Synaptotagmins 1 and 7 Play Complementary Roles in Somatodendritic Dopamine Release.

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.

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

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.

TRPM2 channels are required for NMDA-induced burst firing and contribute to H(2)O(2)-dependent modulation in substantia nigra pars reticulata GABAergic neurons.

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.

GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABAA receptors.

Striatal dopamine axons co-release dopamine and gamma-aminobutyric acid (GABA), using GABA provided by uptake via GABA transporter-1 (GAT1). Functions of GABA co-release are poorly understood. We asked whether co-released GABA autoinhibits dopamine release via axonal GABA type A receptors (GABAARs), complementing established inhibition by dopamine acting at axonal D2 autoreceptors. We show that dopamine axons express α3-GABAAR subunits in mouse striatum. Enhanced dopamine release evoked by single-pulse optical stimulation in striatal slices with GABAAR antagonism confirms that an endogenous GABA tone limits dopamine release. Strikingly, an additional inhibitory component is seen when multiple pulses are used to mimic phasic axonal activity, revealing the role of GABAAR-mediated autoinhibition of dopamine release. This autoregulation is lost in conditional GAT1-knockout mice lacking GABA co-release. Given the faster kinetics of ionotropic GABAARs than G-protein-coupled D2 autoreceptors, our data reveal a mechanism whereby co-released GABA acts as a first responder to dampen phasic-to-tonic dopamine signaling.

The polymodal ion channel transient receptor potential vanilloid 4 modulates calcium flux, spiking rate, and apoptosis of mouse retinal ganglion cells.

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. Müller glial cells, but not retinal astrocytes or microglia, also expressed TRPV4 immunoreactivity. The selective TRPV4 agonists 4α-PDD and GSK1016790A elevated [Ca2+]i in dissociated RGCs in a dose-dependent manner, whereas the TRPV1 agonist capsaicin had no effect on [Ca2+](RGC). Exposure to hypotonic stimulation evoked robust increases in [Ca2+](RGC). RGC responses to TRPV4-selective agonists and hypotonic stimulation were absent in Ca2+ -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 [Ca2+]i and augmented excitability. Excessive Ca2+ influx through TRPV4 predisposes RGCs to activation of Ca2+ -dependent proapoptotic signaling pathways, indicating that TRPV4 is a component of the response mechanism to pathological elevations of intraocular pressure.

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

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 ∼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.

Subsecond regulation of striatal dopamine release by pre-synaptic KATP channels.

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.

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

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.

Mobilization of calcium from intracellular stores facilitates somatodendritic dopamine release.

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.

Targeted mutation of the calbindin D28K gene disrupts circadian rhythmicity and entrainment.

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker in mammals. A salient feature of the SCN is that cells of a particular phenotype are topographically organized; this organization defines functionally distinct subregions that interact to generate coherent rhythmicity. In Syrian hamsters (Mesocricetus auratus), a dense population of directly retinorecipient calbindin D(28K) (CalB) neurons in the caudal SCN marks a subregion critical for circadian rhythmicity. In mouse SCN, a dense cluster of CalB neurons occurs during early postnatal development, but in the adult CalB neurons are dispersed through the SCN. In the adult retina CalB colocalizes with melanopsin-expressing ganglion cells. In the present study, we explored the role of CalB in modulating circadian function and photic entrainment by investigating mice with a targeted mutation of the CalB gene (CalB-/- mice). In constant darkness (DD), CalB-/- animals either become arrhythmic (40%) or exhibit low-amplitude locomotor rhythms with marked activity during subjective day (60%). Rhythmic clock gene expression is blunted in these latter animals. Importantly, CalB-/- mice exhibit anomalies in entrainment revealed following transfer from a light : dark cycle to DD. Paradoxically, responses to acute light pulses measured by behavioral phase shifts, SCN FOS protein and Period1 mRNA expression are normal. Together, the developmental pattern of CalB expression in mouse SCN, the presence of CalB in photoresponsive ganglion cells and the abnormalities seen in CalB-/- mice suggest an important role for CalB in mouse circadian function.

Differential distribution of voltage-gated calcium channels in dopaminergic neurons of the rat retina.

