RESUMO
The subcellular localization of membrane Ca2+ channels is crucial for their functioning, but is difficult to study because channels may be distributed more closely than the resolution of conventional microscopy is able to detect. We describe a technique, stochastic channel Ca2+ nanoscale resolution (SCCaNR), employing Ca2+-sensitive fluorescent dyes to localize stochastic openings and closings of single Ca2+-permeable channels within <50 nm, and apply it to examine the clustered arrangement of inositol trisphosphate receptor (IP3R) channels underlying local Ca2+ puffs. Fluorescence signals (blips) arising from single functional IP3Rs are almost immotile (diffusion coefficient<0.003 microm2 s(-1)), as are puff sites over prolonged periods, suggesting that the architecture of this signaling system is stable and not subject to rapid, dynamic rearrangement. However, rapid stepwise changes in centroid position of fluorescence are evident within the durations of individual puffs. These apparent movements likely result from asynchronous gating of IP3Rs distributed within clusters that have an overall diameter of approximately 400 nm, indicating that the nanoscale architecture of IP3R clusters is important in shaping local Ca2+ signals. We anticipate that SCCaNR will complement superresolution techniques such as PALM and STORM for studies of Ca2+ channels as it obviates the need for photoswitchable labels and provides functional as well as spatial information.
Assuntos
Sinalização do Cálcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Difusão , Humanos , Ativação do Canal Iônico , Microscopia de Fluorescência , Transporte Proteico , Fatores de Tempo , Xenopus/metabolismoRESUMO
RATIONALE: Pavlovian feature negative discriminations have been widely used to understand inhibitory conditioning processes using exteroceptive stimuli. Comparatively little is known about inhibitory conditioning processes using a drug state as a negative feature. A negative feature signals that presentation of a conditional stimulus (CS) will not be paired with an unconditioned stimulus. OBJECTIVES: The present research examined whether nicotine served as a negative feature and started characterizing its properties. METHODS AND RESULTS: In acquisition, rats received intermixed saline and nicotine (0.4 mg/kg, base) sessions. On saline sessions, a 15-s light CS was paired with 4-s access to sucrose; the CS was presented on nicotine sessions, but sucrose was withheld. The discrimination was acquired with more goal tracking during the CS on saline sessions. Nicotine's inhibition of this conditioned response (CR) was sensitive to nicotine dose (ED50=0.225) and injection to testing interval (CR returned at 200 min). Mecamylamine pretreatment, but not hexamethonium, produced a loss of inhibitory control by nicotine suggesting a role for central nicotinic acetylcholine receptors. Amphetamine, bupropion, arecoline, and chlordiazepoxide, but not caffeine, substituted for the nicotine feature. However, in locomotor tests, amphetamine and bupropion increased activity; arecoline and chlordiazepoxide decreased activity. For this reason, the motor effects of these ligands could not be dissociated from substitution via shared stimulus properties. CONCLUSIONS: This feature negative task provides a preclinical model for studying how drug states inhibit responding, although identifying the process(es) mediating CR inhibition will require further research.
Assuntos
Condicionamento Clássico/efeitos dos fármacos , Condicionamento Operante/efeitos dos fármacos , Nicotina/farmacologia , Reforço Psicológico , Anfetamina/farmacologia , Animais , Arecolina/farmacologia , Aprendizagem por Associação/efeitos dos fármacos , Bupropiona/farmacologia , Cafeína/farmacologia , Clordiazepóxido/farmacologia , Aprendizagem por Discriminação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Hexametônio/farmacologia , Masculino , Mecamilamina/farmacologia , Atividade Motora/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/efeitos dos fármacos , Sacarose/administração & dosagemRESUMO
Calcium puffs are localized Ca(2+) signals mediated by Ca(2+) release from the endoplasmic reticulum (ER) through clusters of inositol trisphosphate receptor (IP3R) channels. The recruitment of IP3R channels during puffs depends on Ca(2+)-induced Ca(2+) release, a regenerative process that must be terminated to maintain control of cell signaling and prevent Ca(2+) cytotoxicity. Here, we studied puff termination using total internal reflection microscopy to resolve the gating of individual IP3R channels during puffs in intact SH-SY5Y neuroblastoma cells. We find that the kinetics of IP3R channel closing differ from that expected for independent, stochastic gating, in that multiple channels tend to remain open together longer than predicted from their individual open lifetimes and then close in near-synchrony. This behavior cannot readily be explained by previously proposed termination mechanisms, including Ca(2+)-inhibition of IP3Rs and local depletion of Ca(2+) in the ER lumen. Instead, we postulate that the gating of closely adjacent IP3Rs is coupled, possibly via allosteric interactions, suggesting an important mechanism to ensure robust puff termination in addition to Ca(2+)-inactivation.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Microscopia de Fluorescência/métodos , Neuroblastoma/metabolismo , Fluorescência , Humanos , Cinética , Microscopia de Fluorescência/instrumentação , Neuroblastoma/patologia , Células Tumorais CultivadasRESUMO
