Retinotectal Projection Research Articles

Serotonin Strengthens a Developing Glutamatergic Synapse through a PI3K-Dependent Mechanism.

It is well established that, during neural circuit development, glutamatergic synapses become strengthened via NMDA receptor (NMDAR)-dependent upregulation of AMPA receptor (AMPAR)-mediated currents. In addition, however, it is known that the neuromodulator serotonin is present throughout most regions of the vertebrate brain while synapses are forming and being shaped by activity-dependent processes. This suggests that serotonin may modulate or contribute to these processes. Here, we investigate the role of serotonin in the developing retinotectal projection of the Xenopus tadpole. We altered endogenous serotonin transmission in stage 48/49 (∼10-21 days postfertilization) Xenopus tadpoles and then carried out a set of whole-cell electrophysiological recordings from tectal neurons to assess retinotectal synaptic transmission. Because tadpole sex is indeterminate at these early stages of development, experimental groups were composed of randomly chosen tadpoles. We found that pharmacologically enhancing and reducing serotonin transmission for 24 h up- and downregulates, respectively, AMPAR-mediated currents at individual retinotectal synapses. Inhibiting 5-HT2 receptors also significantly weakened AMPAR-mediated currents and abolished the synapse strengthening effect seen with enhanced serotonin transmission, indicating a 5-HT2 receptor-dependent effect. We also determine that the serotonin-dependent upregulation of synaptic AMPAR currents was mediated via an NMDAR-independent, PI3K-dependent mechanism. Altogether, these findings indicate that serotonin regulates AMPAR currents at developing synapses independent of NMDA transmission, which may explain its role as an enabler of activity-dependent plasticity.

Bilateral Retinofugal Pathfinding Impairments Limit Behavioral Compensation in Near-Congenital One-Eyed Xenopus laevis.

To generate a coherent visual percept, information from both eyes must be appropriately transmitted into the brain, where binocular integration forms the substrate for visuomotor behaviors. To establish the anatomical substrate for binocular integration, the presence of bilateral eyes and interaction of both optic nerves during retinotectal development play a key role. However, the extent to which embryonic monocularly derived visual circuits can convey visuomotor behaviors is unknown. In this study, we assessed the retinotectal anatomy and visuomotor performance of embryonically generated one-eyed tadpoles. In one-eyed animals, the axons of retinal ganglion cells from the singular remaining eye exhibited striking irregularities in their central projections in the brain, generating a noncanonical ipsilateral retinotectal projection. This data is indicative of impaired pathfinding abilities. We further show that these novel projections are correlated with an impairment of behavioral compensation for the loss of one eye.

Alzheimer PHF-tau aggregates do not spread tau pathology to the brain via the Retino-tectal projection after intraocular injection in male mouse models

Neurofibrillary tangles (NFT), a neuronal lesion found in Alzheimer's disease (AD), are composed of fibrillary aggregates of modified forms of tau proteins. The propagation of NFT follows neuroanatomical pathways suggesting that synaptically connected neurons could transmit tau pathology by the recruitment of normal tau in a prion-like manner. Moreover, the intracerebral injection of pathological tau from AD brains induces the seeding of normal tau in mouse brain. Creutzfeldt-Jacob disease has been transmitted after ocular transplants of cornea or sclera and the scrapie agent can spread across the retino-tectal pathway after intraocular injection of scrapie mouse brain homogenates. In AD, a tau pathology has been detected in the retina. To investigate the potential risk of tau pathology transmission during eye surgery using AD tissue material, we have analysed the development of tau pathology in the visual pathway of mice models expressing murine tau, wild-type or mutant human tau after intraocular injection of pathological tau proteins from AD brains. Although these pathological tau proteins were internalized in retinal ganglion cells, they did not induce aggregation of endogenous tau nor propagation of a tau pathology in the retino-tectal pathway after a 6-month incubation period. These results suggest that retinal ganglion cells exhibit a resistance to develop a tau pathology, and that eye surgery is not a major iatrogenic risk of transmission of tau pathology, contrary to what has been observed for transmission of infectious prions in prion diseases.

