Genetically encoded sensors of glutamate concentration are based on FRET between cyan and yellow fluorescent proteins bracketing a bacterial glutamate-binding protein. Such have yet to find quantitative applications in neurons, because poor response amplitude physiological buffers or when expressed the neuronal cell surface. We improved our glutamate-sensing reporter (GluSnFR) by systematic optimization linker sequences affinities. Using SuperGluSnFR, which exhibits 6.2-fold increase...

The mechanisms that contribute to the extinction of previously acquired memories are not well understood. These processes, often referred as inhibitory learning, thought be parallel learning require a reacquisition new information and suppression experiences in order adapt novel situations. Using newly generated metabotropic glutamate receptor 5 (mGluR5) knock-out mice, we investigated role mGluR5 acquisition reversal an associative conditioned task spatial reference task. We found fear...

Decades of research have focused on the circuit connectivity between retinal neurons, but only a handful amacrine cells been described functionally and placed in context specific circuit. Here, we identify where inhibition from cell plays vital role shaping feature selectivity postsynaptic ganglion cell. We record transgenically labeled CRH-1 target for an atypical (RGC) mouse retina, Suppressed-by-Contrast (SbC) RGC. Unlike other RGC types, SbC RGCs spike tonically steady illumination are...

Neuronal nicotinic acetylcholine receptors (nAChRs) belong to a family of ligand-gated ion channels that play important roles in central and peripheral nervous systems. The subcellular distribution neuronal nAChRs has implications for function is not well understood. Here, we analyzed the targeting two major types by expressing epitope-tagged subunits cultured hippocampal neurons. Surprisingly, α7 nAChR (α7) α4/β2 (α4β2) displayed distinct patterns expression, with targeted preferentially...

Metaplasticity regulates the threshold for modification of synaptic strength and is an important regulator learning rules; however, it not known whether these cellular mechanisms homeostatic regulation synapses contribute to particular forms learning. Conditional ablation mGluR5 in CA1 pyramidal neurons resulted inability low-frequency trains afferent activation prime subsequent theta burst potentiation. Priming-induced metaplasticity requires mGluR5-mediated mobilization endocannabinoids...

Recent developments in intersectional strategies have greatly advanced our ability to precisely target brain cell types based on unique co-expression patterns. To accelerate the application of genetics, we perform a brain-wide characterization 13 Flp and tTA mouse driver lines selected seven for further analysis expression vesicular neurotransmitter transporters. Using selective Cre lines, created more than 10 Cre/tTA combinational type targeting circuit analysis. We then used...

In daylight vision, parallel processing starts at the cone synapse. Cone signals flow to On and Off bipolar cells, which are further divided into types according morphology, immunocytochemistry, function. The axons of cell stratify different levels in inner plexiform layer (IPL) can interact with costratifying amacrine ganglion cells. These interactions endow unique functional properties. wiring that underlies among bipolar, amacrine, cells is poorly understood. It may be easier elucidate...

We developed two-photon scanning patterned illumination microscopy (2P-SPIM) for super-resolution imaging. Our approach used a traditional setup with temporally modulated excitation to create fields. Combing nine different illuminations and structured reconstruction, imaging was achieved in microscopy. Using 2P-SPIM we lateral resolution of 141 nm, which represents an improvement by factor 1.9 over the corresponding diffraction limit. further demonstrated cellular image actin cytoskeleton...

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such have been localized to the cytoplasmic carboxyl-terminal domain, but other domains subunit proteins can play roles modulating surface expression. Here we demonstrate that formation an intact binding site also acts as additional quality-control check for expression homomeric and heteromeric kainate receptors. A key ligand-binding residue KA2 subunit,...

Intracellular trafficking of ionotropic glutamate receptors is regulated predominantly by determinants in the cytoplasmic C-terminal domain subunit proteins. Although AMPA are found at vast majority excitatory synapses, synaptic kainate exhibit a much more restricted distribution, suggesting that specific mechanisms exist for selective these receptor In this report, we define critical forward motif necessary surface expression 6 (GluR6) as well chimeric proteins containing only GluR6 domain....

Metabotropic glutamate receptor 5 (mGluR5) plays important roles in modulating neural activity and plasticity has been associated with several neuropathological disorders. Previous work shown that genetic ablation or pharmacological inhibition of mGluR5 disrupts fear extinction spatial reversal learning, suggesting signaling is required for different forms adaptive learning. Here, we tested whether ADX47273, a selective positive allosteric modulator (PAM) mGluR5, can enhance learning mice....

Highlights•Mice with loss of all five kainate receptor genes have striatal-dependent phenotypes•Mutant mice display perseverative and compulsive behavior locomotor deficits•Synapses onto striatal neurons morphological functional deficitsSummaryKainate receptors are members the glutamate family that regulate synaptic function in brain. They modulate transmission excitability neurons; however, their contributions to neural circuits underlie unclear. To understand net impact signaling, we...

The mammalian retina harbors over 100 different cell types. To understand how retinal circuits work, it is essential to systematically access each type. A widely used approach for achieving targeted transgene expression exploits promoter-driven Cre lines. However, in a given transgenic line the and elsewhere brain rarely confined single type, contributing ambiguity interpretation of results from broadly applied manipulations. obtain unambiguous information about processing, desirable have...

Abstract A fundamental organizing plan of the retina is that visual information divided into ON and OFF streams are processed in separate layers. This functional dichotomy originates bipolar cells, which then make excitatory glutamatergic synapses onto amacrine ganglion cells inner plexiform layer. We have identified an cell (AC), sign-inverting (SI) AC, challenges this plan. The glycinergic, ON-stratifying SI-AC has light responses. In opposition to classical wiring diagrams, it receives...

Because synaptic vesicle exocytosis is a nano-mechanical process, it should be influenced by the mechanical properties of cell membrane to which fuses. By dissolving surfactants at various concentrations in neuronal membrane, we have perturbed and found that dissolved lower probability will open its fusion pore when machinery activated binding calcium. using standard theories from physics chemistry surfaces, can account for this decrease infer vesicle, activated, opens approximately 3 times...

Direct gene delivery to the neurons of interest, without affecting other neuron populations in cerebral cortex, represent a challenge owing heterogeneity and cellular complexity brain. Genetic modulation corticospinal motor (CSMN) is required for developing effective long-term treatment strategies diseases, which voluntary movement impaired. Adeno-associated viruses (AAV) have been widely used neuronal transduction studies stable expression as well low immunoreactivity humans. Here we report...

Adeno-associated virus (AAV) is the most commonly used viral vector for both biological and gene therapeutic applications. Although many methods have been developed to measure quantity attributes of AAV, they are often technically challenging time-consuming. Here, we report a method titer AAV with GelGreen® dye, safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred as GelGreen provides fast (∼30 min) reliable strategy...

Although it is now known that certain neurons can produce, store, and release multiple neurotransmitters, their locations, abundance, functions remain elusive. We developed intersectional genetic strategies to identify multi-transmitter based on the expression of neurotransmitter-specific genes. Here we present our procedures for whole-brain mapping GABA/glutamate co-releasing neurons. also detail technique labeling in specific brain regions with adeno-associated virus (AAV). Our protocol be...

At nerve terminals, neurotransmitter release is mediated by exocytosis of synaptic vesicles at active zone. After exocytosis, vesicular components are efficiently retrieved endocytosis. Tight coupling between vesicle and endocytosis critical for the maintenance neurotransmission central synapses. Recently, we have developed a new fluorescent pH reporter that permits us to examine exocytosis-endocytosis level individual hippocampal synapses.1 To our surprise, observed tight broke down very...