Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore.

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.

Reelin signaling modulates GABAB receptor function in the neocortex.

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.

Adult loss of Cacna1a in mice recapitulates childhood absence epilepsy by distinct thalamic bursting mechanisms.

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.

Lipoprotein receptor loss in forebrain radial glia results in neurological deficits and severe seizures.

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.

Opsins for vision restoration.

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.

Lamprey Parapinopsin ("UVLamP"): a Bistable UV-Sensitive Optogenetic Switch for Ultrafast Control of GPCR Pathways.

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.

Serotonin neurons in the dorsal raphe mediate the anticataplectic action of orexin neurons by reducing amygdala activity.

Narcolepsy is a sleep disorder caused by the loss of orexin (hypocretin)-producing neurons and marked by excessive daytime sleepiness and a sudden weakening of muscle tone, or cataplexy, often triggered by strong emotions. In a mouse model for narcolepsy, we previously demonstrated that serotonin neurons of the dorsal raphe nucleus (DRN) mediate the suppression of cataplexy-like episodes (CLEs) by orexin neurons. Using an optogenetic tool, in this paper we show that the acute activation of DRN serotonin neuron terminals in the amygdala, but not in nuclei involved in regulating rapid eye-movement sleep and atonia, suppressed CLEs. Not only did stimulating serotonin nerve terminals reduce amygdala activity, but the chemogenetic inhibition of the amygdala using designer receptors exclusively activated by designer drugs also drastically decreased CLEs, whereas chemogenetic activation increased them. Moreover, the optogenetic inhibition of serotonin nerve terminals in the amygdala blocked the anticataplectic effects of orexin signaling in DRN serotonin neurons. Taken together, the results suggest that DRN serotonin neurons, as a downstream target of orexin neurons, inhibit cataplexy by reducing the activity of amygdala as a center for emotional processing.

Design of an Ultrafast G Protein Switch Based on a Mouse Melanopsin Variant.

The primary goal of optogenetics is the light-controlled noninvasive and specific manipulation of various cellular processes. Herein, we present a hybrid strategy for targeted protein engineering combining computational techniques with electrophysiological and UV/visible spectroscopic experiments. We validated our concept for channelrhodopsin-2 and applied it to modify the less-well-studied vertebrate opsin melanopsin. Melanopsin is a promising optogenetic tool that functions as a selective molecular light switch for G protein-coupled receptor pathways. Thus, we constructed a model of the melanopsin Gq protein complex and predicted an absorption maximum shift of the Y211F variant. This variant displays a narrow blue-shifted action spectrum and twofold faster deactivation kinetics compared to wild-type melanopsin on G protein-coupled inward rectifying K+ (GIRK) channels in HEK293 cells. Furthermore, we verified the in vivo activity and optogenetic potential for the variant in mice. Thus, we propose that our developed concept will be generally applicable to designing optogenetic tools.

Optogenetic Destabilization of the Memory Trace in CA1: Insights into Reconsolidation and Retrieval Processes.

Reactivation of memory can cause instability necessitating the reconsolidation of the trace. This process can be blocked by amnestic treatments administered after memory reactivation resulting in subsequent memory deficits. While the basolateral amygdala (BLA) is known to be crucial for reconsolidation, evidence for a contribution of the hippocampal CA1 region has only started to accumulate. Moreover, the effect of a reconsolidation blockade in CA1 has only been evaluated behaviorally, and it is unknown whether this manipulation has a long-term effect on neuronal activity. We combined optogenetic and high-resolution molecular imaging techniques to inhibit cell firing in CA1 following the reactivation of a fear memory in mice, evaluated memory performance and imaged neuronal activity the next day upon reexposure to the conditioning context. Blocking memory reconsolidation led to severe memory impairments that were associated with reduced neuronal activity not only in CA1 but also in CA3 and the BLA. Thus, our results indicate that CA1 is necessary for reconsolidation and suggest the involvement of a CA3-CA1-BLA network in the retrieval of contextual fear memory. Further investigations of this network might contribute to the validation of new brain targets for the treatment of pathologies such as posttraumatic stress disorders.

