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BACKGROUND: The light organs of the splitfin flashlight fish Anomalops katoptron are necessary for schooling behavior, to determine nearest neighbor distance, and to feed on zooplankton under dim light conditions. Each behavior is coupled to context-dependent blink frequencies and can be regulated via mechanical occlusion of light organs. During shoaling in the laboratory individuals show moderate blink frequencies around 100 blinks per minute. In this study, we correlated bioluminescent blinks with the spatio-temporal dynamics of swimming profiles in three dimensions, using a stereoscopic, infrared camera system. RESULTS: Groups of flashlight fish showed intermediate levels of polarization and distances to the group centroid. Individuals showed higher swimming speeds and curved swimming profiles during light organ occlusion. The largest changes in swimming direction occurred when darkening the light organs. Before A. katoptron exposed light organs again, they adapted a nearly straight movement direction. CONCLUSIONS: We conclude that a change in movement direction coupled to light organ occlusion in A. katoptron is an important behavioral trait in shoaling of flashlight fish.
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Natación , Animales , Natación/fisiología , Luminiscencia , Peces/fisiología , Conducta Animal/fisiologíaRESUMEN
Fluorescence is a widespread phenomenon found in animals, bacteria, fungi, and plants. In marine environments fluorescence has been proposed to play a role in physiological and behavioral responses. Many fluorescent proteins and other molecules have been described in jellyfish, corals, and fish. Here we describe fluorescence in marine species, which we observed and photographed during night dives in the Banda Sea, Indonesia, and in the Red Sea, Egypt. Among various phyla we found fluorescence in sponges, molluscs, tunicates, and fish. Our study extends the knowledge on how many different organisms fluoresce in marine environments. We describe the occurrence of fluorescence in 27 species, in which fluorescence has not been described yet in peer-reviewed literature. It especially extends the knowledge beyond Scleractinia, the so far best described taxon regarding diversity in fluorescent proteins.
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Organismos Acuáticos , Océano Índico , Animales , Fluorescencia , Indonesia , Egipto , Peces , Océanos y MaresRESUMEN
Fluorescence in marine animals has mainly been studied in Cnidaria but is found in many different phyla such as Annelida, Crustacea, Mollusca, and Chordata. While many fluorescent proteins and molecules have been identified, very little information is available about the biological functions of fluorescence. In this review, we focus on describing the occurrence of fluorescence in marine animals and the behavioural and physiological functions of fluorescent molecules based on experimental approaches. These biological functions of fluorescence range from prey and symbiont attraction, photoprotection, photoenhancement, stress mitigation, mimicry, and aposematism to inter- and intraspecific communication. We provide a comprehensive list of marine taxa that utilise fluorescence, including demonstrated effects on behavioural or physiological responses. We describe the numerous known functions of fluorescence in anthozoans and their underlying molecular mechanisms. We also highlight that other marine taxa should be studied regarding the functions of fluorescence. We suggest that an increase in research effort in this field could contribute to understanding the capacity of marine animals to respond to negative effects of climate change, such as rising sea temperatures and increasing intensities of solar irradiation.
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Organismos Acuáticos , Animales , Organismos Acuáticos/fisiología , Fluorescencia , Invertebrados/fisiología , Conducta Animal/fisiologíaRESUMEN
Neuronal plasticity underlying cerebellar learning behavior is strongly associated with type 1 metabotropic glutamate receptor (mGluR1) signaling. Activation of mGluR1 leads to activation of the Gq/11 pathway, which is involved in inducing synaptic plasticity at the parallel fiber-Purkinje cell synapse (PF-PC) in form of long-term depression (LTD). To optogenetically modulate mGluR1 signaling we fused mouse melanopsin (OPN4) that activates the Gq/11 pathway to the C-termini of mGluR1 splice variants (OPN4-mGluR1a and OPN4-mGluR1b). Activation of both OPN4-mGluR1 variants showed robust Ca2+ increase in HEK cells and PCs of cerebellar slices. We provide the prove-of-concept approach to modulate synaptic plasticity via optogenetic activation of OPN4-mGluR1a inducing LTD at the PF-PC synapse in vitro. Moreover, we demonstrate that light activation of mGluR1a signaling pathway by OPN4-mGluR1a in PCs leads to an increase in intrinsic activity of PCs in vivo and improved cerebellum driven learning behavior.
