RESUMEN
Light-controllable tools provide powerful means to manipulate and interrogate brain function with relatively low invasiveness and high spatiotemporal precision. Although optogenetic approaches permit neuronal excitation or inhibition at the network level, other technologies, such as optopharmacology (also known as photopharmacology) have emerged that provide molecular-level control by endowing light sensitivity to endogenous biomolecules. In this Review, we discuss the challenges and opportunities of photocontrolling native neuronal signalling pathways, focusing on ion channels and neurotransmitter receptors. We describe existing strategies for rendering receptors and channels light sensitive and provide an overview of the neuroscientific insights gained from such approaches. At the crossroads of chemistry, protein engineering and neuroscience, optopharmacology offers great potential for understanding the molecular basis of brain function and behaviour, with promises for future therapeutics.
Asunto(s)
Canales Iónicos/metabolismo , Neuronas/metabolismo , Optogenética/tendencias , Procesos Fotoquímicos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/química , Moduladores del Transporte de Membrana/farmacología , Neuronas/química , Neuronas/efectos de los fármacos , Optogenética/métodos , Procesos Fotoquímicos/efectos de los fármacos , Receptores Acoplados a Proteínas G/químicaRESUMEN
We have developed a photochemical protecting group that enables wavelength selective uncaging using green versus violet light. Change of the exocyclic oxygen of the laser dye coumarin-102 to sulfur, gave thio-coumarin-102, a new chromophore with an absorption ratio at 503/402â nm of 37. Photolysis of thio-coumarin-102 caged γ-aminobutyric acid was found to be highly wavelength selective on neurons, with normalized electrical responses >100-fold higher in the green versus violet channel. When partnered with coumarin-102 caged glutamate, we could use whole cell violet and green irradiation to fire and block neuronal action potentials with complete orthogonality. Localized irradiation of different dendritic segments, each connected to a neuronal cell body, in concert with 3-dimenional Ca2+ imaging, revealed that such inputs could function independently. Chemical signaling in living cells always involves a complex balance of multiple pathways, use of (thio)-coumarin-102 caged compounds will enable arbitrarily timed flashes of green and violet light to interrogate two independent pathways simultaneously.
Asunto(s)
Luz Verde , Neuronas , Neuronas/metabolismo , Fotólisis , Cumarinas/química , Ácido Glutámico/metabolismoRESUMEN
Glutamate is the major excitatory neurotransmitter in the brain, and photochemical release of glutamate (or uncaging) is a chemical technique widely used by biologists to interrogate its physiology. A basic prerequisite of these optical probes is bio-inertness before photolysis. However, all caged glutamates are known to have strong antagonism toward receptors of γ-aminobutyric acid, the major inhibitory transmitter. We have developed a caged glutamate probe that is inert toward these receptors at concentrations that are effective for photolysis with violet light. Pharmacological tests in vitro revealed that attachment of a fifth-generation (G5) dendrimer (i.e., cloaking) to the widely used 4-methoxy-7-nitro-indolinyl(MNI)-Glu probe prevented such off-target effects while not changing the photochemical properties of MNI-Glu significantly. G5-MNI-Glu was used with optofluidic delivery to stimulate dopamine neurons of the ventral tegmental area of freely moving mice in a conditioned place-preference protocol so as to mediate Pavlovian conditioning.
Asunto(s)
Glutamatos/farmacología , Indoles/farmacología , Aprendizaje/fisiología , Microfluídica , Neuronas/fisiología , Neurotransmisores/farmacología , Animales , Aprendizaje/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Neuroquímica , Neuronas/efectos de los fármacos , Fotoquímica , Fotólisis , Receptores de GABA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Light passes through biological tissue, and so it is used for imaging biological processes in situ. Such observation is part of the very essence of science, but mechanistic understanding requires intervention. For more than 50â years a "second function" for light has emerged; namely, that of photochemical control. Caged compounds are biologically inert signaling molecules that are activated by light. These optical probes enable external instruction of biological processes by stimulation of an individual element in complex signaling cascades in its native environment. Cause and effect are linked directly in spatial, temporal, and frequency domains in a quantitative manner by their use. I provide a guide to the basic properties required to make effective caged compounds for the biological sciences.