We studied by immunocytochemistry and Western blots the identity and cellular distribution of voltage-gated calcium channels within dopaminergic neurons of the rat retina. The aim was to associate particular calcium channel subtypes with known activities of the neuron (e.g., transmitter release from axon terminals). Five voltage-gated calcium channels were identified: alpha1A, alpha1B, alpha1E, alpha1F, and alpha1H. All of these, except the alpha1B subtype, were found within dopaminergic perikarya. The alpha1B channels were concentrated at axon terminal rings, together with alpha1A calcium channels. In contrast, alpha1H calcium channels were most abundant in the dendrites, and alpha1F calcium channels were restricted to the perikaryon. The alpha1E calcium channel was present at such a low density that its cellular distribution beyond the perikaryon could not be determined. Our findings are consistent with the available pharmacological data indicating that alpha1A and alpha1B calcium channels control the major fraction of dopamine release in the rat retina.

Synaptic transmission at retinal ribbon synapses.

The molecular organization of ribbon synapses in photoreceptors and ON bipolar cells is reviewed in relation to the process of neurotransmitter release. The interactions between ribbon synapse-associated proteins, synaptic vesicle fusion machinery and the voltage-gated calcium channels that gate transmitter release at ribbon synapses are discussed in relation to the process of synaptic vesicle exocytosis. We describe structural and mechanistic specializations that permit the ON bipolar cell to release transmitter at a much higher rate than the photoreceptor does, under in vivo conditions. We also consider the modulation of exocytosis at photoreceptor synapses, with an emphasis on the regulation of calcium channels.

Activity-dependent phosphorylation of tyrosine hydroxylase in dopaminergic neurons of the rat retina.

We studied in vivo activity-dependent phosphorylation of tyrosine hydroxylase (TH) in dopaminergic (DA) neurons of the rat retina. TH phosphorylation (TH-P) was evaluated by immunocytochemistry, using antibodies specific for each of three regulated phosphorylation sites. TH synthesis rate was measured by dihydroxyphenylalanine (DOPA) accumulation in the presence of NSD-1015, an inhibitor of aromatic amino acid decarboxylase. TH-P was increased markedly by light or after intraocular injection of GABA(A) and glycine inhibitors. All three phosphospecific antibodies responded similarly to test drugs or light. A 30 min exposure to light increased DOPA accumulation by threefold over that seen after 30 min in darkness. Immunostaining to an anti-panNa channel antibody was found in all parts of the DA neuron. TTX blocked TH-P induced by light or GABA/glycine inhibitors but only in varicosities of the DA axon plexus, not in perikarya or dendrites. Veratridine increased TH-P in all parts of the DA neuron. The distribution of the monoamine vesicular transporter 2 was shown by immunocytochemistry to reside in varicosities of the DA plexus but not in dendrites, indicating that the varicosities are sites of dopamine release. Collectively, these data indicate that, in the retina, dopamine synthesis in varicosities is affected by the spiking activity of retinal neurons, possibly including that of the DA neurons themselves.

Cellular location and circadian rhythm of expression of the biological clock gene Period 1 in the mouse retina.

The cellular location and rhythmic expression of Period 1 (Per1) circadian clock gene were examined in the retina of a Per1::GFP transgenic mouse. Mouse Per1 (mPer1) RNA was localized to inner nuclear and ganglion cell layers but was absent in the outer nuclear (photoreceptor) layer. Green fluorescent protein (GFP), which was shown to colocalize with PER1 protein, was found in a few subtypes of amacrine neuron, including those containing tyrosine hydroxylase, calbindin, and calretinin, but not in cholinergic amacrine cells. A small subset of ganglion cells also contained GFP immunoreactivity (GFP-IR), but the melanopsin-containing subtype, which projects to the suprachiasmatic nuclei (SCN), lacked GFP-IR. Although the intensity of GFP-IR varied among the populations of amacrine cells at each time point that was examined, both diurnal and circadian rhythms were found for the fraction of neurons showing strong GFP-IR, with peak expression between Zeitgeber/circadian (ZT/CT) times 10 and 14. In SCNs that were examined in the same mice used for the retinal measures, the peak in GFP-IR also occurred at approximately ZT/CT 10. Our results are the first to demonstrate a circadian rhythm of a biological clock component in identified neurons of a mammalian retina.

