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Caffeine has significant effects on neurovascular activity and behavior throughout the sleep-wake cycle. We used a minimally invasive microchip/video system to continuously record effects of caffeine in the drinking water of freely behaving mice. Chronic caffeine shifted both rest and active phases by up to 2â h relative to the light-dark cycle in a dose-dependent fashion. There was a particular delay in the onset of rapid eye movement (REM) sleep as compared with non-REM sleep during the rest phase. Chronic caffeine increased wakefulness during the active phase and consolidated sleep during the rest phase; overall, there was no net change in the amount of time spent in the wake, sleep, or REM sleep states during caffeine administration. Despite these effects on wakefulness and sleep, chronic caffeine decreased mean cerebral blood volume (CBV) during the active phase and increased mean CBV during the rest phase. Chronic caffeine also increased heart rate variability in both the sleep and wake states. These results provide new insight into the effects of caffeine on the biology of the sleep-wake cycle. Increased blood flow during sleep caused by chronic caffeine may have implications for its potential neuroprotective effects through vascular mechanisms of brain waste clearance.
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Transcranial ultrasound stimulation (tUS) shows potential as a noninvasive brain stimulation (NIBS) technique, offering increased spatial precision compared to other NIBS techniques. However, its reported effects on primary motor cortex (M1) are limited. We aimed to better understand tUS effects in human M1 by performing tUS of the hand area of M1 (M1hand) during tonic muscle contraction of the index finger. Stimulation during muscle contraction was chosen because of the transcranial magnetic stimulation-induced phenomenon known as cortical silent period (cSP), in which transcranial magnetic stimulation (TMS) of M1hand involuntarily suppresses voluntary motor activity. Since cSP is widely considered an inhibitory phenomenon, it presents an ideal parallel for tUS, which has often been proposed to preferentially influence inhibitory interneurons. Recording electromyography (EMG) of the first dorsal interosseous (FDI) muscle, we investigated effects on muscle activity both during and after tUS. We found no change in FDI EMG activity concurrent with tUS stimulation. Using single-pulse TMS, we found no difference in M1 excitability before versus after sparsely repetitive tUS exposure. Using acoustic simulations in models made from structural MRI of the participants that matched the experimental setups, we estimated in-brain pressures and generated an estimate of cumulative tUS exposure experienced by M1hand for each subject. We were unable to find any correlation between cumulative M1hand exposure and M1 excitability change. We also present data that suggest a TMS-induced MEP always preceded a near-threshold cSP.
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Corteza Motora , Estimulación Eléctrica , Electromiografía , Potenciales Evocados Motores/fisiología , Humanos , Corteza Motora/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Estimulación Magnética Transcraneal/métodosRESUMEN
OBJECTIVE: The objective of this study is to characterize the effects of the sleep-wake cycle on neurovascular and behavioral characteristics of cortical spreading depression (CSD). BACKGROUND: There is an important bi-directional relationship between migraine and the sleep-wake cycle, but the basic mechanisms of this relationship are poorly understood. METHODS: We have developed a minimally invasive microchip system to continuously monitor cerebral blood volume (CBV) with optical intrinsic signal (OIS), head movement, and multiple other physiological and behavioral parameters in freely behaving mice over weeks. Behavior is also monitored with simultaneous video recording. This system can also be used to intermittently trigger and record CSD and accompanying neurovascular and behavioral responses. CSD was triggered optically in different stages of the sleep-wake cycle. RESULTS: The optical stimulus threshold to trigger CSD was significantly higher in the wake state compared to sleep (stimulation duration = 16.4 ± 9.7 s vs. 10.8 ± 5.8 s, p = 0.037, n = 6 mice). CSD evoked in the wake versus sleep state produced changes in CBV that were smaller (largest relative change -4.5 ± 5.0% ∆OIS vs. -14.3 ± 8.5% ∆OIS, p = 0.001) and shorter in duration (33:22 ± 6:37 vs. 49:42 ± 8:05 min:s, p = 0.012, n = 6 mice). The threshold for CSD and kinetics of associated CBV changes were correlated with the time since falling asleep or awakening (n = 47 CSDs in 6 mice). CSD triggered in the wake state was associated with a transient freezing behavior. CSD triggered during sleep typically caused a transient awakening and behavioral response. This was followed by a return to sleep until recovery from the sustained phase of decreased CBV that occurred 30-60 min later, at which time there was consistent awakening with behaviors similar to those that occurred at CSD onset. CSD triggered in the wake state evoked a transient decrease in heart rate (from 11.9 ± 0.8 to 9.6 ± 0.8 Hz, p = 0.002, n = 5), whereas when triggered in the sleep state there was a transient increase in HR (from 7.5 ± 0.4 Hz to 9.3 ± 1.1 Hz, p = 0.016, n = 5). CONCLUSIONS: The sleep-wake cycle has significant effects on CSD that may have relevance to the clinical presentations of migraine and brain injury.