Asunto(s)
Biología , Transducción de Señal , Fotoquímica/métodosRESUMEN
KEY POINTS: In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery. Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias. We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two-photon photolysis of a new ultra-effective caged calcium compound. The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation. Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types. ABSTRACT: In cardiac myocytes Ca2+ -induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca2+ release occurs via subcellular Ca2+ signalling events, Ca2+ sparks. Local recovery of Ca2+ release depends on both SR refilling and restoration of Ca2+ sensitivity of the RyRs. We used two-photon (2P) photolysis of the ultra-effective caged Ca2+ compound BIST-2EGTA and laser-scanning confocal Ca2+ imaging to probe refractoriness of local Ca2+ release in control conditions and in the presence of cAMP or low-dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST-2EGTA and rhod-2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca2+ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self-learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyRR420Q mutation were used for further validation and revealed a higher Ca2+ sensitivity of CICR. This new 2P approach provides composite information of Ca2+ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca2+ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.
Asunto(s)
Calcio , Retículo Sarcoplasmático , Animales , Calcio/metabolismo , Señalización del Calcio , Ratones , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Light has been instrumental in the study of living cells since its use helped in their discovery in the late 17th century. Further, combining chemical technology with light microscopy was an essential part of the Nobel Prize for Physiology in 1906. Such landmark scientific findings involved passive observation of cells. However, over the past 50 years, a "second use" of light has emerged in cell physiology, namely one of rational control. The seminal method for this emerged in late 1970s with the invention of caged compounds. This was the point when "caged compounds" were defined as optical probes in which the active functionality of a physiological signaling molecule was blocked with a photochemical protecting group. Caged compounds are analogous to prodrugs; in both, the activity of the effector is latent. However, caged compounds, unlike prodrugs, use a trigger that confers the power of full temporal and spatial manipulation of the effects of release of its latent biological cargo. Light is distinct because it is bio-orthogonal, passes through living tissue (even into the cell interior), and initiates rapid release of the "caged" biomolecule. Further, because light can be directed to broad areas or focused to small points, caged compounds offer an array of timing scenarios for physiologists to dissect virtually any type of cellular process.The collaborative interaction between chemists and physiologists plays a fundamental role in the development of caged compounds. First, the physiologists must define the problem to be addressed; then, with the help of chemists, decide if a caged compound would be useful. For this, structure-activity relationships of the potential optical probe and receptor must be determined. If rational targets seem feasible, synthetic organic chemistry is used to make the caged compound. The crucial property of prephotolysis bio-inertness relies on physiological or biochemical assays. Second, detailed optical characterization of the caged compound requires the skill of photochemists because the rate and efficiency of uncaging are also crucial properties for a useful caged compound. Often, these studies reveal limitations in the caged compound which has been developed; thus, chemists and physiologists use their abilities for iterative development of even more powerful optical probes. A similar dynamic will be familiar to scientists in the pharmaceutical industry. Therefore, caged compound development provides an excellent training framework for (young) chemists both intellectually and professionally. In this Account, I draw on my long experience in the field of making useful caged compounds for cell physiology by showing how each probe I have developed has been defined by an important physiological problem. Fundamental to this process has been my initial training by the pioneers in aromatic photochemistry, Derek Bryce-Smith and Andrew Gilbert. I discuss making a range of "caged calcium" probes, ones which went on to be the most widely used of all caged compounds. Then, I describe the development of caged neurotransmitters for two-photon uncaging microscopy. Finally, I survey recent work on making new photochemical protecting groups for wavelength orthogonal, two-color, and ultraefficient two-photon uncaging.
Asunto(s)
Quelantes/química , Neuronas/fisiología , Acetatos/química , Potenciales de Acción , Animales , Calcio/química , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Etilenodiaminas/química , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Neuronas/citología , Neuronas/efectos de los fármacos , Neurotransmisores/química , Neurotransmisores/metabolismo , Ácido gamma-Aminobutírico/química , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
We have developed a caged neurotransmitter using an extended π-electron chromophore for efficient multiphoton uncaging on living neurons. Widely studied in a chemical context, such chromophores are inherently bioincompatible due to their highly lipophilic character. Attachment of two polycarboxylate dendrimers, a method we call "cloaking", to a bisstyrylthiophene (or BIST) core effectively transformed the chromophore into a water-soluble optical probe, whilst maintaining the high two-photon absorption of over 500â GM. Importantly, the cloaked caged compound was biologically inert at the high concentrations required for multiphoton chemical physiology. Thus, in contrast to non-cloaked BIST compounds, the BIST-caged neurotransmitter can be safely delivered onto neurons in acutely isolated brain slices, thereby enabling high-resolution two-photon uncaging without any side effects. We expect that our cloaking method will enable the development of new classes of cell-compatible photolabile probes using a wide variety of extended π-electron caging chromophores.
