, we provide the first conclusive demonstration that mesolimbic DAergic neurons in mice release glutamate and elicit excitatory postsynaptic responses in projection neurons of the nucleus accumbens. In addition, we describe the properties of the postsynaptic glutamatergic responses of these neurons during experimentally evoked burst firing of DAergic axons that reproduce the reward-related phasic population activity of the mesolimbic projection. These observations indicate that, in addition to DAergic mechanisms, mesolimbic reward signaling may involve glutamatergic transmission

PARK8/LRRK2 (leucine-rich repeat kinase 2) was recently identified as a causative gene for autosomal dominant Parkinson's disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Emerging in vitro evidence indicates that aberrant enzymatic activity of LRRK2 protein carrying this mutation can cause neurotoxicity. However, the physiological and pathophysiological functions of LRRK2 in vivo remain elusive. Here we characterize two bacterial artificial chromosome (BAC) transgenic mouse strains overexpressing LRRK2 wild-type (Wt) or mutant G2019S. Transgenic LRRK2-Wt mice had elevated striatal dopamine (DA) release with unaltered DA uptake or tissue content. Consistent with this result, LRRK2-Wt mice were hyperactive and showed enhanced performance in motor function tests. These results suggest a role for LRRK2 in striatal DA transmission and the consequent motor function. In contrast, LRRK2-G2019S mice showed an age-dependent decrease in striatal DA content, as well as decreased striatal DA release and uptake. Despite increased brain kinase activity, LRRK2-G2019S overexpression was not associated with loss of DAergic neurons in substantia nigra or degeneration of nigrostriatal terminals at 12 months. Our results thus reveal a pivotal role for LRRK2 in regulating striatal DA transmission and consequent control of motor function. The PD-associated mutation G2019S may exert pathogenic effects by impairing these functions of LRRK2. Our LRRK2 BAC transgenic mice, therefore, could provide a useful model for understanding early PD pathological events

We examined the somatodendritic compartment of nigral dopaminergic neurons by immunocytochemistry and confocal microscopy, with the aim of identifying proteins that participate in dopamine packaging and release. Nigral dopaminergic neurons were identified by location, cellular features and tyrosine hydroxylase immunoreactivity. Immunoreactive puncta of vesicular monoamine transporter type 2 and proton ATPase, both involved in the packaging of dopamine for release, were located primarily in dopaminergic cell bodies, but were absent in distal dopaminergic dendrites. Many presynaptic proteins associated with transmitter release at fast synapses were absent in nigral dopaminergic neurons, including synaptotagmin 1, syntaxin1, synaptic vesicle proteins 2a and 2b, synaptophysin and synaptobrevin 1 (VAMP 1). On the other hand, syntaxin 3, synaptobrevin 2 (VAMP 2) and SNAP-25-immunoreactivities were found in dopaminergic somata and dendrites Our data imply that the storage and exocytosis of dopamine from the somatodendritic compartment of nigral dopaminergic neurons is mechanistically distinct from transmitter release at axon terminals utilizing amino acid neurotransmitters

Hydrogen peroxide (H(2)O(2)) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H(2)O(2) signaling, which activates ATP-sensitive potassium (K(ATP)) channels to inhibit dopamine release. However, the origin of this modulatory H(2)O(2) has been elusive. Here we addressed three possible sources of H(2)O(2) produced for rapid neuronal signaling in striatum: mitochondrial respiration, monoamine oxidase (MAO), and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H(2)O(2) and tissue analysis of ATP, we found that coapplication of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H(2)O(2) production, but maintained tissue ATP content. Strikingly, coapplication of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H(2)O(2) in release modulation. In contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H(2)O(2) production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H(2)O(2) generation for dynamic neuronal signaling

Somatodendritic dopamine (DA) release in the substantia nigra pars compacta (SNc) shows a limited dependence on extracellular calcium concentration ([Ca(2+)](o)), suggesting the involvement of intracellular Ca(2+) stores. Here, using immunocytochemistry we demonstrate the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) that sequesters cytosolic Ca(2+) into the endoplasmic reticulum (ER), as well as inositol 1,4,5-triphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in DAergic neurons. Notably, RyRs were clustered at the plasma membrane, poised for activation by Ca(2+) entry. Using fast-scan cyclic voltammetry to monitor evoked extracellular DA concentration ([DA](o)) in midbrain slices, we found that SERCA inhibition by cyclopiazonic acid (CPA) decreased evoked [DA](o) in the SNc, indicating a functional role for ER Ca(2+) stores in somatodendritic DA release. Implicating IP(3)R-dependent stores, an IP(3)R antagonist, 2-APB, also decreased evoked [DA](o). Moreover, DHPG, an agonist of group I metabotropic glutamate receptors (mGluR1s, which couple to IP(3) production), increased somatodendritic DA release, whereas CPCCOEt, an mGluR1 antagonist, suppressed it. Release suppression by mGluR1 blockade was prevented by 2-APB or CPA, indicating facilitation of DA release by endogenous glutamate acting via mGluR1s and IP(3)R-gated Ca(2+) stores. Similarly, activation of RyRs by caffeine increased [Ca(2+)](i) and elevated evoked [DA](o). The increase in DA release was prevented by a RyR blocker, dantrolene, and by CPA. Importantly, the efficacy of dantrolene was enhanced in low [Ca(2+)](o), suggesting a mechanism for maintenance of somatodendritic DA release with limited Ca(2+) entry. Thus, both mGluR1-linked IP(3)R- and RyR-dependent ER Ca(2+) stores facilitate somatodendritic DA release in the SNc

Dopamine-glutamate interactions in the striatum are critical for normal basal ganglia-mediated control of movement. Although regulation of glutamatergic transmission by dopamine is increasingly well understood, regulation of dopaminergic transmission by glutamate remains uncertain given the apparent absence of ionotropic glutamate receptors on dopaminergic axons in dorsal striatum. Indirect evidence suggests glutamatergic regulation of striatal dopamine release is mediated by a diffusible messenger, hydrogen peroxide (H2O2), generated downstream from glutamatergic AMPA receptors (AMPARs). The mechanism of H2O2-dependent inhibition of dopamine release involves activation of ATP-sensitive K+ (KATP) channels. However, the source of modulatory H2O2 is unknown. Here, we used whole cell recording, fluorescence imaging of H2O2, and voltammetric detection of evoked dopamine release in guinea pig striatal slices to examine contributions from medium spiny neurons (MSNs), the principal neurons of striatum, and dopamine axons to AMPAR-dependent H2O2 generation. Imaging studies of H2O2 generation in MSNs provide the first demonstration of AMPAR-dependent H2O2 generation in neurons in the complex brain-cell microenvironment of brain slices. Stimulation-induced increases in H2O2 in MSNs were prevented by GYKI-52466, an AMPAR antagonist, or catalase, an H2O2 metabolizing enzyme, but amplified by mercaptosuccinate (MCS), a glutathione peroxidase inhibitor. By contrast, dopamine release evoked by selective stimulation of dopamine axons was unaffected by GYKI-52466 or MCS, arguing against dopamine axons as a significant source of modulatory H2O2. Together, these findings suggest that glutamatergic regulation of dopamine release via AMPARs is mediated through retrograde signaling by diffusible H2O2 generated in striatal cells, including medium spiny neurons, rather than in dopamine axons