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Depresión de Propagación Cortical , Trastornos Migrañosos , Animales , Depresión de Propagación Cortical/fisiología , Humanos , Ratones , SueñoRESUMEN
A minimally invasive, microchip-based approach enables continuous long-term recording of brain neurovascular activity, heart rate, and head movement in freely behaving rodents. This approach can also be used for transcranial optical triggering of cortical activity in mice expressing channelrhodopsin. The system uses optical intrinsic signal recording to measure cerebral blood volume, which under baseline conditions is correlated with spontaneous neuronal activity. The arterial pulse and breathing can be quantified as a component of the optical intrinsic signal. Multi-directional head movement is measured simultaneously with a movement sensor. A separate movement tracking element through a camera enables precise mapping of overall movement within an enclosure. Data is processed by a dedicated single board computer, and streamed from multiple enclosures to a central server, enabling simultaneous remote monitoring and triggering in many subjects. One application of this system described here is the characterization of changes in of cerebral blood volume, heart rate and behaviour that occur with the sleep-wake cycle over weeks. Another application is optical triggering and recording of cortical spreading depression (CSD), the slowly propagated wave of neurovascular activity that occurs in the setting of brain injury and migraine aura. The neurovascular features of CSD are remarkably different in the awake vs. anaesthetized state in the same mouse. With its capacity to continuously and synchronously record multiple types of physiological and behavioural data over extended time periods in combination with intermittent triggering of brain activity, this inexpensive method has the potential for widespread practical application in rodent research. KEY POINTS: Recording and triggering of brain activity in mice and rats has typically required breaching the skull, and experiments are often performed under anaesthesia A minimally invasive microchip system enables continuous recording and triggering of neurovascular activity, and analysis of heart rate and behaviour in freely behaving rodents over weeks This system can be used to characterize physiological and behavioural changes associated with the sleep-wake cycle over extended time periods This approach can also be used with mice expressing channelrhodopsin to trigger and record cortical spreading depression (CSD) in freely behaving subjects. The neurovascular responses to CSD are remarkably different under anaesthesia compared with the awake state. The method is inexpensive and straightforward to employ at a relatively large scale. It enables translational investigation of a wide range of physiological and pathological conditions in rodent models of neurological and systemic diseases.
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Depresión de Propagación Cortical , Roedores , Animales , Encéfalo , Channelrhodopsins , Ratones , RatasRESUMEN
Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Most genetically encodable voltage indicators (GEVI) have drawbacks including slow response, low fluorescence, or excessive bleaching. Here we present a dark quencher GEVI approach (dqGEVI) using a Förster resonance energy transfer pair between a fluorophore glycosylphosphatidylinositol-enhanced green fluorescent protein (GPI-eGFP) on the outer surface of the neuronal membrane and an azo-benzene dye quencher (D3) that rapidly moves in the membrane driven by voltage. In contrast to previous probes, the sensor has a single photon bleaching time constant of â¼40 min, has a high temporal resolution and fidelity for detecting action potential firing at 100 Hz, resolves membrane de- and hyperpolarizations of a few millivolts, and has negligible effects on passive membrane properties or synaptic events. The dqGEVI approach should be a valuable tool for optical recordings of subcellular or population membrane potential changes in nerve cells.
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Potenciales de Acción/fisiología , Potenciales de la Membrana/fisiología , Memoria/fisiología , Neuronas/fisiología , Potenciales de Acción/genética , Animales , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Células HEK293 , Humanos , Potenciales de la Membrana/genéticaRESUMEN
Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.
