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Abstract Neural computations arise from highly precise connections between specific types of neurons. Retinal ganglion cells (RGCs) with similar stratification patterns are positioned to receive similar inputs but often display different response properties. In this study, we used intersectional mouse genetics to achieve single-cell type labeling and identified an object motion sensitive (OMS) AC type, COMS-AC(counter-OMS AC). Optogenetic stimulation revealed that COMS-AC makes glycinergic synapses with the OMS-insensitive HD2p-RGC, while chemogenetic inactivation showed that COMS-AC provides inhibitory control to HD2p-RGC during local motion. This local inhibition, combined with the inhibitory drive from TH2-AC during global motion, explains the OMS-insensitive feature of HD2p-RGC. In contrast, COMS-AC fails to make synapses with W3(UHD)-RGC, allowing it to exhibit OMS under the control of VGlut3-AC and TH2-AC. These findings reveal modular interneuron circuits that endow structurally similar RGC types with different responses and present a mechanism for redundancy-reduction in the retina to expand coding capacity.

Abstract A fundamental organizing plan of the retina is that visual information is divided into ON and OFF streams that are processed in separate layers. This functional dichotomy originates in the ON and OFF bipolar cells, which then make excitatory glutamatergic synapses onto amacrine and ganglion cells in the inner plexiform layer. We have identified an amacrine cell (AC), the sign-inverting (SI) AC, that challenges this fundamental plan. The glycinergic, ON-stratifying SI-AC has OFF light responses. In opposition to the classical wiring diagrams, it receives inhibitory inputs from glutamatergic ON bipolar cells at mGluR8 synapses, and excitatory inputs from an OFF wide-field AC at electrical synapses. This “inhibitory ON center - excitatory OFF surround” receptive-field of the SI-AC allows it to use monostratified dendrites to conduct crossover inhibition and push-pull activation to enhance light detection by ACs and RGCs in the dark and feature discrimination in the light.

Summary: Although it is now known that certain neurons can produce, store, and release multiple neurotransmitters, their locations, abundance, and functions remain elusive. We developed intersectional genetic strategies to identify multi-transmitter neurons based on the expression of neurotransmitter-specific genes. Here we present our procedures for whole-brain mapping of GABA/glutamate co-releasing neurons. We also detail our technique for labeling GABA/glutamate neurons in specific brain regions with adeno-associated virus (AAV). Our protocol can be readily extended to other types of multi-transmitter neurons.For complete details on the use and execution of this protocol, please refer to Xu et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: 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 intersectional genetics, we perform a brain-wide characterization of 13 Flp and tTA mouse driver lines and selected seven for further analysis based on expression of vesicular neurotransmitter transporters. Using selective Cre driver lines, we created more than 10 Cre/tTA combinational lines for cell type targeting and circuit analysis. We then used VGLUT-Cre/VGAT-Flp combinational lines to identify and map 30 brain regions containing neurons that co-express vesicular glutamate and gamma-aminobutyric acid (GABA) transporters, followed by tracing their projections with intersectional viral vectors. Focusing on the lateral habenula (LHb) as a target, we identified glutamatergic, GABAergic, or co-glutamatergic/GABAergic innervations from ∼40 brain regions. These data provide an important resource for the future application of intersectional strategies and expand our understanding of the neuronal subtypes in the brain.

Abstract Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants.

Kainate receptors are members of the glutamate receptor family that regulate synaptic function in the brain. They modulate synaptic transmission and the excitability of neurons; however, their contributions to neural circuits that underlie behavior are unclear. To understand the net impact of kainate receptor signaling, we generated knockout mice in which all five kainate receptor subunits were ablated (5ko). These mice displayed compulsive and perseverative behaviors, including over-grooming, as well as motor problems, indicative of alterations in striatal circuits. There were deficits in corticostriatal input to spiny projection neurons (SPNs) in the dorsal striatum and correlated reductions in spine density. The behavioral alterations were not present in mice only lacking the primary receptor subunit expressed in adult striatum (GluK2 KO), suggesting that signaling through multiple receptor types is required for proper striatal function. This demonstrates that alterations in striatal function dominate the behavioral phenotype in mice without kainate receptors.

Decades of research have focused on the circuit connectivity between retinal neurons, but only a handful of amacrine cells have been described functionally and placed in the context of a specific retinal circuit. Here, we identify a circuit where inhibition from a specific amacrine cell plays a vital role in shaping the feature selectivity of a postsynaptic ganglion cell. We record from transgenically labeled CRH-1 amacrine cells and identify a postsynaptic target for CRH-1 amacrine cell inhibition in an atypical retinal ganglion cell (RGC) in mouse retina, the Suppressed-by-Contrast (SbC) RGC. Unlike other RGC types, SbC RGCs spike tonically in steady illumination and are suppressed by both increases and decreases in illumination. Inhibition from GABAergic CRH-1 amacrine cells shapes this unique contrast response profile to positive contrast. We show the existence and impact of this circuit, with both paired recordings and cell-type-specific ablation.