Topographic map formation and the effects of NMDA receptor blockade in the developing visual system

The development of functional topography in the developing brain follows a progression from initially coarse to more precisely organized maps. To examine the emergence of topographically organized maps in the retinotectal system, we performed longitudinal visual receptive field mapping by calcium imaging in the optic tectum of GCaMP6-expressing transgenic Xenopus laevis tadpoles. At stage 42, just 1 d after retinal axons arrived in the optic tectum, a clear retinotopic azimuth map was evident. Animals were imaged over the following week at stages 45 and 48, over which time the tectal neuropil nearly doubled in length and exhibited more precise retinotopic organization. By microinjecting GCaMP6s messenger ribonucleic acid (mRNA) into one blastomere of two-cell stage embryos, we acquired bilateral mosaic tadpoles with GCaMP6s expression in postsynaptic tectal neurons on one side of the animal and in retinal ganglion cell axons crossing to the tectum on the opposite side. Longitudinal observation of retinotopic map emergence revealed the presence of orderly representations of azimuth and elevation as early as stage 42, although presynaptic inputs exhibited relatively less topographic organization than the postsynaptic component for the azimuth axis. Retinotopic gradients in the tectum became smoother between stages 42 and 45. Blocking N-methyl-D-aspartate (NMDA) receptor conductance by rearing tadpoles in MK-801 did not prevent the emergence of retinotopic maps, but it produced more discontinuous topographic gradients and altered receptive field characteristics. These results provide evidence that current through NMDA receptors is dispensable for coarse topographic ordering of retinotectal inputs but does contribute to the fine-scale organization of the retinotectal projection.

Electrophysiological Approaches to Studying Normal and Abnormal Retinotectal Circuit Development in the Xenopus Tadpole.

The Xenopus tadpole retinotectal projection is the main component of the amphibian visual system. It comprises the retinal ganglion cells (RGCs) in the eye, which project an axon to synapse onto tectal neurons in the optic tectum. There are many attributes of this relatively simple projection that render it uniquely well-suited for studying the functional development of neural circuits. One major experimental advantage of this circuit is that it can be genetically or pharmacologically altered and then assessed at high resolution via whole-cell electrophysiological recordings using an ex vivo isolated brain preparation. This protocol provides instructions for performing such electrophysiological investigations using the ex-vivo-isolated brain preparation. It allows one to measure many different aspects of synaptic transmission between the RGC axons and individual postsynaptic tectal neurons, including AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) to NMDA (N-methyl-d-aspartate) ratios, strength of individual RGC axons, paired pulse facilitation, and strength of individual synapses.

Shootin-1 is required for nervous system development in zebrafish.

Semaphorin6A (Sema6A) and its PlexinA2 (PlxnA2) receptor canonically function as repulsive axon guidance cues. To understand downstream signaling mechanisms, we performed a microarray screen and identified the "clutch molecule" shootin-1 (shtn-1) as a transcriptionally repressed target. Shtn-1 is a key proponent of cell migration and neuronal polarization and must be regulated during nervous system development. The mechanisms of Shtn-1 regulation and the phenotypic consequences of loss of repression are poorly understood. We demonstrate shtn-1 overexpression results in impaired migration of the optic vesicles, lack of retinal pigmented epithelium, and pathfinding errors of retinotectal projections. We also observed patterning defects in the peripheral nervous system. Importantly, these phenotypes were rescued by overexpressing PlxnA2. We demonstrate a functional role for repression of shtn-1 by PlxnA2 in development of the eyes and peripheral nervous system in zebrafish. These results demonstrate that careful regulation of shtn-1 is critical for development of the nervous system.

Genetic developmental timing revealed by inter-species transplantations in fish.

