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Leptin promotes striatal dopamine release via cholinergic interneurons and regionally distinct signaling pathways

Mancini, Maria; Patel, Jyoti C; Affinati, Alison H; Witkovsky, Paul; Rice, Margaret E
Dopamine (DA) is a critical regulator of striatal network activity and is essential for motor activation and reward-associated behaviors. Previous work has shown that DA is influenced by the reward value of food, as well as by hormonal factors implicated in the regulation of food intake and energy expenditure. Changes in striatal DA signaling also have been linked to aberrant eating patterns. Here we test the effect of leptin, an adipocyte-derived hormone involved in feeding and energy homeostasis regulation, on striatal DA release and uptake. Immunohistochemical evaluation identified leptin receptor expression throughout mouse striatum, including on striatal cholinergic interneurons and their extensive processes. Using fast-scan cyclic voltammetry, we found that leptin causes a concentration-dependent increase in evoked extracellular DA concentration ([DA]o) in dorsal striatum and nucleus accumbens (NAc) core and shell in male mouse striatal slices, and also an increase in the rate of DA uptake. Further, we found that leptin increases cholinergic interneuron excitability, and that the enhancing effect of leptin on evoked [DA]o is lost when nicotinic acetylcholine (ACh) receptors are antagonized or when examined in striatal slices from mice lacking ACh synthesis. Evaluation of signaling pathways underlying leptin's action revealed a requirement for intracellular Ca2+, and the involvement of different downstream pathways in dorsal striatum and NAc core versus NAc shell. These results provide the first evidence for dynamic regulation of DA release and uptake by leptin within brain motor and reward pathways, and highlight the involvement of cholinergic interneurons in this process.SIGNIFICANCE STATEMENTGiven the importance of striatal dopamine in reward, motivation, motor behavior and food intake, identifying the actions of metabolic hormones on dopamine release in striatal subregions should provide new insight into factors that influence dopamine-dependent motivated behaviors. We find that one of these hormones, leptin, boosts striatal dopamine release through a process involving striatal cholinergic interneurons and nicotinic acetylcholine receptors. Moreover, we find that the intracellular cascades downstream from leptin receptor activation underlying enhanced dopamine release differ among striatal subregions. Thus, we not only show that leptin regulates dopamine release, but also identify characteristics of this process that could be harnessed to alter pathological eating behaviors.
PMID: 35906070
ISSN: 1529-2401
CID: 5277032

Synaptotagmins 1 and 7 Play Complementary Roles in Somatodendritic Dopamine Release

Hikima, Takuya; Witkovsky, Paul; Khatri, Latika; Chao, Moses V; Rice, Margaret E
The molecular mechanisms underlying somatodendritic dopamine (DA) release remain unresolved, despite the passing of decades since its discovery. Our previous work showed robust release of somatodendritic DA in submillimolar extracellular Ca2+ concentration ([Ca2+]o). Here we tested the hypothesis that the high-affinity Ca2+ sensor synaptotagmin 7 (Syt7), is a key determinant of somatodendritic DA release and its Ca2+ dependence. Somatodendritic DA release from SNc DA neurons was assessed using whole-cell recording in midbrain slices from male and female mice to monitor evoked DA-dependent D2 receptor-mediated inhibitory currents (D2ICs). Single-cell application of an antibody to Syt7 (Syt7 Ab) decreased pulse train-evoked D2ICs, revealing a functional role for Syt7. The assessment of the Ca2+ dependence of pulse train-evoked D2ICs confirmed robust DA release in submillimolar [Ca2+]o in wild-type (WT) neurons, but loss of this sensitivity with intracellular Syt7 Ab or in Syt7 knock-out (KO) mice. In millimolar [Ca2+]o, pulse train-evoked D2ICs in Syt7 KOs showed a greater reduction in decreased [Ca2+]o than seen in WT mice; the effect on single pulse-evoked DA release, however, did not differ between genotypes. Single-cell application of a Syt1 Ab had no effect on train-evoked D2ICs in WT SNc DA neurons, but did cause a decrease in D2IC amplitude in Syt7 KOs, indicating a functional substitution of Syt1 for Syt7. In addition, Syt1 Ab decreased single pulse-evoked D2ICs in WT cells, indicating the involvement of Syt1 in tonic DA release. Thus, Syt7 and Syt1 play complementary roles in somatodendritic DA release from SNc DA neurons.SIGNIFICANCE STATEMENT The respective Ca2+ dependence of somatodendritic and axonal dopamine (DA) release differs, resulting in the persistence of somatodendritic DA release in submillimolar Ca2+ concentrations too low to support axonal release. We demonstrate that synaptotagmin7 (Syt7), a high-affinity Ca2+ sensor, underlies phasic somatodendritic DA release and its Ca2+ sensitivity in the substantia nigra pars compacta. In contrast, we found that synaptotagmin 1 (Syt1), the Ca2+ sensor underlying axonal DA release, plays a role in tonic, but not phasic, somatodendritic DA release in wild-type mice. However, Syt1 can facilitate phasic DA release after Syt7 deletion. Thus, we show that both Syt1 and Syt7 act as Ca2+ sensors subserving different aspects of somatodendritic DA release processes.
PMCID:9097777
PMID: 35361702
ISSN: 1529-2401
CID: 5220042