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Serotonin 2C receptors (5-HT2CRs) are widely distributed throughout the brain and are strongly implicated in the pathophysiology of anxiety disorders such as post-traumatic stress disorder (PTSD). Although in recent years, a considerable amount of evidence supports 5-HT2CRs facilitating effect on anxiety behavior, the involvement in learned fear responses and fear extinction is rather unexplored. Here, we used a 5-HT2CR knock-out mouse line (2CKO) to gain new insights into the involvement of 5-HT2CRs in the neuronal fear circuitry. Using a cued fear conditioning paradigm, our results revealed that global loss of 5-HT2CRs exclusively accelerates fear extinction, without affecting fear acquisition and fear expression. To investigate the neuronal substrates underlying the extinction enhancing effect, we mapped the immediate-early gene product cFos, a marker for neuronal activity, in the dorsal raphe nucleus (DRN), amygdala and bed nucleus of the stria terminalis (BNST). Surprisingly, besides extinction-associated changes, our results revealed alterations in neuronal activity even under basal home cage conditions in specific subregions of the DRN and the BNST in 2CKO mice. Neuronal activity in the dorsal BNST was shifted in an extinction-supporting direction due to 5-HT2CR knock-out. Finally, the assessment of DRN-BNST connectivity using antero- and retrograde tracing techniques uncovered a discrete serotonergic pathway projecting from the most caudal subregion of the DRN (DRC) to the anterodorsal portion of the BNST (BNSTad). This serotonergic DRC-BNSTad pathway showed increased neuronal activity in 2CKO mice. Thus, our results provide new insights for the fear extinction network by revealing a specific serotonergic DRC-BNSTad pathway underlying a 5-HT2CR-sensitive mechanism with high significance in the treatment of PTSD.
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Miedo , Núcleos Septales , Ratones , Animales , Miedo/fisiología , Núcleos Septales/metabolismo , Núcleo Dorsal del Rafe/metabolismo , Extinción Psicológica , Receptor de Serotonina 5-HT2C/genética , Receptor de Serotonina 5-HT2C/metabolismoRESUMEN
Electrical activity is considered a key driver for the neurochemical and morphological maturation of neurons and the formation of neuronal networks. Designer receptors exclusively activated by designer drugs (DREADDs) are tools for controlling neuronal activity at the single cell level by triggering specific G protein signaling. Our objective was to investigate if prolonged silencing of differentiating cortical neurons can influence dendritic and axonal maturation. The DREADD hM4Di couples to Gi/o signaling and evokes hyperpolarization via GIRK channels. HM4Di was biolistically transfected into neurons in organotypic slice cultures of rat visual cortex, and activated by clozapine-N-oxide (CNO) dissolved in H2O; controls expressed hM4Di, but were mock-stimulated with H2O. Neurons were analyzed after treatment for two postnatal time periods, DIV 5-10 and 10-20. We found that CNO treatment delays the maturation of apical dendrites of L2/3 pyramidal cells. Further, the number of collaterals arising from the main axon was significantly lower, as was the number of bouton terminaux along pyramidal cell and basket cell axons. The dendritic maturation of L5/6 pyramidal cells and of multipolar interneurons (basket cells and bitufted cells) was not altered by CNO treatment. Returning CNO-treated cultures to CNO-free medium for 7 days was sufficient to recover dendritic and axonal complexity. Our findings add to the view that activity is a key driver in particular of postnatal L2/3 pyramidal cell maturation. Our results further suggest that inhibitory G protein signaling may represent a factor balancing the strong driving force of neurotrophic factors, electrical activity and calcium signaling.
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Ionophores are small molecules or peptides that transport metal ions across biological membranes. Their transport capabilities are typically characterized in vitro using vesicles and single ion species. It is difficult to infer from these data which effects ionophores have on living cells in a complex environment (e.g., culture medium), since net ion movement is influenced by many factors including ion composition of the medium, concentration gradients, pH gradient, and protein-mediated transport processes across the membrane. To gain insights into the antibacterial mechanism of action of the semisynthetic polyether ionophore 4-Br-A23187, known to efficiently transport zinc and manganese in vitro, we investigated its effects on the gram-positive model organism Bacillus subtilis. In addition to monitoring cellular ion concentrations, the physiological impact of treatment was assessed on the proteome level. 4-Br-A23187 treatment resulted in an increase in intracellular copper levels, the extent of which depended on the copper concentration of the medium. Effects of copper accumulation mirrored by the proteomic response included oxidative stress, disturbance of proteostasis, metal and sulfur homeostasis. The antibiotic effect of 4-Br-A23187 is further aggravated by a decrease in intracellular manganese and magnesium. A liposome model confirmed that 4-Br-A23187 acts as copper ionophore in vitro.