The path from a fertilised egg to an embryo involves the coordinated formation of cell types, tissues and organs. Developmental modules comprise discrete units specified by self-sufficient genetic programs that can interact with each other during embryogenesis. Here, we have taken advantage of the different span of embryonic development between two distantly related teleosts, zebrafish (Danio rerio) and medaka (Oryzias latipes) (3 and 9 days, respectively), to explore modularity principles. We report that inter-species blastula transplantations result in the ectopic formation of a retina formed by donor cells - a module. We show that the time taken for the retina to develop follows a genetic program: an ectopic zebrafish retina in medaka develops with zebrafish dynamics. Heterologous transplantation results in a temporal decoupling between the donor retina and host organism, illustrated by two paradigms that require retina-host interactions: lens recruitment and retino-tectal projections. Our results uncover a new experimental system for addressing temporal decoupling along embryonic development, and highlight the presence of largely autonomous but interconnected developmental modules that orchestrate organogenesis.

An interhemispheric neural circuit allowing binocular integration in the optic tectum

Binocular stereopsis requires the convergence of visual information from corresponding points in visual space seen by two different lines of sight. This may be achieved by superposition of retinal input from each eye onto the same downstream neurons via ipsi- and contralaterally projecting optic nerve fibers. Zebrafish larvae can perceive binocular cues during prey hunting but have exclusively contralateral retinotectal projections. Here we report brain activity in the tectal neuropil ipsilateral to the visually stimulated eye, despite the absence of ipsilateral retinotectal projections. This activity colocalizes with arbors of commissural neurons, termed intertectal neurons (ITNs), that connect the tectal hemispheres. ITNs are GABAergic, establish tectal synapses bilaterally and respond to small moving stimuli. ITN-ablation impairs capture swim initiation when prey is positioned in the binocular strike zone. We propose an intertectal circuit that controls execution of the prey-capture motor program following binocular localization of prey, without requiring ipsilateral retinotectal projections.

ADHD AND MIGRAINE: INVESTIGATING SHARED GENETIC MECHANISM IN A POPULATION-BASED CASE-COHORT SAMPLE

Animals, interacting with the environment, learn and exploit the consequences of their movements. Fundamental to this is the pairing of salient sensory input with recent motor output to form an action–outcome pair linking a performed movement with its outcome. Short-latency dopamine (DA) signalling in the basal ganglia has been proposed to support this crucial task. For visual stimuli, this DA signalling is triggered at short latency by input from the superior colliculus (SC). While some aspects of the visual signal (e.g. luminance), are relayed directly to the SC via the retinotectal projection, other information unavailable to this subcortical pathway must take a more circuitous route to the SC, first submitting to early visual processing in cortex. By comparing action–outcome pairing when the visual stimulus denoting success was immediately available to the SC, via the retinotectal pathway, against that when cortical processing of the signal was required, the impact this additional sensory processing has on action–outcome learning can be established. We found that action acquisition was significantly impaired when the action was reinforced by a stimulus ineligible for the retinotectal pathway. Furthermore, we found that when the stimulus was eligible for the retinotectal pathway but evoked an increased latency, action acquisition was not impaired. These results suggest that the afferent sensory pathway via the SC is certainly primary and possibly instrumental to the DA neurons’ role in the discovery of novel actions and that the differences found are not due to simple sensory latency.

Bioenergy with Carbon Capture and Sequestration (BECCS): The Distracting Injustice of an Infeasible and Unlikely Technofix

The retinotectal projections from double-nasal (NN), double-temporal (TT) and double-ventral (VV) compound eyes in Xenopus were studied autoradiographically and electrophysiologically during development. Early TT projections were confined to rostrolateral tectum and spread with advancing age to cover most of the tectum by shortly after metamorphosis. Early VV projections showed a decreased density of label on lateral tectum. Early NN projections appeared to extend across the entire rostrocaudal length of the available tectum at all stages of development, but showed a decrease in label density on rostral tectum. The results are discussed in relation to various hypotheses about the formation of retinotectal connexions.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles.