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, Cre expression in a given transgenic line in the retina and elsewhere in the brain is rarely confined to a single cell type, contributing ambiguity to the interpretation of results from broadly applied manipulations. To obtain unambiguous information about retinal processing, it is desirable to have strategies for further restricting transgene expression to a few or even to a single cell type. We employed an intersectional strategy based on a Cre/Flp double recombinase system to target amacrine and ganglion cell types in the inner retina. We analyzed expression patterns in seven Flp drivers and then created combinational mouse lines by selective cross breeding with Cre drivers. Breeding with Flp drivers can routinely remove labeling from more than 90% of the cells in Cre drivers, leading to only a handful cell types, typically 2–3, remaining in the intersection. Cre/Flp combinatorial mouse lines enabled us to identify and anatomically characterize retinal cell types with greater ease and demonstrated the feasibility of intersectional strategies in retinal research. In addition to the retina, we examined Flp expression in the lateral geniculate nucleus and superior colliculus. Our results establish a foundation for future application of intersectional strategies in the retina and retino-recipient regions.

The single-channel kinetics of extracellular [Mg.sup.2+] block was used to probe [K.sup.+] binding sites in the permeation pathway of rat recombinant NR1/NR2B NMDA receptor channels. [K.sup.+] binds to three sites: two that are external and one that is internal to the site of [Mg.sup.2+] block. The internal site is ~0.84 through the electric field from the extracellular surface. The equilibrium dissociation constant for this site for [K.sup.+] is 304 mM at 0 mV and with [Mg.sup.2+] in the pore. The occupancy of any one of the three sites by K+ effectively prevents the association of extracellular [Mg.sup.2+]. Occupancy of the internal site also prevents [Mg.sup.2+] permeation and increases (by approximately sevenfold) the rate constant for [Mg.sup.2+] dissociation back to the extracellular solution. Under physiological intracellular ionic conditions and at -60 mV, there is ~l,400-fold apparent decrease in the affinity of the channel for extracellular [Mg.sup.2+] and ~2-fold enhancement of the apparent voltage dependence of [Mg.sup.2+] block caused by the voltage dependence of [K.sup.+] occupancy of the external and internal sites. KEY WORDS: ion binding sites * magnesium * channel block * permeation * selectivity

The effect of extracellular and intracellular [Na.sup.+] on the single-channel kinetics of [Mg.sup.2+] block was studied in recombinant NR1-NR2B NMDA receptor channels. [Na.sup.+] prevents [Mg.sup.2+] access to its blocking site by occupying two sites in the external portion of the permeation pathway. The occupancy of these sites by intracellular, but not extracellular, [Na.sup.+] is voltage-dependent. In the absence of competing ions, [Mg.sup.2+] binds rapidly ([is greater than] [10.sup.8] [M.sup.-1][s.sup.-1], with no membrane potential) to a site that is located 0.60 through the electric field from the extracellular surface. Occupancy of one of the external sites by [Na.sup.+] may be sufficient to prevent [Mg.sup.2+] dissociation from the channel back to the extracellular compartment. With no membrane potential; and in the absence of competing ions, the [Mg.sup.2+] dissociation rate constant is [is greater than] 10 times greater than the [Mg.sup.2+] permeation rate constant, and the [Mg.sup.2+] equilibrium dissociation constant is ~12 [micro]M. Physiological concentrations of extracellular [Na.sup.+] reduce the [Mg.sup.2+] association rate constant ~40-fold but, because of the 'lock-in' effect, reduce the [Mg.sup.2+] equilibrium dissociation constant only ~18-fold. KEY WORDS: ion binding sites magnesium * channel blockade * permeation selectivity

Metaplasticity regulates the threshold for modification of synaptic strength and is an important regulator of learning rules; however, it is not known whether these cellular mechanisms for homeostatic regulation of synapses contribute to particular forms of learning. Conditional ablation of mGluR5 in CAI pyramidal neurons resulted in the inability of low-frequency trains of afferent activation to prime synapses for subsequent theta burst potentiation. Priming-induced metaplasticity requires mGluR5-mediated mobilization of endocannabinoids during the priming train to induce long-term depression of inhibition (I-LTD). Mice lacking priming-induced plasticity had no deficit in spatial reference memory tasks, but were impaired in an associative task with a temporal component. Conversely, enhancing endocannabinoid signaling facilitated temporal associative memory acquisition and, after training animals in these tasks, ex vivo I-LTD was partially occluded and theta burst LTP was enhanced. Together, these results suggest a link between metaplasticity mechanisms in the hippocampus and the formation of temporal associative memories. [ABSTRACT FROM AUTHOR]