Rat retinal dopaminergic neurons: differential maturation of somatodendritic and axonal compartments.

We examined developmental changes in dopaminergic (DA) neurons of rat pups between postnatal (P) days 3 and 21. DA cell bodies and dendrites grew progressively between P3-15. Voltage-sensitive sodium channels were present in axons at P11, but the ring-like DA axon terminals appeared only during the third postnatal week. The density of ring terminals increased markedly between P15 and P21. The vesicular monoamine transporter (VMAT2) was absent before P13 and became concentrated in DA ring terminals after P17. A steady increase in VMAT2-containing rings around AII amacrine cells occurred during the third postnatal week. The presynaptic membrane protein SNAP-25 colocalized with DA terminals, but several other presynaptic proteins tested, including synaptotagmin I, synapsin, bassoon, syntaxin, and synaptogyrin, appeared not to be associated with DA neurons. Our study shows that the somatodendritic compartment of DA neurons matures before the DA axon terminals do. Maturation of DA axons during the third postnatal week corresponds to the period of onset of visual function.

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

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.

Calcium and retinal function.

We survey the primary roles of calcium in retinal function, including photoreceptor transduction, transmitter release by different classes of retinal neuron, calcium-mediated regulation of gap-junctional conductance, activation of certain voltage-gated channels for K+ and Cl-, and modulation of postsynaptic potentials in retinal ganglion cells. We discuss three mechanisms for changing [Ca2+]i, which include flux through voltage-gated calcium channels, through ligand-gated channels, and by release from stores. The neuromodulatory pathways affecting each of these routes of entry are considered. The many neuromodulatory mechanisms in which calcium is a player are described and their effects upon retinal function discussed.

Association of the AMPA receptor-related postsynaptic density proteins GRIP and ABP with subsets of glutamate-sensitive neurons in the rat retina.

We used specific antibodies against two postsynaptic density proteins, GRIP (glutamate receptor interacting protein) and ABP (AMPA receptor-binding protein), to study their distribution in the rat retina. In the central nervous system, it has been shown that both proteins bind strongly to the AMPA glutamate receptor (GluR) 2/3 subunits, but not other GluRs, through a set of three PDZ domains. Western blots detected a single GRIP protein that was virtually identical in retina and brain, whereas retinal ABP corresponded to only one of three ABP peptides found in brain. The retinal distributions of GluR2/3, GRIP, and ABP immunoreactivity (IR) were similar but not identical. GluR2/3 immunoreactivity (IR) was abundant in both plexiform layers and in large perikarya. ABP IR was concentrated in large perikarya but was sparse in the plexiform layers, whereas GRIP IR was relatively more abundant in the plexiform layers than in perikarya. Immunolabel for these three antibodies consisted of puncta < or = 0.2 microm in diameter. The cellular localization of GRIP and ABP IR was examined by double labeling subclasses of retinal neuron with characteristic marker proteins, e.g., calbindin. GRIP, ABP, and GluR2/3 IR were detected in horizontal cells, dopaminergic and glycinergic AII amacrine cells and large ganglion cells. Immunolabel was absent in rod bipolar and weak or absent in cholinergic amacrine cells. By using the tyramide method of signal amplification, a colocalization of GluR2/3 was found with either GRIP or ABP in horizontal cell terminals, and perikarya of amacrine and ganglion cells. Our results show that ABP and GRIP colocalize with GluR2/3 in particular subsets of retinal neuron, as was previously established for certain neurons in the brain.

Regulation of Substantia Nigra Pars Reticulata GABAergic Neuron Activity by H2O2 via Flufenamic Acid-Sensitive Channels and KATP Channels.

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 (H2O2), 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 H2O2 affects the activity of these neurons and to explore the classes of ion channels underlying those effects. Elevation of H2O2 levels caused an increase in the spontaneous firing rate of SNr GABAergic neurons, whether by application of exogenous H2O2 or amplification of endogenous H2O2 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 H2O2 by catalase, a peroxidase enzyme, decreased spontaneous firing rate and firing precision of SNr neurons, demonstrating tonic control of firing rate by H2O2. Elevation of H2O2 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 H2O2 elevation in mouse SNr GABAergic neurons was hyperpolarization, indicating a species difference in H2O2-dependent regulation. Thus, H2O2 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.