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Bacillus subtilis , Liposomas Unilamelares , Antibacterianos/farmacología , Calcimicina/análogos & derivados , Calcimicina/farmacología , Calcio , Cobre/farmacología , Ionóforos/farmacología , Manganeso/farmacología , ProteómicaRESUMEN
What is the effect of activating a single modulatory neuronal receptor type on entire brain network dynamics? Can such effect be isolated at all? These are important questions because characterizing elementary neuronal processes that influence network activity across the given anatomical backbone is fundamental to guide theories of brain function. Here, we introduce the concept of the cortical 'receptome' taking into account the distribution and densities of expression of different modulatory receptor types across the brain's anatomical connectivity matrix. By modelling whole-brain dynamics in silico, we suggest a bidirectional coupling between modulatory neurotransmission and neuronal connectivity hardware exemplified by the impact of single serotonergic (5-HT) receptor types on cortical dynamics. As experimental support of this concept, we show how optogenetic tools enable specific activation of a single 5-HT receptor type across the cortex as well as in vivo measurement of its distinct effects on cortical processing. Altogether, we demonstrate how the structural neuronal connectivity backbone and its modulation by a single neurotransmitter system allow access to a rich repertoire of different brain states that are fundamental for flexible behaviour. We further propose that irregular receptor expression patterns-genetically predisposed or acquired during a lifetime-may predispose for neuropsychiatric disorders like addiction, depression and anxiety along with distinct changes in brain state. Our long-term vision is that such diseases could be treated through rationally targeted therapeutic interventions of high specificity to eventually recover natural transitions of brain states.
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Encéfalo , Receptores de Serotonina , Encéfalo/metabolismo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Optogenética , Receptores de Serotonina/metabolismoRESUMEN
Opn7b is a non-visual G protein-coupled receptor expressed in zebrafish. Here we find that Opn7b expressed in HEK cells constitutively activates the Gi/o pathway and illumination with blue/green light inactivates G protein-coupled inwardly rectifying potassium channels. This suggests that light acts as an inverse agonist for Opn7b and can be used as an optogenetic tool to inhibit neuronal networks in the dark and interrupt constitutive inhibition in the light. Consistent with this prediction, illumination of recombinant expressed Opn7b in cortical pyramidal cells results in increased neuronal activity. In awake mice, light stimulation of Opn7b expressed in pyramidal cells of somatosensory cortex reliably induces generalized epileptiform activity within a short (<10 s) delay after onset of stimulation. Our study demonstrates a reversed mechanism for G protein-coupled receptor control and Opn7b as a tool for controlling neural circuit properties.
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Proteínas de Unión al GTP/metabolismo , Neuronas/metabolismo , Opsinas/metabolismo , Optogenética/métodos , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Proteínas de Unión al GTP/genética , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Neuronas/fisiología , Opsinas/genética , Células Piramidales/metabolismo , Células Piramidales/fisiología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Corteza Somatosensorial/citología , Corteza Somatosensorial/metabolismo , Sinapsis/genética , Sinapsis/fisiología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The schooling flashlight fish Anomalops katoptron can be found at dark nights at the water surface in the Indo-Pacific. Schools are characterized by bioluminescent blink patterns of sub-ocular light organs densely-packed with bioluminescent, symbiotic bacteria. Here we analyzed how blink patterns of A. katoptron are used in social interactions. We demonstrate that isolated specimen of A. katoptron showed a high motivation to align with fixed or moving artificial light organs in an experimental tank. This intraspecific recognition of A. katoptron is mediated by blinking light and not the body shape. In addition, A. katoptron adjusts its blinking frequencies according to the light intensities. LED pulse frequencies determine the swimming speed and the blink frequency response of A. katoptron, which is modified by light organ occlusion and not exposure. In the natural environment A. katoptron is changing its blink frequencies and nearest neighbor distance in a context specific manner. Blink frequencies are also modified by changes in the occlusion time and are increased from day to night and during avoidance behavior, while group cohesion is higher with increasing blink frequencies. Our results suggest that specific blink patterns in schooling flashlight fish A. katoptron define nearest neighbor distance and determine intraspecific communication.