Activity-dependent somatodendritic dopamine release in the substantia nigra autoinhibits the releasing neuron

Hikima, Takuya; Lee, Christian R; Witkovsky, Paul; Chesler, Julia; Ichtchenko, Konstantin; Rice, Margaret E
Somatodendritic dopamine (DA) release from midbrain DA neurons activates D2 autoreceptors on these cells to regulate their activity. However, the source of autoregulatory DA remains controversial. Here, we test the hypothesis that D2 autoreceptors on a given DA neuron in the substantia nigra pars compacta (SNc) are activated primarily by DA released from that same cell, rather than from its neighbors. Voltage-clamp recording allows monitoring of evoked D2-receptor-mediated inhibitory currents (D2ICs) in SNc DA neurons as an index of DA release. Single-cell application of antibodies to Na+ channels via the recording pipette decreases spontaneous activity of recorded neurons and attenuates evoked D2ICs; antibodies to SNAP-25, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, also decrease D2IC amplitude. Evoked D2ICs are nearly abolished by the light chain of botulinum neurotoxin A, which cleaves SNAP-25, whereas synaptically activated GABAB-receptor-mediated currents are unaffected. Thus, somatodendritic DA release in the SNc autoinhibits the neuron that releases it.
PMID: 33826884
ISSN: 2211-1247
CID: 4839342

Insulin enhances striatal dopamine release by activating cholinergic interneurons and thereby signals reward

Stouffer, Melissa A; Woods, Catherine A; Patel, Jyoti C; Lee, Christian R; Witkovsky, Paul; Bao, Li; Machold, Robert P; Jones, Kymry T; de Vaca, Soledad Cabeza; Reith, Maarten E A; Carr, Kenneth D; Rice, Margaret E
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.
PMCID:4624275
PMID: 26503322
ISSN: 2041-1723
CID: 1816772

TRPM2 Channels Are Required for NMDA-Induced Burst Firing and Contribute to H2O2-Dependent Modulation in Substantia Nigra Pars Reticulata GABAergic Neurons

Lee, Christian R; Machold, Robert P; Witkovsky, Paul; Rice, Margaret E
Substantia nigra pars reticulata (SNr) GABAergic neurons are projection neurons that convey output from the basal ganglia to target structures. These neurons exhibit spontaneous regular firing, but also exhibit burst firing in the presence of NMDA or when excitatory glutamatergic input to the SNr is activated. Notably, an increase in burst firing is also seen in Parkinson's disease. Therefore, elucidating conductances that mediate spontaneous activity and changes of firing pattern in these neurons is essential for understanding how the basal ganglia control movement. Using ex vivo slices of guinea pig midbrain, we show that SNr GABAergic neurons express transient receptor potential melastatin 2 (TRPM2) channels that underlie NMDA-induced burst firing. Furthermore, we show that spontaneous firing rate and burst activity are modulated by the reactive oxygen species H(2)O(2) acting via TRPM2 channels. Thus, our results indicate that activation of TRPM2 channels is necessary for burst firing in SNr GABAergic neurons and their responsiveness to modulatory H(2)O(2). These findings have implications not only for normal regulation, but also for Parkinson's disease, which involves excitotoxicity and oxidative stress.
PMCID:3705724
PMID: 23325252
ISSN: 0270-6474
CID: 213462

Phase waves in the suprachiasmatic nucleus (commentary on Hong et al.) [Comment]

Silver, Rae; Witkovsky, Paul
PMID: 22564072
ISSN: 0953-816x
CID: 902322

Subsecond regulation of striatal dopamine release by pre-synaptic K(ATP) channels

Patel, Jyoti C; Witkovsky, Paul; Coetzee, William A; Rice, Margaret E
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
PMCID:3369699
PMID: 21689107
ISSN: 1471-4159
CID: 136636

Light exposure induces short- and long-term changes in the excitability of retinorecipient neurons in suprachiasmatic nucleus

LeSauter, Joseph; Silver, Rae; Cloues, Robin; Witkovsky, Paul
The suprachiasmatic nucleus (SCN) is the locus of a hypothalamic circadian clock that synchronizes physiological and behavioral responses to the daily light-dark cycle. The nucleus is composed of functionally and peptidergically diverse populations of cells for which distinct electrochemical properties are largely unstudied. SCN neurons containing gastrin-releasing peptide (GRP) receive direct retinal input via the retinohypothalamic tract. We targeted GRP neurons with a green fluorescent protein (GFP) marker for whole cell patch-clamping. In these neurons, we studied short (0.5-1.5 h)- and long-term (2-6 h) effects of a 1-h light pulse (LP) given 2 h after lights off [Zeitgeber time (ZT) 14:00-15:00] on membrane potential and spike firing. In brain slices taken from light-exposed animals, cells were depolarized, and spike firing rate increased between ZT 15:30 and 16:30. During a subsequent 4-h period beginning around ZT 17:00, GRP neurons from light-exposed animals were hyperpolarized by approximately 15 mV. None of these effects was observed in GRP neurons from animals not exposed to light or in immediately adjacent non-GRP neurons whether or not exposed to light. Depolarization of GRP neurons was associated with a reduction in GABA(A)-dependent synaptic noise, whereas hyperpolarization was accompanied both by a loss of GABA(A) drive and suppression of a TTX-resistant leakage current carried primarily by Na. This suggests that, in the SCN, exposure to light may induce a short-term increase in GRP neuron excitability mediated by retinal neurotransmitters and neuropeptides, followed by long-term membrane hyperpolarization resulting from suppression of a leakage current, possibly resulting from genomic signals
PMCID:3154817
PMID: 21593396
ISSN: 1522-1598
CID: 137872