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Protein Tyrosine Phosphatase Receptor Type J (PTPRJ) Regulates Retinal Axonal Projections by Inhibiting Eph and Abl Kinases in Mice.

Eph receptors play pivotal roles in the axon guidance of retinal ganglion cells (RGCs) at the optic chiasm and the establishment of the topographic retinocollicular map. We previously demonstrated that protein tyrosine phosphatase receptor type O (PTPRO) is specifically involved in the control of retinotectal projections in chicks through the dephosphorylation of EphA and EphB receptors. We subsequently revealed that all the mouse R3 subfamily members (PTPRB, PTPRH, PTPRJ, and PTPRO) of the receptor protein tyrosine phosphatase (RPTP) family inhibited Eph receptors as their substrates in cultured mammalian cells. We herein investigated the functional roles of R3 RPTPs in the projection of mouse retinal axon of both sexes. Ptpro and Ptprj were expressed in mouse RGCs; however, Ptprj expression levels were markedly higher than those of Ptpro Consistent with their expression levels, Eph receptor activity was significantly enhanced in Ptprj-knock-out (Ptprj-KO) retinas. In Ptprj-KO and Ptprj/Ptpro-double-KO (DKO) mice, the number of retinal axons that projected ipsilaterally or to the contralateral eye was significantly increased. Furthermore, retinal axons in Ptprj-KO and DKO mice formed anteriorly shifted ectopic terminal zones in the superior colliculus (SC). We found that c-Abl (Abelson tyrosine kinase) was downstream of ephrin-Eph signaling for the repulsion of retinal axons at the optic chiasm and in the SC. c-Abl was identified as a novel substrate for PTPRJ and PTPRO, and the phosphorylation of c-Abl was upregulated in Ptprj-KO and DKO retinas. Thus, PTPRJ regulates retinocollicular projections in mice by controlling the activity of Eph and c-Abl kinases.SIGNIFICANCE STATEMENT Correct retinocollicular projection is a prerequisite for proper vision. Eph receptors have been implicated in retinal axon guidance at the optic chiasm and the establishment of the topographic retinocollicular map. We herein demonstrated that protein tyrosine phosphatase receptor type J (PTPRJ) regulated retinal axonal projections by controlling Eph activities. The retinas of Ptprj-knock-out (KO) and Ptpro/Ptprj double-KO mice exhibited significantly enhanced Eph activities over those in wild-type mice, and their axons showed defects in pathfinding at the chiasm and retinocollicular topographic map formation. We also revealed that c-Abl (Abelson tyrosine kinase) downstream of Eph receptors was regulated by PTPRJ. These results indicate that the regulation of the ephrin-Eph-c-Abl axis by PTPRJ plays pivotal roles in the proper central projection of retinal axons during development.

The stepwise development of the lamprey visual system and its evolutionary implications.

Lampreys, which represent the oldest group of living vertebrates (cyclostomes), show unique eye development. The lamprey larva has only eyespot-like immature eyes beneath a non-transparent skin, whereas after metamorphosis, the adult has well-developed image-forming camera eyes. To establish a functional visual system, well-organised visual centres as well as motor components (e.g. trunk muscles for locomotion) and interactions between them are needed. Here we review the available knowledge concerning the structure, function and development of the different parts of the lamprey visual system. The lamprey exhibits stepwise development of the visual system during its life cycle. In prolarvae and early larvae, the 'primary' retina does not have horizontal and amacrine cells, but does have photoreceptors, bipolar cells and ganglion cells. At this stage, the optic nerve projects mostly to the pretectum, where the dendrites of neurons in the nucleus of the medial longitudinal fasciculus (nMLF) appear to receive direct visual information and send motor outputs to the neck and trunk muscles. This simple neural circuit may generate negative phototaxis. Through the larval period, the lateral region of the retina grows again to form the 'secondary' retina and the topographic retinotectal projection of the optic nerve is formed, and at the same time, the extra-ocular muscles progressively develop. During metamorphosis, horizontal and amacrine cells differentiate for the first time, and the optic tectum expands and becomes laminated. The adult lamprey then has a sophisticated visual system for image-forming and visual decision-making. In the adult lamprey, the thalamic pathway (retina-thalamus-cortex/pallium) also transmits visual stimuli. Because the primary, simple light-detecting circuit in larval lamprey shares functional and developmental similarities with that of protochordates (amphioxus and tunicates), the visual development of the lamprey provides information regarding the evolutionary transition of the vertebrate visual system from the protochordate-type to the vertebrate-type.