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Comunicación Animal , Peces/fisiología , Luminiscencia , Conducta Social , Animales , Ecosistema , NataciónRESUMEN
Although optogenetics has revolutionized rodent neuroscience, it is still rarely used in other model organisms as the efficiencies of viral gene transfer differ between species and comprehensive viral transduction studies are rare. However, for comparative research, birds offer valuable model organisms as they have excellent visual and cognitive capabilities. Therefore, the following study establishes optogenetics in pigeons on histological, physiological, and behavioral levels. We show that AAV1 is the most efficient viral vector in various brain regions and leads to extensive anterograde and retrograde ChR2 expression when combined with the CAG promoter. Furthermore, transient optical stimulation of ChR2 expressing cells in the entopallium decreases pigeons' contrast sensitivity during a grayscale discrimination task. This finding demonstrates causal evidence for the involvement of the entopallium in contrast perception as well as a proof of principle for optogenetics in pigeons and provides the groundwork for various other methods that rely on viral gene transfer in birds.
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Channelrhodopsins/metabolismo , Columbidae/genética , Dependovirus , Optogenética , Telencéfalo/metabolismo , AnimalesRESUMEN
Reelin is a protein that is best known for its role in controlling neuronal layer formation in the developing cortex. Here, we studied its role for post-natal cortical network function, which is poorly explored. To preclude early cortical migration defects caused by Reelin deficiency, we used a conditional Reelin knock-out (RelncKO ) mouse, and induced Reelin deficiency post-natally. Induced Reelin deficiency caused hyperexcitability of the neocortical network in vitro and ex vivo. Blocking Reelin binding to its receptors ApoER2 and VLDLR resulted in a similar effect. Hyperexcitability in RelncKO organotypic slice cultures could be rescued by co-culture with wild-type organotypic slice cultures. Moreover, the GABAB receptor (GABAB R) agonist baclofen failed to activate and the antagonist CGP35348 failed to block GABAB Rs in RelncKO mice. Immunolabeling of RelncKO cortical slices revealed a reduction in GABAB R1 and GABAB R2 surface expression at the plasma membrane and western blot of RelncKO cortical tissue revealed decreased phosphorylation of the GABAB R2 subunit at serine 892 and increased phosphorylation at serine 783, reflecting receptor deactivation and proteolysis. These data show a role of Reelin in controlling early network activity, by modulating GABAB R function. Cover Image for this issue: https://doi.org/10.1111/jnc.15054.
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Moléculas de Adhesión Celular Neuronal/deficiencia , Proteínas de la Matriz Extracelular/deficiencia , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Receptores de GABA-B/fisiología , Serina Endopeptidasas/deficiencia , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Femenino , Agonistas de Receptores GABA-B/farmacología , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Técnicas de Cultivo de Órganos , Proteína Reelina , Serina Endopeptidasas/genética , Transducción de Señal/efectos de los fármacosRESUMEN
The Alzheimer disease-associated multifunctional low-density lipoprotein receptor-related protein-1 is expressed in the brain. Recent studies uncovered a role of this receptor for the appropriate functioning of neural stem cells, oligodendrocytes, and neurons. The constitutive knock-out (KO) of the receptor is embryonically lethal. To unravel the receptors' role in the developing brain we generated a mouse mutant by specifically targeting radial glia stem cells of the dorsal telencephalon. The low-density lipoprotein receptor-related protein-1 lineage-restricted KO female and male mice, in contrast to available models, developed a severe neurological phenotype with generalized seizures during early postnatal development. The mechanism leading to a buildup of hyperexcitability and emergence of seizures was traced to a failure in adequate astrocyte development and deteriorated postsynaptic density integrity. The detected impairments in the astrocytic lineage: precocious maturation, reactive gliosis, abolished tissue plasminogen activator uptake, and loss of functionality emphasize the importance of this glial cell type for synaptic signaling in the developing brain. Together, the obtained results highlight the relevance of astrocytic low-density lipoprotein receptor-related protein-1 for glutamatergic signaling in the context of neuron-glia interactions and stage this receptor as a contributing factor for epilepsy.
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Células Ependimogliales , Animales , Astrocitos , Femenino , Lipoproteínas LDL , Masculino , Ratones , Prosencéfalo , Receptores de Lipoproteína , Convulsiones , Activador de Tejido PlasminógenoRESUMEN
Controlling gain of cortical activity is essential to modulate weights between internal ongoing communication and external sensory drive. Here, we show that serotonergic input has separable suppressive effects on the gain of ongoing and evoked visual activity. We combined optogenetic stimulation of the dorsal raphe nucleus (DRN) with wide-field calcium imaging, extracellular recordings, and iontophoresis of serotonin (5-HT) receptor antagonists in the mouse visual cortex. 5-HT1A receptors promote divisive suppression of spontaneous activity, while 5-HT2A receptors act divisively on visual response gain and largely account for normalization of population responses over a range of visual contrasts in awake and anesthetized states. Thus, 5-HT input provides balanced but distinct suppressive effects on ongoing and evoked activity components across neuronal populations. Imbalanced 5-HT1A/2A activation, either through receptor-specific drug intake, genetically predisposed irregular 5-HT receptor density, or change in sensory bombardment may enhance internal broadcasts and reduce sensory drive and vice versa.
