RESUMO
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.
Assuntos
Autorreceptores , Dopamina , Camundongos , Animais , Ácido gama-Aminobutírico/farmacologia , Axônios/metabolismo , Corpo Estriado/metabolismo , Receptores de GABA-A/metabolismo , Camundongos Knockout , HomeostaseRESUMO
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.
RESUMO
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.
Assuntos
Dopamina , Substância Negra , Sinaptotagmina I , Sinaptotagminas , Animais , Dendritos , Dopamina/farmacologia , Neurônios Dopaminérgicos , Estimulação Elétrica , Feminino , Masculino , Camundongos , Sinaptotagmina I/genética , Sinaptotagminas/genéticaRESUMO
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.
Assuntos
Dendritos/metabolismo , Dopamina/metabolismo , Substância Negra/metabolismo , Animais , Anticorpos/metabolismo , Estimulação Elétrica , Potenciais Pós-Sinápticos Inibidores , Cinética , Masculino , Camundongos Endogâmicos C57BL , Receptores de Dopamina D2/metabolismo , Análise de Célula Única , Proteína 25 Associada a Sinaptossoma/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
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.
Assuntos
Neurônios Colinérgicos/metabolismo , Dopamina/metabolismo , Insulina/metabolismo , Interneurônios/metabolismo , Núcleo Accumbens/metabolismo , Obesidade/metabolismo , Obesidade/psicologia , Animais , Preferências Alimentares , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Receptor de Insulina/metabolismo , Recompensa , Transdução de SinaisRESUMO
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.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Peróxido de Hidrogênio/farmacologia , N-Metilaspartato/farmacologia , Substância Negra/metabolismo , Canais de Cátion TRPM/metabolismo , Potenciais de Ação/genética , Animais , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/efeitos dos fármacos , Cobaias , Masculino , Técnicas de Patch-Clamp , Substância Negra/citologia , Substância Negra/efeitos dos fármacos , Canais de Cátion TRPM/genéticaRESUMO
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.
Assuntos
Corpo Estriado/citologia , Corpo Estriado/metabolismo , Dopamina/metabolismo , Canais KATP/metabolismo , Neurônios/citologia , Terminações Pré-Sinápticas/fisiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Análise de Variância , Animais , Biofísica/métodos , Diazóxido/farmacologia , Agonistas de Dopamina/farmacologia , Estimulação Elétrica/métodos , Eletroquímica/métodos , Glibureto/farmacologia , Cobaias , Peróxido de Hidrogênio/farmacologia , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Mecamilamina/farmacologia , Antagonistas Nicotínicos/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Quimpirol/farmacologia , Receptores de Droga/metabolismo , Receptores de Sulfonilureias , Tiomalatos/farmacologia , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
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.
Assuntos
Apoptose/fisiologia , Cálcio/metabolismo , Células Ganglionares da Retina/fisiologia , Canais de Cátion TRPV/metabolismo , Animais , Apoptose/efeitos dos fármacos , Axônios/metabolismo , Capsaicina/farmacologia , Dendritos/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Imuno-Histoquímica , Leucina/análogos & derivados , Leucina/farmacologia , Mecanotransdução Celular/fisiologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Sulfonamidas/farmacologia , Canais de Cátion TRPV/genéticaRESUMO
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 â¼15 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.
Assuntos
Potenciais de Ação/fisiologia , Peptídeo Liberador de Gastrina/fisiologia , Estimulação Luminosa/métodos , Fotoperíodo , Retina/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Relógios Circadianos/fisiologia , Cobaias , Hipotálamo/fisiologia , Camundongos , Camundongos Transgênicos , Cifozoários , Fatores de TempoRESUMO
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.
RESUMO
Evidence showing expression of endogenous opioids in the mammalian retina is sparse. In the present study we examined a transgenic mouse line expressing an obligate dimerized form of Discosoma red fluorescent protein (DsRed) under the control of the pro-opiomelanocortin promoter and distal upstream regulatory elements to assess whether pro-opiomelanocortin peptide (POMC), and its opioid cleavage product, beta-endorphin, are expressed in the mouse retina. Using double label immunohistochemistry we found that DsRed fluorescence was restricted to a subset of GAD-67-positive cholinergic amacrine cells of both orthotopic and displaced subtypes. About 50% of cholinergic amacrine cells colocalized DsRed and a large fraction of DsRed-expressing amacrine cells was positive for beta-endorphin immunostaining, whereas beta-endorphin-immunoreactive neurons were absent in retinas of POMC null mice. Our findings contribute to a growing body of evidence demonstrating that opioid peptides are an integral component of vertebrate retinas, including those of mammals.