Isolated P/Q Calcium Channel Deletion in Layer VI Corticothalamic Neurons Generates Absence Epilepsy.

Generalized spike-wave seizures involving abnormal synchronization of cortical and underlying thalamic circuitry represent a major category of childhood epilepsy. Inborn errors of Cacna1a, the P/Q-type voltage-gated calcium channel α subunit gene, expressed throughout the brain destabilize corticothalamic rhythmicity and produce this phenotype. To determine the minimal cellular lesion required for this network disturbance, we used neurotensin receptor 1 (Ntsr1) cre-driver mice to ablate floxed Cacna1a in layer VI pyramidal neurons, which supply the sole descending cortical synaptic input to thalamocortical relay cells and reticular interneurons and activate intrathalamic circuits. Targeted Cacna1a ablation in layer VI cells resulted in mice that display a robust spontaneous spike-wave absence seizure phenotype accompanied by behavioral arrest and inhibited by ethosuximide. To verify the selectivity of the molecular lesion, we determined that P/Q subunit proteins were reduced in corticothalamic relay neuron terminal zones, and confirmed that P/Q-mediated glutamate release was reduced at these synapses. Spike-triggered exocytosis was preserved by N-type calcium channel rescue, demonstrating that evoked release at layer VI terminals relies on both P/Q and N-type channels. Whereas intrinsic excitability of the P/Q channel depleted layer VI neurons was unaltered, T-type calcium currents in the postsynaptic thalamic relay and reticular cells were dramatically elevated, favoring rebound bursting and seizure generation. We find that an early P/Q-type release defect, limited to synapses of a single cell-type within the thalamocortical circuit, is sufficient to remodel synchronized firing behavior and produce a stable generalized epilepsy phenotype. SIGNIFICANCE STATEMENT: This study dissects a critical component of the corticothalamic circuit in spike-wave epilepsy and identifies the developmental importance of P/Q-type calcium channel-mediated presynaptic glutamate release at layer VI pyramidal neuron terminals. Genetic ablation of Cacna1a in layer VI neurons produced synchronous spike-wave discharges in the cortex and thalamus that were inhibited by ethosuximide. These mice also displayed N-type calcium channel compensation at descending thalamic synapses, and consistent with other spike-wave models increased low-threshold T-type calcium currents within postsynaptic thalamic relay and reticular neurons. These results demonstrate, for the first time, that preventing the developmental homeostatic switch from loose to tightly coupled synaptic release at a single class of deep layer cortical excitatory output neurons results in generalized spike-wave epilepsy.

Keeping Our Calcium in Balance to Maintain Our Balance.

Calcium is a key signaling molecule and ion involved in a variety of diverse processes in our central nervous system (CNS) which include gene expression, synaptic transmission and plasticity, neuronal excitability and cell maintenance. Proper control of calcium signaling is not only vital for neuronal physiology but also cell survival. Mutations in fundamental channels, transporters and second messenger proteins involved in orchestrating the balance of our calcium homeostasis can lead to severe neurodegenerative disorders, such as Spinocerebellar (SCA) and Episodic (EA) ataxias. Hereditary ataxias make up a remarkably diverse group of neurological disorders clinically characterized by gait ataxia, nystagmus, dysarthria, trunk and limb ataxia and often atrophy of the cerebellum. The largest family of hereditary ataxias is SCAs which consists of a growing family of 42 members. A relatively smaller family of 8 members compose the EAs. The gene mutations responsible for half of the EA members and over 35 of the SCA subtypes have been identified, and several have been found to be responsible for cerebellar atrophy, abnormal intracellular calcium levels, dysregulation of Purkinje cell pacemaking, altered cerebellar synaptic transmission and/or ataxia in mouse models. Although the genetic diversity and affected cellular pathways of hereditary ataxias are broad, one common theme amongst these genes is their effects on maintaining calcium balance in primarily the cerebellum. There is emerging evidence that the pathogenesis of hereditary ataxias may be caused by imbalances in intracellular calcium due to genetic mutations in calcium-mediating proteins. In this review we will discuss the current evidence supporting the role of deranged calcium as the culprit to neurodegenerative diseases with a primary focus on SCAs and EAs.