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Núcleo Dorsal del Rafe/fisiología , Optogenética/métodos , Neuronas Serotoninérgicas/fisiología , Corteza Visual/fisiología , Animales , Línea Celular , Núcleo Dorsal del Rafe/efectos de los fármacos , Luz , Estudios Longitudinales , Ratones , Ratones Transgénicos , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/fisiología , Serotonina/fisiología , Antagonistas de la Serotonina/administración & dosificación , Corteza Visual/efectos de los fármacosRESUMEN
Carbon-based nanoelectrodes fabricated by means of pyrolysis of an alkane precursor gas purged through a glass capillary and subsequently etched with HF were modified with redox polymer/enzyme films for the detection of glucose at the single-cell level. Glucose oxidase (GOx) was immobilized and electrically wired by means of an Os-complex-modified redox polymer in a sequential dip coating process. For the synthesis of the redox polymer matrix, a poly(1-vinylimidazole-co-acrylamide)-based backbone was used that was first modified with the electron transfer mediator [Os(bpy)2Cl]+ (bpy = 2,2'-bipyridine) followed by the conversion of the amide groups within the acrylamide monomer into hydrazide groups in a polymer-analogue reaction. The hydrazide groups react readily with bifunctional epoxide-based crosslinkers ensuring high film stability. Insertion of the nanometre-sized polymer/enzyme modified electrodes into adherently growing single NG108-15 cells resulted in a positive current response correlating with the intracellular glucose concentration. Moreover, the nanosensors showed a stable current output without significant loss in performance after intracellular measurements.
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Técnicas Biosensibles/instrumentación , Carbono/química , Glucosa/análisis , Polímeros/química , Análisis de la Célula Individual/instrumentación , Animales , Aspergillus niger/enzimología , Línea Celular , Enzimas Inmovilizadas/química , Glucosa Oxidasa/química , Ratones , MicroelectrodosRESUMEN
Carbon nanoelectrodes in the sub-micron range were modified with an enzyme cascade immobilized in a spatially separated polymer double layer system for the detection of glutamate at the cellular level. The enzyme cascade consists of glutamate oxidase (GlutOx) that was immobilized in a hydrophilic redox silent polymer on top of a horseradish peroxidase (HRP)/redox polymer layer. In the presence of O2, glutamate was oxidized under concomitant reduction of O2 to H2O2 at GlutOx. H2O2 is further reduced to water by means of HRP and electrons are shuttled via the redox polymer matrix that wires the HRP to the electrode surface, hence delivering a current response proportional to the glutamate concentration. The nanometer-sized sensors could be successfully used to measure glutamate release from primary mouse astrocytes in 10 mM HEPES buffer.
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Aminoácido Oxidorreductasas/química , Carbono/química , Glutamatos/análisis , Nanopartículas/química , Polímeros/química , Aminoácido Oxidorreductasas/metabolismo , Animales , Astrocitos/química , Técnicas Biosensibles , Carbono/metabolismo , Células Cultivadas , Técnicas Electroquímicas , Electrodos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Glutamatos/metabolismo , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Nanopartículas/metabolismo , Tamaño de la Partícula , Polímeros/metabolismo , Streptomyces/enzimología , Propiedades de SuperficieRESUMEN
Optogenetics is a biological technique that combines the advantageous spatial-temporal resolution of optics and genetic cell targeting to control cellular activity with unprecedented precision. It has found vast applications both in neurosciences and therapy, particularly in view of its application to restore vision in blind patients. Optogenetics requires the ectopic expression of a so-called opsin to render neurons sensitive to light. There are two types of opsins for modulating membrane potential of neurons: (i) microbial opsins from unicellular organisms that respond to a light stimulus by mediating a flow of ions across the membrane (ii) animal opsins that are naturally present in mammalian retinas that initiate G protein coupled signaling in response to light. The former category has been extensively employed for vision restoration in the past decade with two ongoing clinical trials employing microbial opsins to restore light sensation in retinitis pigmentosa patients. The latter subtype of animal opsins is emerging more recently as strong candidates to restore vision with the promise of greater light sensitivity and tolerability. In this review we will discuss each approach in view of its utility for vision restoration in retinal blindness.