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Encéfalo/metabolismo , Estimulación Eléctrica/instrumentación , Microelectrodos , Potasio/metabolismo , Animales , Encéfalo/citología , Estimulación Eléctrica/métodos , Hipocampo/citología , Hipocampo/metabolismo , RatonesRESUMEN
Endothelial cells transduce mechanical forces from blood flow into intracellular signals required for vascular homeostasis. Here we show that endothelial NOTCH1 is responsive to shear stress, and is necessary for the maintenance of junctional integrity, cell elongation, and suppression of proliferation, phenotypes induced by laminar shear stress. NOTCH1 receptor localizes downstream of flow and canonical NOTCH signaling scales with the magnitude of fluid shear stress. Reduction of NOTCH1 destabilizes cellular junctions and triggers endothelial proliferation. NOTCH1 suppression results in changes in expression of genes involved in the regulation of intracellular calcium and proliferation, and preventing the increase of calcium signaling rescues the cell-cell junctional defects. Furthermore, loss of Notch1 in adult endothelium increases hypercholesterolemia-induced atherosclerosis in the descending aorta. We propose that NOTCH1 is atheroprotective and acts as a mechanosensor in adult arteries, where it integrates responses to laminar shear stress and regulates junctional integrity through modulation of calcium signaling.
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Arterias/metabolismo , Mecanotransducción Celular , Receptor Notch1/metabolismo , Animales , Arterias/química , Calcio/metabolismo , Células Endoteliales/química , Células Endoteliales/metabolismo , Endotelio Vascular/química , Endotelio Vascular/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Notch1/genética , Estrés MecánicoRESUMEN
The synapse specificity of long-term potentiation (LTP) ensures that no interference arises from inputs irrelevant to the memory to be encoded. In hippocampi of aged (21-28 months) mice, LTP was relayed to unstimulated synapses, blemishing its synapse specificity. Diminished levels of the K(+)/Cl(-) cotransporter KCC2 and a depolarizing GABAA receptor-mediated synaptic component following LTP were the most likely causes for the spreading of potentiation, unveiling mechanisms hindering information storage in the aged brain and identifying KCC2 as a potential target for intervention.
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Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Simportadores/metabolismo , Sinapsis/metabolismo , Animales , Humanos , Receptores de GABA-A/metabolismo , Cotransportadores de K ClRESUMEN
Fluorescent Ca(2+) indicators are widely used to measure the concentration of free Ca(2+) ([Ca(2+)]free) in biological processes. By calibrating the dye under the same experimental conditions as employed during its planned use, the actual [Ca(2+)] can be calculated from the measured fluorescence. When using non ratiometric dyes, such as the Oregon Green BAPTA (OGB) family of dyes or the Fluo dyes, the steady-state affinity (K(d)) and the ratio between the maximal and minimal fluorescence (F(ratio) = F(max)/F(min)) of the particular dye are needed for this conversion. Although these values are usually given by the manufacturer, we consistently find that the actual values can differ between various batches delivered by the companies that make the dyes. In this protocol, we provide the recipe for a series of solutions with a known and tightly buffered [Ca(2+)](free) and describe how to use these mixtures to determine the exact K(d) and F(ratio) of a fluorescent Ca(2+) dye.
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Calcio/análisis , Técnicas de Química Analítica/métodos , Colorantes Fluorescentes/normas , Calibración , SolucionesRESUMEN
Fluorescent Ca(2+) indicators are widely used to measure the concentration of free Ca(2+) ([Ca(2+)](free)) in biological processes. To determine the exact kinetics of changes in [Ca(2+)](free) and the processes underlying these changes (e.g., Ca(2+) binding to Ca(2+)-binding proteins), it is necessary to know the exact binding properties of the indicator used. Here, we describe how to determine the binding rate constants (k(on) and k(off)) of Ca(2+) indicators.
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Calcio/análisis , Técnicas de Química Analítica , Colorantes Fluorescentes/química , Calcio/químicaRESUMEN
Conventional techniques used to measure Ca(2+) binding are too slow to determine accurately the fast binding kinetics of most molecules such as Ca(2+)-binding proteins (CBPs). We have developed an ultra-fast in vitro technique for measuring the Ca(2+)-binding properties of CBPs following flash photolysis of caged Ca(2+). Although the details of the setup, the mathematics, and the analysis involved in this technique have been published elsewhere, many of the practical details regarding the actual measurements have, until now, only been described minimally. Here, we present a protocol to gather the data necessary to determine the kinetic properties of a caged-Ca(2+) compound and a CBP.