Asunto(s)
Dendrímeros/química , Animales , Dendrímeros/metabolismo , Electrones , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Neuronas/metabolismo , Neurotransmisores/química , Neurotransmisores/metabolismo , Fotones , Tiofenos/química , Ácido gamma-Aminobutírico/química , Ácido gamma-Aminobutírico/metabolismoRESUMEN
We have developed photochromic probes for the nicotinic acetylcholine receptor that exploit the unique chemical properties of the tetrafluoroazobenzene (4FAB) scaffold. Ultraviolet light switching and rapid thermal relaxation of the metastable cis configuration are the main drawbacks associated with standard AB-based switches. We designed our photoprobes to take advantage of the excellent thermodynamic stability of the cis-4FAB configuration (thermal half-life > 12 days at 37 °C in physiological buffer) and cis-trans photostationary states above 84%. Furthermore, the well-separated n-π* absorption bands of trans- and cis-4FAB allow facile photoswitching with visible light in two optical channels. A convergent 11-step synthetic approach allowed the installation of a trimethylammonium (TA) head onto the 4FAB scaffold, by means of an alkyl spacer, to afford a free diffusible 4FABTA probe. TAs are known to agonize nicotinic receptors, so 4FABTA was tested on mouse brain slices and enabled reversible receptor activation with cycles of violet and green light. Due to the very long-lived metastable cis configuration, 4FAB in vivo use could be of great promise for long term biological studies. Further chemical functionalization of this 4FAB probe with a maleimide functionality allowed clean cross-linking with glutathione. However, attempts to conjugate with a cysteine on a genetically modified nicotinic acetylcholine receptor did not afford the expected light-responsive channel. Our data indicate that the 4FAB photoswitch can be derivatized bifunctionally for genetically-targeted photopharmacology whilst preserving all the favorable photophysical properties of the parent 4FAB scaffold, however, the tetrafluoro motif can significantly perturb pharmacophore-protein interactions. In contrast, we found that the freely diffusible 4FABTA probe could be pre-set with green light into an OFF state that was biologically inert, irradiation with violet light effectively "uncaged" agonist activity, but in a photoreversible manner. Since the neurotransmitter acetylcholine has fully saturated heteroatom valences, our photoswitchable 4FABTA probe could be useful for physiological studies of this neurotransmitter.
RESUMEN
KEY POINTS: A new caged nicotinic acetylcholine receptor (nAChR) agonist was developed, ABT594, which is photolysed by one- and two-photon excitation. The caged compound is photolysed with a quantum yield of 0.20. One-photon uncaging of ABT594 elicited large currents and Ca2+ transients at the soma and dendrites of medial habenula (MHb) neurons of mouse brain slices. Unexpectedly, uncaging of ABT594 also revealed highly Ca2+ -permeable nAChRs on axons of MHb neurons. ABSTRACT: Photochemical release of neurotransmitters has been instrumental in the study of their underlying receptors, with acetylcholine being the exception due to its inaccessibility to photochemical protection. We caged a nicotinic acetylcholine receptor (nAChR) agonist, ABT594, via its secondary amine functionality. Effective photolysis could be carried out using either one- or two-photon excitation. Brief flashes (0.5-3.0 ms) of 410 nm light evoked large currents and Ca2+ transients on cell bodies and dendrites of medial habenula (MHb) neurons. Unexpectedly, photorelease of ABT594 also revealed nAChR-mediated Ca2+ signals along the axons of MHb neurons.
Asunto(s)
Azetidinas/farmacología , Habénula/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Piridinas/farmacología , Receptores Nicotínicos/metabolismo , Animales , Habénula/metabolismo , Potenciales de la Membrana/fisiología , Ratones , Neuronas/fisiología , Nicotina/farmacologíaRESUMEN
Photoswitchable bioprobes enable bidirectional control of cell physiology with different wavelengths of light. Many current photoswitches use cytotoxic UV light and are limited by the need for constant illumination owing to thermal relaxation in the dark. Now a photoswitchable tetrafluoroazobenzene(4FAB)-based ion channel antagonist has been developed that can be efficiently isomerized in two separate optical channels with visible light. Importantly, the metastable cis configuration showed very high stability in the dark over the course of days at room temperature. In neurons, the 4FAB antagonist reversibly blocks voltage-gated ion channels with violet and green light. Furthermore, photoswitching could also be achieved with two-photon excitation yielding high spatial resolution. 4FAB probes have the potential to enable long-term biological studies where both ON and OFF states can be maintained in the absence of irradiation.