ERP responses greater for faces in the temporal compared to the nasal visual field.

The distribution of retino-tectal projections is dissimilar depending on whether the receptors are situated in the nasal and temporal visual hemiretinas. Indeed, it has been claimed that the superior colliculus receives a greater proportion of its input from the temporal visual hemifield (nasal hemi-retina) relative to the nasal hemifield (temporal hemi-retina). In order to investigate whether these subcortical projections influence face processing, we investigated the early cortical ERP responses to faces and houses presented in the temporal and nasal retinas using monocular viewing. Neutral or fearful faces were presented concurrently with houses on either side of a central fixation cross, while participants were asked to discriminate changes in luminance at the center. Results showed that the lateralized N170, computed as the contralateral-ipsilateral electrode difference, was greater for faces appearing in the nasal relative to the temporal visual hemifield. This was due to a greater ipsilateral N170 for temporal relative to nasal presentations. By contrast, no difference was found across emotional expressions. The enhanced ERP response to faces appearing in the temporal visual field, suggests that the retinotectal pathway modulates cortical processing, most likely through activation of a colliculo-pulvino-amygdalar pathway, with subsequent back-projections from the amygdala to visual cortical regions. However, unattended facial expressions do not seem to modulate the response, at least at these angles of eccentricity.

Ephrin-A/EphA specific co-adaptation as a novel mechanism in topographic axon guidance.

Genetic hardwiring during brain development provides computational architectures for innate neuronal processing. Thus, the paradigmatic chick retinotectal projection, due to its neighborhood preserving, topographic organization, establishes millions of parallel channels for incremental visual field analysis. Retinal axons receive targeting information from quantitative guidance cue gradients. Surprisingly, novel adaptation assays demonstrate that retinal growth cones robustly adapt towards ephrin-A/EphA forward and reverse signals, which provide the major mapping cues. Computational modeling suggests that topographic accuracy and adaptability, though seemingly incompatible, could be reconciled by a novel mechanism of coupled adaptation of signaling channels. Experimentally, we find such 'co-adaptation' in retinal growth cones specifically for ephrin-A/EphA signaling. Co-adaptation involves trafficking of unliganded sensors between the surface membrane and recycling endosomes, and is presumably triggered by changes in the lipid composition of membrane microdomains. We propose that co-adaptative desensitization eventually relies on guidance sensor translocation into cis-signaling endosomes to outbalance repulsive trans-signaling.

Synchrotron microCT imaging of soft tissue in juvenile zebrafish reveals retinotectal projections.