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Acetatos/metabolismo , Proteínas de Unión al Calcio/metabolismo , Técnicas de Química Analítica/métodos , Etilenodiaminas/metabolismo , Acetatos/química , Proteínas de Unión al Calcio/química , Etilenodiaminas/química , Proteínas/química , Proteínas/metabolismoRESUMEN
Ca(2+)-binding proteins (CBPs) are instrumental in the control of Ca(2+) signaling. For the transduction of a change in intracellular Ca(2+) concentration into a cellular biochemical or biophysical action, it is necessary for Ca(2+) to bind specific Ca(2+)-binding proteins (CBPs) that relay the Ca(2+) signal. The competition for Ca(2+) between the various CBPs plays an essential and direct role in this transduction and in the resulting biochemical message. Therefore, a thorough understanding of Ca(2+) signaling necessitates appreciating the kinetic properties of all the relevant CBPs. Unfortunately, most conventional techniques used to measure Ca(2+)-binding kinetics are too slow to determine accurately the fast binding kinetics of most CBPs. To address this problem, we have developed an ultrafast in vitro technique for measuring the Ca(2+)-binding properties of CBPs following flash photolysis of caged Ca(2+). We introduce here the protocols that are necessary for the data collection associated with this technique.
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Bioquímica/métodos , Calcio/química , Proteínas/química , Calcio/metabolismo , Señalización del Calcio , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Cinética , Modelos Biológicos , Unión Proteica , Proteínas/metabolismo , Factores de TiempoRESUMEN
Huntington's disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, in which mutant huntingtin is expressed in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models was not associated with classical astrogliosis, but was associated with decreased Kir4.1 K(+) channel functional expression, leading to elevated in vivo striatal extracellular K(+), which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K(+), ameliorated aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte-mediated K(+) homeostasis, revealing astrocytes and Kir4.1 channels as therapeutic targets.
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Astrocitos/metabolismo , Enfermedad de Huntington/patología , Neuronas/patología , Canales de Potasio de Rectificación Interna/deficiencia , Factores de Edad , Animales , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Suspensión Trasera/fisiología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/mortalidad , Enfermedad de Huntington/fisiopatología , Técnicas In Vitro , Locomoción/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Proteínas Nucleares/genética , Canales de Potasio de Rectificación Interna/genética , Análisis de Supervivencia , Repeticiones de Trinucleótidos/genéticaRESUMEN
Bicarbonate (HCO3(-)) is an abundant anion that regulates extracellular and intracellular pH. Here, we use patch-clamp techniques to assess regulation of hippocampal CA3 pyramidal cell excitability by HCO3(-) in acute brain slices from C57BL/6 mice. We found that increasing HCO3(-) levels enhances action potential (AP) generation in both the soma and axon initial segment (AIS) by reducing Kv7/KCNQ channel activity, independent of pH (i.e., at a constant pH of 7.3). Conversely, decreasing intracellular HCO3(-) leads to attenuation of AP firing. We show that HCO3(-) interferes with Kv7/KCNQ channel activation by phosphatidylinositol-4,5-biphosphate. Consequently, we propose that, even in the presence of a local depolarizing Cl(-) gradient, HCO3(-) efflux through GABAA receptors may ensure the inhibitory effect of axoaxonic cells at the AIS due to activation of Kv7/KCNQ channels.
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Potenciales de Acción/fisiología , Bicarbonatos/metabolismo , Región CA3 Hipocampal/fisiología , Canales de Potasio KCNQ/metabolismo , Células Piramidales/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Bicarbonatos/farmacología , Región CA3 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/metabolismo , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismoRESUMEN
BACKGROUND: Calcium-binding proteins (CBPs) are instrumental in the control of Ca2+ signaling. They are the fastest players within the Ca2+ toolkit responding within microseconds to [Ca2+] changes. The CBPs compete for Ca2+ which plays a direct role in modulating Ca2+ transients and the resulting biochemical message. The kinetic properties of the CBPs have to be known to have a good understanding of Ca2+ signaling. SCOPE OF REVIEW: Most techniques used to measure binding kinetics are too slow to accurately determine the fast kinetics of most CBP. Furthermore, many CBPs bind Ca2+ in a cooperative way, which should be incorporated in the kinetic modeling. Here we will review a new ultra-fast in vitro technique for measuring Ca2+ binding properties of CBPs following flash photolysis of caged Ca2+. Compartmental modeling is used to resolve the kinetics of fast cooperative Ca2+ binding to CBPs. MAJOR CONCLUSIONS: Currently this technique has only been used to quantify the kinetics of three CBPs (calbindin, calretinin and calmodulin), but has already provided remarkable insights into the specific role that these kinetics in Ca2+ signaling. GENERAL SIGNIFICANCE: The potential to gain novel insights into Ca2+ signaling by quantifying kinetics of other CBPs using this technique is very promising. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.