Asunto(s)
Canales Iónicos/metabolismo , Luz , Neuronas/metabolismo , Animales , Compuestos Azo/química , Femenino , Canales Iónicos/antagonistas & inhibidores , Isomerismo , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Fotones , TermodinámicaRESUMEN
Caged neurotransmitters, in combination with focused light beams, enable precise interrogation of neuronal function, even at the level of single synapses. However, most caged transmitters are, surprisingly, severe antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors. By conjugation of a large, neutral dendrimer to a caged GABA probe we introduce a "cloaking" technology that effectively reduces such antagonism to very low levels. Such cloaked caged compounds will enable the study of the signaling of the inhibitory neurotransmitter GABA in its natural state using two-photon uncaging microscopy for the first time.
Asunto(s)
Dendrímeros/química , Antagonistas de Receptores de GABA-A/química , Neuronas/metabolismo , Imagen Óptica/métodos , Ácido gamma-Aminobutírico/análogos & derivados , Animales , Células Cultivadas , Dendrímeros/metabolismo , Dendrímeros/farmacología , Femenino , Antagonistas de Receptores de GABA-A/metabolismo , Antagonistas de Receptores de GABA-A/farmacología , Masculino , Ratones , Microscopía Fluorescente/métodos , Neuronas/citología , Neuronas/efectos de los fármacos , Fotólisis , Fotones , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
We have designed a nitroaromatic photochemical protecting group that absorbs visible light in the violet-blue range. The chromophore is a dinitro derivative of bisstyrylthiophene (or BIST) that absorbs light very effectively (ε440 = 66,000 M(-1) cm(-1) and two-photon cross section of 350 GM at 775 nm). We developed a "caged calcium" molecule by conjugation of BIST to a Ca(2+) chelator that upon laser flash photolysis rapidly releases Ca(2+) in <0.2 ms. Using the patch-clamp method the optical probe, loaded with Ca(2+), was delivered into acutely isolated mouse cardiac myocytes, where either one- and two-photon uncaging of Ca(2+) induced highly local or cell-wide physiological Ca(2+) signaling events.
Asunto(s)
Compuestos de Calcio/química , Tiofenos/química , Compuestos de Calcio/síntesis química , Quelantes/química , Ácido Egtácico/química , Luz , Procesos Fotoquímicos , Tiofenos/síntesis químicaRESUMEN
Neurotransmitter uncaging, especially that of glutamate, has been used to study synaptic function for over 30â years. One limitation of caged glutamate probes is the blockade of γ-aminobutyric acid (GABA)-A receptor function. This problem comes to the fore when the probes are applied at the high concentrations required for effective two-photon photolysis. To mitigate such problems one could improve the photochemical properties of caging chromophores and/or remove receptor blockade. We show that addition of a dicarboxylate unit to the widely used 4-methoxy-7-nitroindolinyl-Glu (MNI-Glu) system reduced the off-target effects by about 50-70 %. When the same strategy was applied to an electron-rich 2-(p-Phenyl-o-nitrophenyl)propyl (PNPP) caging group, the pharmacological improvements were not as significant as in the MNI case. Finally, we used very extensive biological testing of the PNPP-caged Glu (more than 250 uncaging currents at single dendritic spines) to show that nitro-biphenyl caging chromophores have two-photon uncaging efficacies similar to that of MNI-Glu.