Assuntos
Retina/metabolismo , beta-Endorfina/biossíntese , Hormônio Adrenocorticotrópico/metabolismo , Células Amácrinas/metabolismo , Animais , Especificidade de Anticorpos , Calbindina 2 , Calbindinas , Colina O-Acetiltransferase/metabolismo , Glutamato Descarboxilase/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Proteínas Luminescentes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Sistema Nervoso Parassimpático/metabolismo , Hipófise/metabolismo , Pró-Opiomelanocortina/biossíntese , Pró-Opiomelanocortina/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , alfa-MSH/metabolismo , Proteína Vermelha FluorescenteRESUMO
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.
Assuntos
Axônios/metabolismo , Cálcio/metabolismo , Dendritos/metabolismo , Dopamina/metabolismo , Líquido Intracelular/metabolismo , Neurônios/citologia , Animais , Compostos de Boro/farmacologia , Cádmio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/fisiologia , Quelantes/farmacologia , Cromonas/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estimulação Elétrica , Técnicas Eletroquímicas/métodos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Cobaias , Técnicas In Vitro , Indóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Neurônios/ultraestrutura , Técnicas de Patch-Clamp/métodos , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Substância Negra/citologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Dopaminergic (DA) neurons of mouse and rat retinas are of the interplexiform subtype (DA-IPC), i.e., they send processes distally toward the outer retina, exhibiting numerous varicosities along their course. The primary question we addressed was whether distally located DA-IPC varicosities, identified by tyrosine hydroxylase (TH) immunoreactivity, had the characteristic presynaptic proteins associated with calcium-dependent vesicular release of neurotransmitter. We found that TH immunoreactive varicosities in the outer retina possessed vesicular monoamine transporter 2 and vesicular GABA transporter, but they lacked immunostaining for any of nine subtypes of voltage-dependent calcium channel. Immunoreactivity for other channels that may permit calcium influx such as certain ionotropic glutamate receptors and canonical transient receptor potential channels (TRPCs) was similarly absent, although DA-IPC varicosities did show ryanodine receptor immunoreactivity, indicating the presence of intracellular calcium stores. The synaptic vesicle proteins sv2a and sv2b and certain other proteins associated with the presynaptic membrane were absent from DA-IPC varicosities, but the vesicular SNARE protein, vamp2, was present in a fraction of those varicosities. We identified a presumed second class of IPC that is GABAergic but not dopaminergic. Outer retinal varicosities of this putative GABAergic IPC did colocalize synaptic vesicle protein 2a, suggesting they possessed a conventional vesicular release mechanism.
Assuntos
Dopamina/metabolismo , Neurônios , Retina , Animais , Cálcio/metabolismo , Forma Celular , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/ultraestrutura , Ratos , Ratos Sprague-Dawley , Retina/citologia , Retina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canais de Sódio/metabolismo , Sinapses/química , Sinapses/ultraestrutura , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
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.
Assuntos
Ritmo Circadiano/genética , Mutação/genética , Proteína G de Ligação ao Cálcio S100/genética , Núcleo Supraquiasmático/metabolismo , Adaptação Ocular/genética , Animais , Calbindina 1 , Calbindinas , Sinalização do Cálcio/genética , Proteínas de Ciclo Celular/genética , Ritmo Circadiano/efeitos da radiação , Adaptação à Escuridão/genética , Regulação da Expressão Gênica/genética , Marcação de Genes/métodos , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Atividade Motora/efeitos da radiação , Proteínas Nucleares/genética , Proteínas Circadianas Period , Estimulação Luminosa , Fotoperíodo , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismo , Núcleo Supraquiasmático/efeitos da radiaçãoRESUMO
Genetically modified mice lacking the beta2 laminin chain (beta2null), the gamma3 laminin chain (gamma3 null), or both beta2/gamma3 chains (compound null) were produced. The development of tyrosine hydroxylase (TH) immunoreactive neurons in these mouse lines was studied between birth and postnatal day (P) 20. Compared to wild type mice, no alterations were seen in gamma3 null mice. In beta2 null mice, however, the large, type I TH neurons appeared later in development, were at a lower density and had reduced TH immunoreactivity, although TH process number and size were not altered. In the compound null mouse, the same changes were observed together with reduced TH process outgrowth. Surprisingly, in the smaller, type II TH neurons, TH immunoreactivity was increased in laminin-deficient compared to wild type mice. Other retinal defects we observed were a patchy disruption of the inner limiting retinal basement membrane and a disoriented growth of Müller glial cells. Starburst and AII type amacrine cells were not apparently altered in laminin-deficient relative to wild type mice. We postulate that laminin-dependent developmental signals are conveyed to TH amacrine neurons through intermediate cell types, perhaps the Müller glial cell and/or the retinal ganglion cell.