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Señalización del Calcio , Proteínas de Unión al Calcio/metabolismo , Acetatos/química , Algoritmos , Animales , Proteínas de Unión al Calcio/química , Quelantes/química , Etilenodiaminas/química , Humanos , Cinética , Modelos Biológicos , Fotólisis , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
Many forms of signal transduction occur when Ca(2+) enters the cytoplasm of a cell. It has been generally thought that there is a fast buffer that rapidly reduces the free Ca(2+) level and that it is this buffered level of Ca(2+) that triggers downstream biochemical processes, notably the activation of calmodulin (CaM) and the resulting activation of CaM-dependent enzymes. Given the importance of these transduction processes, it is crucial to understand exactly how Ca(2+) activates CaM. We have determined the rate at which Ca(2+) binds to CaM and found that Ca(2+) binds more rapidly to CaM than to other Ca(2+)-binding proteins. This property of CaM and its high concentration support a new view of signal transduction: CaM directly intercepts incoming Ca(2+) and sets the free Ca(2+) level (that is, it strongly contributes to fast Ca(2+) buffering) rather than responding to the lower Ca(2+) level set by other buffers. This property is crucial for making CaM an efficient transducer. Our results also suggest that other Ca(2+) binding proteins have a previously undescribed role in regulating the lifetime of Ca(2+) bound to CaM and thereby setting the gain of signal transduction.
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Señalización del Calcio/fisiología , Calcio/metabolismo , Calmodulina/metabolismo , Animales , Calbindinas , Espinas Dendríticas/metabolismo , Hipocampo/citología , Modelos Biológicos , Neuronas/citología , Neuronas/metabolismo , Unión Proteica , Proteína G de Unión al Calcio S100/metabolismoRESUMEN
Cooperativity is one of the most important properties of molecular interactions in biological systems. It is the ability to influence ligand binding at one site of a macromolecule by previous ligand binding at another site of the same molecule. As a consequence, the affinity of the macromolecule for the ligand is either decreased (negative cooperativity) or increased (positive cooperativity). Over the last 100 years, O2 binding to hemoglobin has served as the paradigm for cooperative ligand binding and allosteric modulation, and four practical models were developed to quantitatively describe the mechanism: the Hill, the Adair-Klotz, the Monod-Wyman-Changeux, and the Koshland-Némethy-Filmer models. The predictions of these models apply under static conditions when the binding reactions are at equilibrium. However, in a physiological setting, e.g., inside a cell, the timing and dynamics of the binding events are essential. Hence, it is necessary to determine the dynamic properties of cooperative binding to fully understand the physiological implications of cooperativity. To date, the Monod-Wyman-Changeux model was applied to determine the kinetics of cooperative binding to biologically active molecules. In this model, cooperativity is established by postulating two allosteric isoforms with different binding properties. However, these studies were limited to special cases, where transition rates between allosteric isoforms are much slower than the binding rates or where binding and unbinding rates could be measured independently. For all other cases, the complex mathematical description precludes straightforward interpretations. Here, we report on calculating for the first time the fast dynamics of a cooperative binding process, the binding of Ca2+ to calretinin. Calretinin is a Ca2+-binding protein with four cooperative binding sites and one independent binding site. The Ca2+ binding to calretinin was assessed by measuring the decay of free Ca2+ using a fast fluorescent Ca2+ indicator following rapid (<50-mus rise time) Ca2+ concentration jumps induced by uncaging Ca2+ from DM-nitrophen. To unravel the kinetics of cooperative binding, we devised several approaches based on known cooperative binding models, resulting in a novel and relatively simple model. This model revealed unexpected and highly specific nonlinear properties of cellular Ca2+ regulation by calretinin. The association rate of Ca2+ with calretinin speeds up as the free Ca2+ concentration increases from cytoplasmic resting conditions ( approximately 100 nM) to approximately 1 muM. As a consequence, the Ca2+ buffering speed of calretinin highly depends on the prevailing Ca2+ concentration prior to a perturbation. In addition to providing a novel mode of action of cellular Ca2+ buffering, our model extends the analysis of cooperativity beyond the static steady-state condition, providing a powerful tool for the investigation of the dynamics and functional significance of cooperative binding in general.