Asunto(s)
Compuestos de Bifenilo/química , Glutamatos/química , Indoles/química , Neurotransmisores/química , Aniones , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/metabolismo , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/metabolismo , Glutamatos/síntesis química , Glutamatos/metabolismo , Indoles/síntesis química , Indoles/metabolismo , Luz , Microscopía Fluorescente , Neurotransmisores/metabolismo , Fotólisis/efectos de los fármacos , Receptores de GABA-A/química , Receptores de GABA-A/metabolismoRESUMEN
Caged compounds are widely used by neurophysiologists to study many aspects of cellular signaling in glia and neurons. Biologically inert before irradiation, they can be loaded into cells via patch pipette or topically applied in situ to a defined concentration; photolysis releases the caged compound in a very rapid and spatially defined way. As caged compounds are exogenous optical probes, they include not only natural products such neurotransmitters, calcium and IP3 but non-natural products such as fluorophores, drugs and antibodies. In this Technical Spotlight we provide a short introduction to the uncaging technique by discussing the nitroaromatic caging chromophores most widely used in such experiments [e.g. α-carboxy-ortho-nitrobenyl (CNB), dimethoxynitrobenzyl (DMNB), 4-methoxy-7-nitroindolinyl (MNI) and 4-carboxymethoxy-7-nitroindolinyl (CDNI)]. We show that recently developed caging chromophores [rutheniumbipyridial (RuBi) and 7-diethylaminocoumarin (DEAC)450] that are photolyzed with blue light (~ 430-480 nm range) can be combined with traditional nitroaromatic caged compounds to enable two-color optical probing of neuronal function. For example, one-photon uncaging of either RuBi-GABA or DEAC450-GABA with a 473-nm laser is facile, and can block nonlinear currents (dendritic spikes or action potentials) evoked by two-photon uncaging of CDNI-Glu at 720 nm. We also show that two-photon uncaging of DEAC450-Glu and CDNI-GABA at 900 and 720 nm, respectively, can be used to fire and block action potentials. Our experiments illustrate that recently developed chromophores have taken uncaging out of the 'monochrome era', in which it has existed since 1978, so as to enable multichromic interrogation of neuronal function with single-synapse precision.
Asunto(s)
Indicadores y Reactivos , Neuronas/fisiología , Imagen Óptica/métodos , Animales , Procesos FotoquímicosRESUMEN
Calcium signalling in astrocytes couples changes in neural activity to alterations in cerebral blood flow by eliciting vasoconstriction or vasodilation of arterioles. However, the mechanism for how these opposite astrocyte influences provide appropriate changes in vessel tone within an environment that has dynamic metabolic requirements remains unclear. Here we show that the ability of astrocytes to induce vasodilations over vasoconstrictions relies on the metabolic state of the rat brain tissue. When oxygen availability is lowered and astrocyte calcium concentration is elevated, astrocyte glycolysis and lactate release are maximized. External lactate attenuates transporter-mediated uptake from the extracellular space of prostaglandin E(2), leading to accumulation and subsequent vasodilation. In conditions of low oxygen concentration extracellular adenosine also increases, which blocks astrocyte-mediated constriction, facilitating dilation. These data reveal the role of metabolic substrates in regulating brain blood flow and provide a mechanism for differential astrocyte control over cerebrovascular diameter during different states of brain activation.
Asunto(s)
Arteriolas/metabolismo , Astrocitos/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Vasoconstricción/fisiología , Vasodilatación/fisiología , Adenosina/metabolismo , Adenosina/farmacología , Animales , Arteriolas/efectos de los fármacos , Dinoprostona/metabolismo , Glucólisis , Ácido Láctico/metabolismo , Masculino , Transportadores de Anión Orgánico/metabolismo , Oxígeno/metabolismo , Presión , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , Ratas Sprague-Dawley , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Although considerable attention has been paid to the mechanisms underlying synaptic strengthening and new learning, little scrutiny has been given to those involved in the attenuation of synaptic strength that attends suppression of a previously learned association. Our studies revealed a novel, non-Hebbian, long-term, postsynaptic depression of glutamatergic SPN synapses induced by interneuronal nitric oxide (NO) signaling (NO-LTD) that was preferentially engaged at quiescent synapses. This form of plasticity was gated by local Ca 2+ influx through CaV1.3 Ca 2+ channels and stimulation of phosphodiesterase 1 (PDE1), which degraded cyclic guanosine monophosphate (cGMP) and blunted NO signaling. Consistent with this model, mice harboring a gain-of-function mutation in the gene coding for the pore-forming subunit of CaV1.3 channels had elevated depolarization-induced dendritic Ca 2+ entry and impaired NO-LTD. Extracellular uncaging of glutamate and intracellular uncaging of cGMP suggested that this Ca 2+ -dependent regulation of PDE1 activity allowed for local regulation of dendritic NO signaling. This inference was supported by simulation of SPN dendritic integration, which revealed that dendritic spikes engaged PDE1 in a branch-specific manner. In a mouse model of Parkinson's disease (PD), NO-LTD was absent not because of a postsynaptic deficit in NO signaling machinery, but rather due to impaired interneuronal NO release. Re-balancing intrastriatal neuromodulatory signaling in the PD model restored NO release and NO-LTD. Taken together, these studies provide novel insights into the mechanisms governing NO-LTD in SPN and its role in psychomotor disorders, like PD.