Assuntos
Dopamina/fisiologia , Laminina/deficiência , Neurônios/fisiologia , Retina/citologia , Retina/crescimento & desenvolvimento , Animais , Membrana Basal/fisiologia , Western Blotting , Calbindina 2 , Corantes , Interpretação Estatística de Dados , Imunofluorescência , Imuno-Histoquímica , Laminina/fisiologia , Camundongos , Camundongos Knockout , Mutação/fisiologia , Neuroglia/fisiologia , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The goal of the present study was to elucidate the role of DARPP-32 (dopamine- and cyclic adenosine 3'-5'-monophosphate-regulated phosphoprotein, 32 kDa) in retinal function. We examined mouse and rat retinas for the presence of DARPP-32 by immunocytochemistry. In both rodent retinas DARPP-32 immunoreactivity was localized to horizontal and AII amacrine neurons and to the Mueller glial cells, using immuno-double labelling. Additional unidentified neurons in the amacrine cell layer also showed DARPP-32 immunoreactivity. Using mice entrained to a 12-12 h light-dark cycle, we found that exposure to light presented during the dark phase significantly enhanced phosphorylation of DARPP-32 at threonine (Thr) 34 and phosphorylation of the ionotropic glutamate receptor subunit GluR1 at serine (Ser) 845, as measured by immunoblots. However, light also increased Ser 845-GluR1 phosphorylation in DARPP-32-knockout mice. When a dopamine D1 receptor antagonist was injected into the eye prior to light exposure, phosphorylation of both Thr 34-DARPP-32 and Ser 845-GluR1 was significantly reduced. These data indicate that DARPP-32 participates in dopamine-mediated modifications of retinal function. We also tested for a possible circadian rhythm of Thr 34- and Thr 75-DARPP-32 and Ser 845-GluR1 expression. No significant circadian rhythm of either DARPP-32 or GluR1 phosphorylation was found.
Assuntos
Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Retina/citologia , Retina/metabolismo , Animais , Benzazepinas/farmacologia , Ritmo Circadiano/genética , Antagonistas de Dopamina/farmacologia , Fosfoproteína 32 Regulada por cAMP e Dopamina/deficiência , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Neurônios , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Roedores , Serina , TreoninaRESUMO
This essay looks at the historical significance of three APS classic papers that are freely available online: Naka K-I and Nye PW. Role of horizontal cells in organization of the catfish retinal receptive field. J. Neurophysiol 34:785-801, 1971. Marmarelis PZ and Naka K-I. Nonlinear analysis and synthesis of receptive-field responses in the catfish retina. II. One-input white-noise analysis. J. Neurophysiol 36: 619-633, 1973. Naka K-I, Marmarelis PZ, and Chan RY. Morphological and functional identifications of catfish retinal neurons. III. Functional identification. J. Neurophysiol 38: 92-131, 1975.
Assuntos
Retina/fisiologia , Transdução de Sinais/fisiologia , Células Amácrinas/fisiologia , Animais , Axônios/fisiologia , Humanos , Potenciais da Membrana/fisiologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Retina/anatomia & histologia , Retina/citologia , Células Ganglionares da Retina/fisiologiaRESUMO
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.
Assuntos
Canais de Cálcio/metabolismo , Dopamina/metabolismo , Neurônios/metabolismo , Retina/metabolismo , Animais , Western Blotting , Canais de Cálcio/classificação , Imuno-Histoquímica , Neurônios/citologia , Ratos , Retina/citologia , Distribuição TecidualRESUMO
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.