The Polymodal Ion Channel Transient Receptor Potential Vanilloid 4 Modulates Calcium Flux, Spiking Rate, and Apoptosis of Mouse Retinal Ganglion Cells

Ryskamp DA; Witkovsky P; Barabas P; Huang W; Koehler C; Akimov NP; Lee SH; Chauhan S; Xing W; Renteria RC; Liedtke W; Krizaj D
Sustained increase in intraocular pressure represents a major risk factor for eye disease, yet the cellular mechanisms of pressure transduction in the posterior eye are essentially unknown. Here we show that the mouse retina expresses mRNA and protein for the polymodal transient receptor potential vanilloid 4 (TRPV4) cation channel known to mediate osmotransduction and mechanotransduction. TRPV4 antibodies labeled perikarya, axons, and dendrites of retinal ganglion cells (RGCs) and intensely immunostained the optic nerve head. Muller glial cells, but not retinal astrocytes or microglia, also expressed TRPV4 immunoreactivity. The selective TRPV4 agonists 4alpha-PDD and GSK1016790A elevated [Ca(2+)](i) in dissociated RGCs in a dose-dependent manner, whereas the TRPV1 agonist capsaicin had no effect on [Ca(2+)](RGC). Exposure to hypotonic stimulation evoked robust increases in [Ca(2+)](RGC). RGC responses to TRPV4-selective agonists and hypotonic stimulation were absent in Ca(2+)-free saline and were antagonized by the nonselective TRP channel antagonists Ruthenium Red and gadolinium, but were unaffected by the TRPV1 antagonist capsazepine. TRPV4-selective agonists increased the spiking frequency recorded from intact retinas recorded with multielectrode arrays. Sustained exposure to TRPV4 agonists evoked dose-dependent apoptosis of RGCs. Our results demonstrate functional TRPV4 expression in RGCs and suggest that its activation mediates response to membrane stretch leading to elevated [Ca(2+)](i) and augmented excitability. Excessive Ca(2+) influx through TRPV4 predisposes RGCs to activation of Ca(2+)-dependent proapoptotic signaling pathways, indicating that TRPV4 is a component of the response mechanism to pathological elevations of intraocular pressure
PMCID:3109951
PMID: 21562271
ISSN: 1529-2401
CID: 134279

Regulation of Substantia Nigra Pars Reticulata GABAergic Neuron Activity by H(2)O(2) via Flufenamic Acid-Sensitive Channels and K(ATP) Channels

Lee, Christian R; Witkovsky, Paul; Rice, Margaret E
Substantia nigra pars reticulata (SNr) GABAergic neurons are key output neurons of the basal ganglia. Given the role of these neurons in motor control, it is important to understand factors that regulate their firing rate and pattern. One potential regulator is hydrogen peroxide (H(2)O(2)), a reactive oxygen species that is increasingly recognized as a neuromodulator. We used whole-cell current clamp recordings of SNr GABAergic neurons in guinea-pig midbrain slices to determine how H(2)O(2) affects the activity of these neurons and to explore the classes of ion channels underlying those effects. Elevation of H(2)O(2) levels caused an increase in the spontaneous firing rate of SNr GABAergic neurons, whether by application of exogenous H(2)O(2) or amplification of endogenous H(2)O(2) through inhibition of glutathione peroxidase with mercaptosuccinate. This effect was reversed by flufenamic acid (FFA), implicating transient receptor potential (TRP) channels. Conversely, depletion of endogenous H(2)O(2) by catalase, a peroxidase enzyme, decreased spontaneous firing rate and firing precision of SNr neurons, demonstrating tonic control of firing rate by H(2)O(2). Elevation of H(2)O(2) in the presence of FFA revealed an inhibition of tonic firing that was prevented by blockade of ATP-sensitive K(+) (K(ATP)) channels with glibenclamide. In contrast to guinea-pig SNr neurons, the dominant effect of H(2)O(2) elevation in mouse SNr GABAergic neurons was hyperpolarization, indicating a species difference in H(2)O(2)-dependent regulation. Thus, H(2)O(2) is an endogenous modulator of SNr GABAergic neurons, acting primarily through presumed TRP channels in guinea-pig SNr, with additional modulation via K(ATP) channels to regulate SNr output
PMCID:3074506
PMID: 21503158
ISSN: 1662-5137
CID: 131804