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Señalización del Calcio/fisiología , Calcio/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Sitios de Unión , Tampones (Química) , Calbindina 2 , Escherichia coli/metabolismo , Humanos , Cinética , Ligandos , Modelos Biológicos , Proteínas Recombinantes/metabolismoRESUMEN
Caged-Ca(2+) compounds such as nitrophenyl-EGTA (NP-EGTA) and DM-nitrophen (DMn) are extremely useful in biological research, but their use in live cells is hampered by cytoplasmic [Mg(2+)]. We determined the properties of Ca(2+) release from NP-EGTA and DMn by using Oregon green BAPTA-5N to measure changes in [Ca(2+)] after ultraviolet flash photolysis in vitro, with or without Mg(2+) present. A large fraction (65%) of NP-EGTA, which has a negligible Mg(2+) affinity, uncages with a time constant of 10.3 ms, resulting in relatively slow increases in [Ca(2+)]. Uncaging of DMn is considerably faster, but DMn has a significant affinity for Mg(2+) to complicate the uncaging process. With experimentally determined values for the Ca(2+) and Mg(2+) binding/unbinding rates of DMn and NP-EGTA, we built a mathematical model to assess the utility of NP-EGTA and DMn in rapid Ca(2+)-uncaging experiments in the presence of Mg(2+). We discuss the advantages and disadvantages of using each compound under different conditions. To determine the kinetics of Ca(2+) binding to biologically relevant Ca(2+) buffers, such as Ca(2+)-binding proteins, the use of DMn is preferable even in the presence of Mg(2+).
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Acetatos/metabolismo , Calcio/metabolismo , Etilenodiaminas/metabolismo , Magnesio/metabolismo , Fenómenos Biofísicos , Biofisica , Señalización del Calcio , Ácido Egtácico/análogos & derivados , Ácido Egtácico/metabolismo , Técnicas In Vitro , Cinética , Modelos Biológicos , FotólisisRESUMEN
Synaptic mechanisms of plasticity are calcium-dependent processes that are affected by dysfunction of mitochondrial calcium buffering. Recently, we observed that mice deficient in mitochondrial voltage-dependent anion channels, the outer component of the mitochondrial permeability transition pore, have impairments in learning and hippocampal synaptic plasticity, suggesting that the mitochondrial permeability transition pore is involved in hippocampal synaptic plasticity. In this study, we examined the effect on synaptic transmission and plasticity of blocking the permeability transition pore with low doses of cyclosporin A and found a deficit in synaptic plasticity and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals revealed a transient increase in the resting calcium concentration immediately upon incubation with cyclosporin A that correlated with the changes in synaptic transmission and plasticity. The effect of cyclosporin A on presynaptic calcium was abolished when mitochondria were depolarized prior to cyclosporin A exposure, and the effects of cyclosporin A and mitochondrial depolarization on presynaptic resting calcium were similar, suggesting a mitochondrial locus of action of cyclosporin A. To further characterize the calcium dynamics of the mitochondrial permeability transition pore, we used an in vitro assay of calcium handling by isolated brain mitochondria. Cyclosporin A-exposed mitochondria buffered calcium more rapidly and subsequently triggered a more rapid mitochondrial depolarization. Similarly, mitochondria lacking the voltage-dependent anion channel 1 isoform depolarized more readily than littermate controls. The data suggest a role for the mitochondrial permeability transition pore and voltage-dependent anion channels in mitochondrial synaptic calcium buffering and in hippocampal synaptic plasticity.
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Hipocampo/metabolismo , Mitocondrias/metabolismo , Animales , Aniones , Calcio/metabolismo , Ciclosporina/farmacología , Hipocampo/patología , Hipocampo/ultraestructura , Ratones , Microscopía Electrónica , Isoformas de Proteínas , Sinapsinas/metabolismo , Factores de TiempoRESUMEN
Activation of group I metabotropic glutamate receptors (mGluRs) can induce acute depression of excitatory synaptic transmission and long-term depression (LTD) in area CA1 of the rat hippocampus. The underlying mechanisms for both forms of depression are unknown. By measuring presynaptic calcium transients, we show that a reduction in the stimulation-induced presynaptic calcium rise that triggers vesicular release causes the acute depression of transmission by group I mGluRs. In contrast, the mechanism underlying mGluR-induced LTD does not involve a persistent change in stimulation-induced calcium influx. However, analysis of paired-pulse facilitation experiments suggests a presynaptic location for expression of this form of LTD. Furthermore, we show that mGluR-induced LTD can be completely blocked by a specific mGluR5 antagonist, whereas mGluR1 antagonists strongly attenuate the acute depression of transmission. These results support the hypothesis that the acute depression of transmission caused by activation of group I mGluRs involves regulation of stimulation-induced presynaptic calcium transients, whereas mGluR-induced LTD involves a distinct presynaptic modulation downstream of calcium influx.