A major stumbling block to understanding neural circuits is the extreme anatomical and functional diversity of interneurons. Subsets of interneurons can be targeted for manipulation using Cre mouse lines, but Cre expression is rarely confined to a single interneuron type. It is essential to have a strategy that further restricts labeling in Cre driver lines. We now describe an approach that combines Cre driver mice, recombinant adeno-associated virus, and rabies virus to produce sparse but binary labeling of select interneurons--frequently only a single cell in a large region. We used this approach to characterize the retinal amacrine and ganglion cell types in five GABAergic Cre mouse (Mus musculus) lines, and identified two new amacrine cell types: an asymmetric medium-field type and a wide-field type. We also labeled several wide-field amacrine cell types that have been previously identified based on morphology but whose connectivity and function had not been systematically studied due to lack of genetic markers. All Cre-expressing amacrine cells labeled with an antibody to GABA. Cre-expressing RGCs lacked GABA labeling and included classically defined as well as recently identified types. In addition to the retina, our technique leads to sparse labeling of neurons in the cortex, lateral geniculate nucleus, and superior colliculus, and can be used to express optogenetic tools such as channelrhodopsin and protein sensors such as GCaMP. The Cre drivers identified in this study provide genetic access to otherwise hard to access cell types for systematic analysis including anatomical characterization, physiological recording, optogenetic and/or chemical manipulation, and circuit mapping. [ABSTRACT FROM AUTHOR]

The mechanisms that contribute to the extinction of previously acquired memories are not well understood. These processes, often referred to as inhibitory learning, are thought to be parallel learning mechanisms that require a reacquisition of new information and suppression of previously acquired experiences in order to adapt to novel situations. Using newly generated metabotropic glutamate receptor 5 (mGluR5) knock-out mice,weinvestigated the role ofmGluR5in the acquisition and reversal of an associative conditioned task and a spatial reference task.Wefound that acquisition of fear conditioning is partially impaired in mice lacking mGluR5. More markedly, we found that extinction of both contextual and auditory fear was completely abolished in mGluR5 knock-out mice. In the Morris Water Maze test (MWM), mGluR5 knock-out mice exhibited mild deficits in the rate of acquisition of the regular water maze task, but again had significant deficits in the reversal task, despite overall spatial memory being intact. Together, these results demonstrate that mGluR5 is critical to the function of neural circuits that are required for inhibitory learning mechanisms, and suggest that targeting metabotropic receptors may be useful in treating psychiatric disorders in which aversive memories are inappropriately retained. [ABSTRACT FROM AUTHOR]

Because synaptic vesicle exocytosis is a nano-mechanical process. it should be influenced by the mechanical properties of the cell membrane to which the vesicle fuses. By dissolving surfactants at various concentrations in the neuronal membrane, we have perturbed mechanical properties of the membrane and have found that dissolved surfactants lower the probability that a synaptic vesicle will open its fusion pore when the fusion machinery of the vesicle is activated by binding calcium. By using standard theories from the physics and chemistry of surfaces, we can account for this decrease in fusion probability and can infer that a vesicle, when activated, opens its fusion pore ∼3 times out of 4 and that the area of the fusion pore is ∼4 nm². [ABSTRACT FROM AUTHOR]

Genetically encoded sensors of glutamate concentration are based on FRET between cyan and yellow fluorescent proteins bracketing a bacterial glutamate-binding protein. Such sensors have yet to find quantitative applications in neurons, because of poor response amplitude in physiological buffers or when expressed on the neuronal cell surface. We have improved our glutamate-sensing fluorescent reporter (GIuSnFR) by systematic optimization of linker sequences and glutamate affinities. Using SuperGluSnFR. which exhibits a 6.2-fold increase in response magnitude over the original GIuSnFR. we demonstrate quantitative optical measurements of the time course of synaptic glutamate release, spillover, and reuptake in cultured hippocampal neurons with centisecond temporal and spine-sized spatial resolution. During burst firing, functionally significant spillover persists for hundreds of milliseconds. These glutamate levels appear sufficient to prime NMDA receptors, potentially affecting dendritic spike initiation and computation. Stimulation frequency-dependent modulation of spillover suggests a mechanism for nonsynaptic neuronal communication. [ABSTRACT FROM AUTHOR]