Gq/5-HT2c receptor signals activate a local GABAergic inhibitory feedback circuit to modulate serotonergic firing and anxiety in mice.

Serotonin 2c receptors (5-HT2c-Rs) are drug targets for certain mental disorders, including schizophrenia, depression, and anxiety. 5-HT2c-Rs are expressed throughout the brain, making it difficult to link behavioral changes to circuit specific receptor expression. Various 5-HT-Rs, including 5-HT2c-Rs, are found in the dorsal raphe nucleus (DRN); however, the function of 5-HT2c-Rs and their influence on the serotonergic signals mediating mood disorders remain unclear. To investigate the role of 5-HT2c-Rs in the DRN in mice, we developed a melanopsin-based optogenetic probe for activation of Gq signals in cellular domains, where 5-HT2c-Rs are localized. Our results demonstrate that precise temporal control of Gq signals in 5-HT2c-R domains in GABAergic neurons upstream of 5-HT neurons provides negative feedback regulation of serotonergic firing to modulate anxiety-like behavior in mice.

Spinocerebellar ataxia type 6 protein aggregates cause deficits in motor learning and cerebellar plasticity.

Spinocerebellar ataxia type 6 (SCA6) is linked to poly-glutamine (polyQ) within the C terminus (CT) of the pore-forming subunits of P/Q-type Ca(2+) channels (Cav2.1) and is characterized by CT protein aggregates found in cerebellar Purkinje cells (PCs). One hypothesis regarding SCA6 disease is that a CT fragment of the Cav2.1 channel, which is detected specifically in cytosolic and nuclear fractions in SCA6 patients, is associated with the SCA6 pathogenesis. To test this hypothesis, we expressed P/Q-type channel protein fragments from two different human CT splice variants, as predicted from SCA6 patients, in PCs of mice using viral and transgenic approaches. These splice variants represent a short (CT-short without polyQs) and a long (CT-long with 27 polyQs) CT fragment. Our results show that the different splice variants of the CTs differentially distribute within PCs, i.e., the short CTs reveal predominantly nuclear inclusions, whereas the long CTs prominently reveal both nuclear and cytoplasmic aggregates. Postnatal expression of CTs in PCs in mice reveals that only CT-long causes SCA6-like symptoms, i.e., deficits in eyeblink conditioning (EBC), ataxia, and PC degeneration. The physiological phenotypes associated specifically with the long CT fragment can be explained by an impairment of LTD and LTP at the parallel fiber-to-PC synapse and alteration in spontaneous PC activity. Thus, our results suggest that the polyQ carrying the CT fragment of the P/Q-type channel is sufficient to cause SCA6 pathogenesis in mice and identifies EBC as a new diagnostic strategy to evaluate Ca(2+) channel-mediated human diseases.

The requirement of BDNF for hippocampal synaptic plasticity is experience-dependent.

Brain-derived neurotrophic factor (BDNF) supports neuronal survival, growth, and differentiation and has been implicated in forms of hippocampus-dependent learning. In vitro, a specific role in hippocampal synaptic plasticity has been described, although not all experience-dependent forms of synaptic plasticity critically depend on BDNF. Synaptic plasticity is likely to enable long-term synaptic information storage and memory, and the induction of persistent (>24 h) forms, such as long-term potentiation (LTP) and long-term depression (LTD) is tightly associated with learning specific aspects of a spatial representation. Whether BDNF is required for persistent (>24 h) forms of LTP and LTD, and how it contributes to synaptic plasticity in the freely behaving rodent has never been explored. We examined LTP, LTD, and related forms of learning in the CA1 region of freely dependent mice that have a partial knockdown of BDNF (BDNF(+/-) ). We show that whereas early-LTD (<90min) requires BDNF, short-term depression (<45 min) does not. Furthermore, BDNF is required for LTP that is induced by mild, but not strong short afferent stimulation protocols. Object-place learning triggers LTD in the CA1 region of mice. We observed that object-place memory was impaired and the object-place exploration failed to induce LTD in BDNF(+/-) mice. Furthermore, spatial reference memory, that is believed to be enabled by LTP, was also impaired. Taken together, these data indicate that BDNF is required for specific, but not all, forms of hippocampal-dependent information storage and memory. Thus, very robust forms of synaptic plasticity may circumvent the need for BDNF, rather it may play a specific role in the optimization of weaker forms of plasticity. The finding that both learning-facilitated LTD and spatial reference memory are both impaired in BDNF(+/-) mice, suggests moreover, that it is critically required for the physiological encoding of hippocampus-dependent memory. © 2015 The Authors Hippocampus Published by Wiley Periodicals, Inc.