The Xenopus oocyte has been a favored model system in which to study spatio-temporal mechanisms of intracellular Ca2+ dynamics, in large part because this giant cell facilitates intracellular injections of Ca2+ indicator dyes, buffers and caged compounds. However, the recent commercial availability of membrane-permeant ester forms of caged IP3 (ci-IP3) and EGTA, now allows for facile loading of these compounds into smaller mammalian cells, permitting control of [IP3]i and cytosolic Ca2+ buffering. Here, we establish the human neuroblastoma SH-SY5Y cell line as an advantageous experimental system for imaging Ca2+ signaling, and characterize IP3-mediated Ca2+ signaling mechanisms in these cells. Flash photo-release of increasing amounts of i-IP3 evokes Ca2+ puffs that transition to waves, but intracellular loading of EGTA decouples release sites, allowing discrete puffs to be studied over a wide range of [IP3]. Puff activity persists for minutes following a single photo-release, pointing to a slow rate of i-IP3 turnover in these cells and suggesting that repetitive Ca2+ spikes with periods of 20-30s are not driven by oscillations in [IP3]. Puff amplitudes are independent of [IP3], whereas their frequencies increase with increasing photo-release. Puff sites in SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated close to the plasma membrane where they can be visualized by total internal reflection microscopy, offering the potential for unprecedented spatio-temporal resolution of Ca2+ puff kinetics.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Microscopia de Interferência/métodos , Neuroblastoma/metabolismo , Oócitos/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Membrana Celular/patologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ácido Egtázico/farmacologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neuroblastoma/patologia , Oócitos/citologia , Técnicas de Patch-Clamp , Transporte Proteico , XenopusRESUMO
Intracellular calcium ion (Ca(2+)) signaling crucially depends on the clustered organization of inositol trisphosphate receptors (IP(3)Rs) in the endoplasmic reticulum (ER) membrane. These ligand-gated ion channels liberate Ca(2+) to generate local signals known as Ca(2+) puffs. We tested the hypothesis that IP(3) itself elicits rapid clustering of IP(3)Rs by using flash photolysis of caged IP(3) in conjunction with high-resolution Ca(2+) imaging to monitor the activity and localization of individual IP(3)Rs within intact mammalian cells. Our results indicate that Ca(2+) puffs arising with latencies as short as 100 to 200 ms after photorelease of IP(3) already involve at least four IP(3)R channels, and that this number does not subsequently grow. Moreover, single active IP(3)Rs show limited mobility, and stochastic simulations suggest that aggregation of IP(3)Rs at puff sites by a diffusional trapping mechanism would require many seconds. We thus conclude that puff sites represent preestablished, stable clusters of IP(3)Rs and that functional IP(3)Rs are not readily diffusible within the ER membrane.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/química , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Linhagem Celular Tumoral , Análise por Conglomerados , Simulação por Computador , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Íons , Ligantes , Microscopia de Fluorescência/métodos , Modelos Biológicos , Transdução de Sinais , Processos EstocásticosRESUMO
In rats, the pharmacological (interoceptive) effects of nicotine can serve as a signal (conditional stimulus) in a Pavlovian (classical) conditioning task. In this task, nicotine administration (0.4 mg base/kg, subcutaneous) is typically paired with intermittent access to a liquid sucrose unconditional stimulus; sucrose is withheld on saline sessions. An increase in sucrose receptacle entries (goal tracking) on nicotine sessions indicates conditioning. Given our limited understanding of the functional relationships controlling conditioned responding to a nicotine conditional stimulus, the present research examined nicotine's sensitivity to several manipulations shown to affect the conditioned responding in more widely studied Pavlovian conditioning tasks that use exteroceptive conditional stimuli: number of nicotine conditional stimulus-sucrose unconditional stimulus pairings per session (0, 3, 9, 18, or 36) and the impact of sucrose deliveries in saline sessions. Differential goal tracking developed in fewer sessions and asymptotic conditioned responding magnitude was greater with more nicotine-sucrose pairings. Further, goal tracking was more resistant to extinction (unconditional stimulus withheld) with more conditional-unconditional stimulus pairings during the acquisition phase. The discrimination was not acquired when sucrose presentations (9 or 18) also occurred during saline sessions. Furthermore, expression of the discrimination was disrupted when sucrose was presented in saline sessions; this disruption resulted from goal tracking in saline sessions. These results are consistent with the notion that nicotine-evoked goal tracking results from interoceptive conditioning processes.