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Ceguera/terapia , Opsinas/genética , Optogenética/métodos , Degeneración Retiniana/terapia , Animales , Ceguera/genética , Ceguera/fisiopatología , Humanos , Retina/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/fisiopatología , Visión OcularRESUMEN
Inborn errors of CACNA1A-encoded P/Q-type calcium channels impair synaptic transmission, producing early and lifelong neurological deficits, including childhood absence epilepsy, ataxia and dystonia. Whether these impairments owe their pathologies to defective channel function during the critical period for thalamic network stabilization in immature brain remains unclear. Here we show that mice with tamoxifen-induced adult-onset ablation of P/Q channel alpha subunit (iKOp/q) display identical patterns of dysfunction, replicating the inborn loss-of-function phenotypes and, therefore demonstrate that these neurological defects do not rely upon developmental abnormality. Unexpectedly, unlike the inborn model, the adult-onset pattern of excitability changes believed to be pathogenic within the thalamic network is non-canonical. Specifically, adult ablation of P/Q channels does not promote Cacna1g-mediated burst firing or T-type calcium current (IT) in the thalamocortical relay neurons; however, burst firing in thalamocortical relay neurons remains essential as iKOp/q mice generated on a Cacna1g deleted background show substantially diminished seizure generation. Moreover, in thalamic reticular nucleus neurons, burst firing is impaired accompanied by attenuated IT. Interestingly, inborn deletion of thalamic reticular nucleus-enriched, human childhood absence epilepsy-linked gene Cacna1h in iKOp/q mice reduces thalamic reticular nucleus burst firing and promotes rather than reduces seizure, indicating an epileptogenic role for loss-of-function Cacna1h gene variants reported in human childhood absence epilepsy cases. Together, our results demonstrate that P/Q channels remain critical for maintaining normal thalamocortical oscillations and motor control in the adult brain, and suggest that the developmental plasticity of membrane currents regulating pathological rhythmicity is both degenerate and age-dependent.
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Ataxia/genética , Canales de Calcio Tipo N/genética , Corteza Cerebral/metabolismo , Epilepsia Tipo Ausencia/genética , Neuronas/metabolismo , Tálamo/metabolismo , Potenciales de Acción , Factores de Edad , Animales , Ataxia/metabolismo , Ataxia/fisiopatología , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Corteza Cerebral/fisiopatología , Modelos Animales de Enfermedad , Epilepsia Tipo Ausencia/metabolismo , Epilepsia Tipo Ausencia/fisiopatología , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Inhibidores/genética , Potenciales de la Membrana/genética , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Núcleos Talámicos/citología , Tálamo/fisiopatologíaRESUMEN
Optogenetics uses light-sensitive proteins, so-called optogenetic tools, for highly precise spatiotemporal control of cellular states and signals. The major limitations of such tools include the overlap of excitation spectra, phototoxicity, and lack of sensitivity. The protein characterized in this study, the Japanese lamprey parapinopsin, which we named UVLamP, is a promising optogenetic tool to overcome these limitations. Using a hybrid strategy combining molecular, cellular, electrophysiological, and computational methods we elucidated a structural model of the dark state and probed the optogenetic potential of UVLamP. Interestingly, it is the first described bistable vertebrate opsin that has a charged amino acid interacting with the Schiff base in the dark state, that has no relevance for its photoreaction. UVLamP is a bistable UV-sensitive opsin that allows for precise and sustained optogenetic control of G protein-coupled receptor (GPCR) pathways and can be switched on, but more importantly also off within milliseconds via lowintensity short light pulses. UVLamP exhibits an extremely narrow excitation spectrum in the UV range allowing for sustained activation of the Gi/o pathway with a millisecond UV light pulse. Its sustained pathway activation can be switched off, surprisingly also with a millisecond blue light pulse, minimizing phototoxicity. Thus, UVLamP serves as a minimally invasive, narrow-bandwidth probe for controlling the Gi/o pathway, allowing for combinatorial use with multiple optogenetic tools or sensors. Because UVLamP activated Gi/o signals are generally inhibitory and decrease cellular activity, it has tremendous potential for health-related applications such as relieving pain, blocking seizures, and delaying neurodegeneration.