Postnatal loss of P/Q-type channels confined to rhombic-lip-derived neurons alters synaptic transmission at the parallel fiber to purkinje cell synapse and replicates genomic Cacna1a mutation phenotype of ataxia and seizures in mice.

Ataxia, episodic dyskinesia, and thalamocortical seizures are associated with an inherited loss of P/Q-type voltage-gated Ca(2+) channel function. P/Q-type channels are widely expressed throughout the neuraxis, obscuring identification of the critical networks underlying these complex neurological disorders. We showed recently that the conditional postnatal loss of P/Q-type channels in cerebellar Purkinje cells (PCs) in mice (purky) leads to these aberrant phenotypes, suggesting that intrinsic alteration in PC output is a sufficient pathogenic factor for disease initiation. The question arises whether P/Q-type channel deletion confined to a single upstream cerebellar synapse might induce the pathophysiological abnormality of genomically inherited P/Q-type channel disorders. PCs integrate two excitatory inputs, climbing fibers from inferior olive and parallel fibers (PFs) from granule cells (GCs) that receive mossy fiber (MF) input derived from precerebellar nuclei. In this study, we introduce a new mouse model with a selective knock-out of P/Q-type channels in rhombic-lip-derived neurons including the PF and MF pathways (quirky). We found that in quirky mice, PF-PC synaptic transmission is reduced during low-frequency stimulation. Using focal light stimulation of GCs that express optogenetic light-sensitive channels, channelrhodopsin-2, we found that modulation of PC firing via GC input is reduced in quirky mice. Phenotypic analysis revealed that quirky mice display ataxia, dyskinesia, and absence epilepsy. These results suggest that developmental alteration of patterned input confined to only one of the main afferent cerebellar excitatory synaptic pathways has a significant role in generating the neurological phenotype associated with the global genomic loss of P/Q-type channel function.

Correlation between bioluminescent blinks and swimming behavior in the splitfin flashlight fish Anomalops katoptron.

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.

Diversity and function of fluorescent molecules in marine animals.

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.

New observations of fluorescent organisms in the Banda Sea and in the Red Sea.

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.

Optogenetic activation of mGluR1 signaling in the cerebellum induces synaptic plasticity.

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.

Delayed postnatal loss of P/Q-type calcium channels recapitulates the absence epilepsy, dyskinesia, and ataxia phenotypes of genomic Cacna1a mutations.

Inherited loss of P/Q-type calcium channel function causes human absence epilepsy, episodic dyskinesia, and ataxia, but the molecular "birthdate" of the neurological syndrome and its dependence on prenatal pathophysiology is unknown. Since these channels mediate transmitter release at synapses throughout the brain and are expressed early in embryonic development, delineating the critical circuitry and onset underlying each of the emergent phenotypes requires targeted control of gene expression. To visualize P/Q-type Ca(2+) channels and dissect their role in neuronal networks at distinct developmental stages, we created a novel conditional Cacna1a knock-in mouse by inserting the floxed green fluorescent protein derivative Citrine into the first exon of Cacna1a and then crossed it with a postnatally expressing PCP2-Cre line for delayed Purkinje cell (PC) gene deletion within the cerebellum and sparsely in forebrain (purky). PCs in purky mice lacked P/Q-type calcium channel protein and currents within the first month after birth, displayed altered spontaneous firing, and showed impaired neurotransmission. Unexpectedly, adult purky mice exhibited the full spectrum of neurological deficits seen in mice with genomic Cacna1a ablation. Our results show that the ataxia, dyskinesia, and absence epilepsy caused by inherited disorders of the P/Q-type channel arise from signaling defects beginning in late infancy, revealing an early window of opportunity for therapeutic intervention.