Biomedical research and clinical diagnosis would benefit greatly from full volume determinations of anatomical phenotype. Comprehensive tools for morphological phenotyping are central for the emerging field of phenomics, which requires high-throughput, systematic, accurate, and reproducible data collection from organisms affected by genetic, disease, or environmental variables. Theoretically, complete anatomical phenotyping requires the assessment of every cell type in the whole organism, but this ideal is presently untenable due to the lack of an unbiased 3D imaging method that allows histopathological assessment of any cell type despite optical opacity. Histopathology, the current clinical standard for diagnostic phenotyping, involves the microscopic study of tissue sections to assess qualitative aspects of tissue architecture, disease mechanisms, and physiological state. However, quantitative features of tissue architecture such as cellular composition and cell counting in tissue volumes can only be approximated due to characteristics of tissue sectioning, including incomplete sampling and the constraints of 2D imaging of 5 micron thick tissue slabs. We have used a small, vertebrate organism, the zebrafish, to test the potential of microCT for systematic macroscopic and microscopic morphological phenotyping. While cell resolution is routinely achieved using methods such as light sheet fluorescence microscopy and optical tomography, these methods do not provide the pancellular perspective characteristic of histology, and are constrained by the limited penetration of visible light through pigmented and opaque specimens, as characterizes zebrafish juveniles. Here, we provide an example of neuroanatomy that can be studied by microCT of stained soft tissue at 1.43 micron isotropic voxel resolution. We conclude that synchrotron microCT is a form of 3D imaging that may potentially be adopted towards more reproducible, large-scale, morphological phenotyping of optically opaque tissues. Further development of soft tissue microCT, visualization and quantitative tools will enhance its utility.

Functional validation of ABHD12 mutations in the neurodegenerative disease PHARC

ABHD12 mutations have been linked to neurodegenerative PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract), a rare, progressive, autosomal, recessive disease. Although ABHD12 is suspected to play a role in the lysophosphatidylserine and/or endocannabinoid pathways, its precise functional role(s) leading to PHARC disease had not previously been characterized. Cell and zebrafish models were designed to demonstrate the causal link between an identified new missense mutation p.T253R, characterized in ABHD12 from a young patient, the previously characterized p.T202I and p.R352* mutations, and the associated PHARC. Measuring ABHD12 monoacylglycerol lipase activity in transfected HEK293 cells demonstrated inhibition with mutated isoforms. Both the expression pattern of zebrafish abhd12 and the phenotype of specific antisense morpholino oligonucleotide gene knockdown morphants were consistent with human PHARC hallmarks. High abhd12 transcript levels were found in the optic tectum and tract, colocalized with myelin basic protein, and in the spinal cord. Morphants have myelination defects and concomitant functional deficits, characterized by progressive ataxia and motor skill impairment. A disruption of retina architecture and retinotectal projections was observed, together with an inhibition of lens clarification and a low number of mechanosensory hair cells in the inner ear and lateral line system. The severe phenotypes in abhd12 knockdown morphants were rescued by introducing wild-type human ABHD12 mRNA, but not by mutation-harboring mRNAs. Zebrafish may provide a suitable vertebrate model for ABHD12 insufficiency and the study of functional impairment and potential therapeutic rescue of this rare, neurodegenerative disease.

Rostrocaudal polarity formation of chick optic tectum.

The optic tectum receives retinal fibers in a topographically ordered manner. For the formation of the precise connections, the tectum is believed to be positionally specified by gradients of molecules along axes. Rostrocaudal polarity of the tectum is first detectable at embryonic day 2 (E2) in the chick, by the caudorostral gradient of en expression, then by the rostrocaudal gradient of cytoarchitectonic development. Tectum rotation experiments showed that tectum rostrocaudal polarity is not determined at around 10-somite stage, but is fixed on E3. Ectopic tectum was produced in the diencephalon by transplanting the mesencephalic alar plate heterotopically. In the ectopic tectum, en expression was weakest at the caudal (nearest to the host diencephalo-mesencephalon junction) and strongest at the rostral end. Consequently, the pattern of en expression in the host and ectopic tecta was nearly a mirror image. Retinal fibers projected to the ectopic tectum in a topographic order in accordance with the inverted gradient of the en expression pattern. Ectopic tecta was also produced by heterochronal transplantations between E3 host and E2 donor, where the en pattern was preserved. Retinotectal projection pattern was also preserved, suggesting that en expression patterns are followed by retinotopic order with regard to rostrocaudal polarity.

Dynamic Axonal Translation in Developing and Mature Visual Circuits

SummaryLocal mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.

A family of photoswitchable NMDA receptors.

NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity.