Faculty Bibliography

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

The predominance of dopamine (DA) receptors at extrasynaptic vs. synaptic sites implies that DA signaling is by diffusion-based volume transmission. In this review, we compare characteristics that regulate extracellular DA behavior in substantia nigra pars compacta (SNc) and striatum, including regional differences in structure (a 40% greater extracellular volume fraction in SNc vs. striatum) and in dynamic DA uptake (a 200-fold greater DA uptake rate in striatum vs. SNc). Furthermore, we test the assumption of diffusion-based volume transmission for SNc and striatum by modeling dynamic DA behavior after quantal release using region-specific parameters for diffusion and uptake at 37 degrees C. Our model shows that DA uptake does not affect peak DA concentration within 1 mum of a release site in either SNc or striatum because of the slow kinetics of DATs vs. diffusion. Rather, diffusion and dilution are the dominant factors governing DA concentration after quantal release. In SNc, limited DAT efficacy is reflected in a lack of influence of uptake on either amplitude or time course of DA transients after quantal release up to 10 mum from a release site. In striatum, the lack of effect of the DAT within 1 mum of a release site means that perisynaptic DATs do not 'gate' synaptic spillover. This contrasts with the conventional view of DA synapses, in which DATs efficiently recycle DA by re-uptake into the releasing axon terminal. However, the model also shows that a primary effect of striatal uptake is to curtail DA lifetime after release. In both SNc and striatum, effective DA radius after quantal release is ~2 mum for activation of low-affinity DA receptors and 7-8 mum for high-affinity receptors; the corresponding spheres of influence would encompass tens to thousands of synapses. Thus, the primary mode of intercellular communication by DA, regardless of region, is volume transmission

The main psychoactive component of marijuana, Delta9-tetrahydrocannabinol (THC), acts in the CNS via type 1 cannabinoid receptors (CB1Rs). The behavioral consequences of THC or synthetic CB1R agonists include suppression of motor activity. One explanation for movement suppression might be inhibition of striatal dopamine (DA) release by CB1Rs, which are densely localized in motor striatum; however, data from previous studies are inconclusive. Here we examined the effect of CB1R activation on locally evoked DA release monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry in striatal slices. Consistent with previous reports, DA release evoked by a single stimulus pulse was unaffected by WIN55,212-2, a cannabinoid receptor agonist. However, when DA release was evoked by a train of stimuli, WIN55,212-2 caused a significant decrease in evoked extracellular DA concentration ([DA]o), implicating the involvement of local striatal circuitry, with similar suppression seen in guinea pig, rat, and mouse striatum. Pulse-train evoked [DA]o was not altered by either AM251, an inverse CB1R agonist, or VCHSR1, a neutral antagonist, indicating the absence of DA release regulation by endogenous cannabinoids with the stimulation protocol used. However, both CB1R antagonists prevented and reversed suppression of evoked [DA]o by WIN55,212-2. The effect of WIN55,212-2 was also prevented by picrotoxin, a GABAA receptor antagonist, and by catalase, a metabolizing enzyme for hydrogen peroxide (H2O2). Furthermore, blockade of ATP-sensitive K+ (KATP) channels by tolbutamide or glybenclamide prevented the effect of WIN55,212-2 on DA release. Together, these data indicate that suppression of DA release by CB1R activation within striatum occurs via a novel nonsynaptic mechanism that involves GABA release inhibition, increased generation of the diffusible messenger H2O2, and activation of KATP channels to inhibit DA release. In addition, the findings suggest a possible physiological substrate for the motor effects of cannabinoid agonist administration

Many retinal ganglion cells are coupled via gap junctions with neighboring amacrine cells and ganglion cells. We investigated the extent and dynamics of coupling in one such network, the OFF alpha ganglion cell of rabbit retina and its associated amacrine cells. We also observed the relative spread of Neurobiotin injected into a ganglion cell in the presence of modulators of gap junctional permeability. We found that gap junctions between amacrine cells were closed via stimulation of a D(1) dopamine receptor, while the gap junctions between ganglion cells were closed via stimulation of a D(2) dopamine receptor. The pairs of hemichannels making up the heterologous gap junctions between the ganglion and amacrine cells were modulated independently, so that elevations of cAMP in the ganglion cell open the ganglion cell hemichannels, while elevations of cAMP in the amacrine cell close its hemichannels. We also measured endogenous dopamine release from an eyecup preparation and found a basal release from the dark-adapted retina of approximately 2 pmol/min during the day. Maximal stimulation with light increased the rate of dopamine release from rabbit retina by 66%. The results suggest that coupling between members of the OFF alpha ganglion cell/amacrine cell network is differentially modulated with changing levels of dopamine