RESUMEN
Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Our studies reveal that SPNs manifest a heterosynaptic, nitric oxide (NO)-dependent form of long-term postsynaptic depression of glutamatergic SPN synapses (NO-LTD) that is preferentially engaged at quiescent synapses. Plasticity is gated by Ca2+ entry through CaV1.3 Ca2+ channels and phosphodiesterase 1 (PDE1) activation, which blunts intracellular cyclic guanosine monophosphate (cGMP) and NO signaling. Both experimental and simulation studies suggest that this Ca2+-dependent regulation of PDE1 activity allows for local regulation of dendritic cGMP signaling. In a mouse model of Parkinson disease (PD), NO-LTD is absent because of impaired interneuronal NO release; re-balancing intrastriatal neuromodulatory signaling restores NO release and NO-LTD. Taken together, these studies provide important insights into the mechanisms governing NO-LTD in SPNs and its role in psychomotor disorders such as PD.
RESUMEN
Caged compounds are molecules rendered functionally inert by derivatization with a photochemical protecting group. We describe the design logic behind the development of a diethylaminocoumarin (DEAC) caging chromophore, DEAC450, that absorbs blue light strongly (ε450 = 43,000 M(-1) cm(-1)) and violet light 11-fold more weakly. The absorption minimum is in the wavelength range (340-360 nm) that is traditionally used for photolysis of many widely used nitroaromatic caged compounds (e.g., 4-carboxymethoxy-5,7-dinitroindolinyl(CDNI)-GABA). We used this chromophore to synthesize DEAC450-caged cAMP and found this probe was very stable toward aqueous hydrolysis in the electronic ground state but was photolyzed with a quantum efficiency of 0.78. When DEAC450-cAMP and CDNI-GABA where co-applied to striatal cholinergic interneurons, the caged compounds were photolyzed in an chromatically orthogonal manner using blue and violet light so as to modulate the neuronal firing rate in a bidirectional way.
Asunto(s)
Aminocumarinas/química , Color , Cumarinas/química , Luz , AMP Cíclico/química , Hidrólisis , Estructura Molecular , Procesos Fotoquímicos , Ácido gamma-Aminobutírico/químicaRESUMEN
We have synthesized a 7-diethylaminocoumarin (DEAC) derivative that allows wavelength-selective two-photon uncaging at 900 nm versus 720 nm. This new caging chromophore, called DEAC450, has an extended π-electron moiety at the 3-position that shifts the absorption spectrum maximum of DEAC from 375 to 450 nm. Two-photon excitation at 900 nm was more than 60-fold greater than at 720 nm. Two-photon uncaging of DEAC450-Glu at 900 nm at spine heads on pyramidal neurons in acutely isolated brain slices generated postsynaptic responses that were similar to spontaneous postsynaptic excitatory miniature currents, whereas significantly higher energies at 720 nm evoked no currents. Since many nitroaromatic caged compounds are two-photon active at 720 nm, optically selective uncaging of DEAC450-caged biomolecules at 900 nm may allow facile two-color optical interrogation of bimodal signaling pathways in living tissue with high resolution for the first time.
Asunto(s)
Ácido Glutámico/química , Neuroimagen/métodos , Animales , Química Encefálica , Cumarinas/química , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Indicadores y Reactivos , Ratones , Técnicas de Placa-Clamp , Fotólisis , Células Piramidales/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Espectrofotometría UltravioletaRESUMEN
We developed a caged GABA (gamma-aminobutyric acid), which, when combined with an appropriate caged glutamate, allows bimodal control of neuronal membrane potential with subcellular resolution using optically independent two-photon uncaging of each neurotransmitter. We used two-color, two-photon uncaging to fire and block action potentials from rat hippocampal CA1 neurons in brain slices with 720-nm and 830-nm light, respectively. Our method should be generalizable to other chemical messenger pairs.