Intracellular trafficking of ionotropic glutamate receptors is regulated predominantly by determinants in the cytoplasmic C-terminal domain of the subunit proteins. Although AMPA receptors are found at the vast majority of excitatory synapses, synaptic kainate receptors exhibit a much more restricted distribution, suggesting that specific mechanisms exist for selective trafficking of these receptor proteins. In this report, we define a critical forward trafficking motif that is necessary for surface expression of the glutamate receptor 6 (GluR6) kainate receptor as well as chimeric proteins containing only the GluR6 C-terminal domain. The trafficking determinant was identified by tracking surface expression of green fluorescent protein-tagged GluR6 receptors with confocal immunofluorescence in COS-7 cells and cultured neurons and patch-clamp electrophysiology in human embryonic kidney 293 cells. Serial truncation and alanine site mutagenesis of the GluR6 subunit C terminus localized the critical motif to a seven amino acid stretch of predominantly basic residues. Alanine mutation of the trafficking motif reduced kainate receptor current amplitudes by >90% and resulted in retention of the mutated receptors in the endoplasmic reticulum. This forward trafficking domain is the first such identified for kainate receptors. [ABSTRACT FROM AUTHOR]

Investigates the potassium (K)-binding sites in the permeation pathway of rat recombinant NR1/NR2B NMDA receptor channels through the use of single-channel kinetics of extracellular magnesium (Mg) block. Effects of intracellular K on the apparent rates of Mg dissociation and permeation; Equilibrium dissociation constant and rate constants for Mg dissociation and permeation; Qualitative assessment of the affinity and selectivity of the internal site.

Investigates the effect of extracellular and intracellular sodium (Na) on the single-channel kinetics of magnesium (Mg) block in recombinant NR1-NR2B NMDA receptor channels. Association rate constant of Mg; Locations and apparent affinities of the Na-binding sites; Dissociation rate constant of Mg.

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 of neuronal nAChRs has important implications for function and is not well understood. Here, we analyzed the targeting of two major types of neuronal nAChRs by expressing epitope-tagged subunits in cultured hippocampal neurons. Surprisingly, the α7 nAChR (α7) and α4/β2 nAChR (α4β2) displayed distinct patterns of expression, with α7 targeted preferentially to the somatodendritic compartments, whereas α4β2 was localized to both axonal and dendritic domains. When fused to CD4 or IL2RA (interleukin 2 receptor α subunit) proteins, which are normally distributed ubiquitously, the M3-M4 intracellular loop from the α7 subunit promoted dendritic expression, whereas the homologous M3-M4 loop from the α4 subunit led to surface axonal expression. Systemic screening and alanine substitution further identified a 25-residue leucine motif ([DE]XXXL[LI]) containing an axonal targeting sequence within the α4 loop and a 48-residue dileucine and tyrosine motif (YXXØ) containing a dendritic targeting sequence from the α7 loop. These results provide valuable information in understanding diverse roles of neuronal nAChRs in mediating and modulating synaptic transmission, synaptic plasticity, and nicotine addiction. [ABSTRACT FROM AUTHOR]

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-bindlng residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/ KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafticking of intracellular kainate receptors following multimeric assembly. [ABSTRACT FROM AUTHOR]

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

Objective: To investigate the efficacy and safety of febuxostat on renal function in CKD stage 3 diabetic nephropathy patients., Methods: Patients in our hospital with chronic kidney disease (CKD) stage 3 diabetic nephropathy (DN) complicated by high serum uric acid (360 µmol/L) were recruited. Patients were then divided into treatment group and control group according to the random number table method. All the patients received low purine diet, renin-angiotensin-aldosterone system (RAAS) inhibitors, and adequate routine hypoglycemic treatment. Febuxostat was employed only in the treatment group. The levels of blood uric acid (sUA), serum creatinine (Scr), cystatin C (cys-c), eGFR, 24-hour urine protein quantification, albuminuria, and creatinine ratio (ACR) were evaluated in all patients before and after treatment at 4, 8, 12, and 24 week., Results: No difference was found before treatment between the two groups. After treatment at 4, 8, 12, and 24 week, the levels of sUA, SCr, cys-c, and eGFR between the two groups were significant different (P<0.05). There was no difference in 24-hour urine protein quantification, albuminuria, and creatinine ratio between two groups before treatment, and significant differences were observed after treatment. Fifty percent of patients from the treatment group achieved the treatment goal with 20 mg febuxostat at 4 weeks. Tubular markers were also decreased with the treatment., Conclusions: Febuxostat can reduce uric acid and improve renal function effectively in patients with CKD stage 3 diabetic nephropathy, while being well tolerated. However, the conclusion is still uncertain due to the short term of the study.