Modular interneuron circuits control motion sensitivity in the mouse retina.

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.

A sign-inverted receptive field of inhibitory interneurons provides a pathway for ON-OFF interactions in the retina.

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.

Intersectional mapping of multi-transmitter neurons and other cell types in the brain.

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.

Upper motor neurons are a target for gene therapy and UCHL1 is necessary and sufficient to improve cellular integrity of diseased upper motor neurons.

There are no effective cures for upper motor neuron (UMN) diseases, such as amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, and hereditary spastic paraplegia. Here, we show UMN loss occurs independent of spinal motor neuron degeneration and that UMNs are indeed effective cellular targets for gene therapy, which offers a potential solution especially for UMN disease patients. UCHL1 (ubiquitin C-terminal hydrolase-L1) is a deubiquitinating enzyme crucial for maintaining free ubiquitin levels. Corticospinal motor neurons (CSMN, a.k.a UMNs in mice) show early, selective, and profound degeneration in Uchl1nm3419 (UCHL1-/-) mice, which lack all UCHL1 function. When UCHL1 activity is ablated only from spinal motor neurons, CSMN remained intact. However, restoring UCHL1 specifically in CSMN of UCHL1-/- mice via directed gene delivery was sufficient to improve CSMN integrity to the healthy control levels. In addition, when UCHL1 gene was delivered selectively to CSMN that are diseased due to misfolded SOD1 toxicity and TDP-43 pathology via AAV-mediated retrograde transduction, the disease causing misfolded SOD1 and mutant human TDP-43 were reduced in hSOD1G93A and prpTDP-43A315T models, respectively. Diseased CSMN retained their neuronal integrity and cytoarchitectural stability in two different mouse models that represent two distinct causes of neurodegeneration in ALS.

Protocol to map multi-transmitter neurons in the mouse brain using intersectional strategy.

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.

Genetic disruption of Grm5 causes complex alterations in motor activity, anxiety and social behaviors.

Autism is a neurodevelopmental disorder characterized by impaired social interactions and restricted and repetitive behaviors. Although group 1 metabotropic glutamate receptors (mGluRs), and in particular mGluR5, have been extensively proposed as potential targets for intervention in autism and other neurodevelopmental disorders, there has not been a comprehensive analysis of the effect of mGluR5 loss on behaviors typically assessed in autism mouse models thought to be correlates of behavioral symptoms of human disorders. Here we present a behavioral characterization of mice with complete or partial loss of mGluR5 (homozygous or heterozygous null mutations in Grm5 gene). We tested several autism related behaviors including social interaction, repetitive grooming, digging and locomotor behaviors. We found that digging and marble burying behaviors were almost completely abolished in mGluR5 ko mice, although self-grooming was not altered. Social interaction was impaired in ko but not in heterozygote (het) mice. In tests of locomotor activity and anxiety related behaviors, mGluR5 ko mice exhibited hyperactivity and reduced anxiety in the open field test but unexpectedly, showed hypoactivity in the elevated zero-maze test. There was no impairment in motor learning in the accelerating rotarod in both ko and het mutant. Together these results provide support for the importance of mGluR5 in motor and social behaviors that are specifically affected in autism disorders.

Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes.

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 and time-consuming. Here, we report a method to titer AAV with GelGreen® dye, a safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred to as GelGreen method, provides a fast (∼30 min) and reliable strategy for AAV titration. To validate GelGreen method, we measured genome titer of an AAV reference material AAV8RSM and compared our titration results with those determined by Reference Material Working Group (ARMWG). We showed that GelGreen results and capsid enzyme-linked immunosorbent assay results are comparable with each other. We also showed that GelRed® dye, a red fluorescence dye from Biotium, can be used to directly "visualize" AAV genome titer on a conventional gel imager, presenting an especially direct approach to estimate viral quantity. Finally, we showed that GelGreen and GelRed dyes can also be used to quantify self-complementary AAV (scAAV) and crudely purified AAV samples. In summary, we described a technique to titer AAV by using new generation of safe DNA dyes. This technique is simple, safe, reliable, and cost efficient. It has potential to be broadly applied for quantifying and normalizing AAV viral vectors.

Intersectional Strategies for Targeting Amacrine and Ganglion Cell Types in the Mouse Retina.

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.

A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid.

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.

Complete Disruption of the Kainate Receptor Gene Family Results in Corticostriatal Dysfunction in Mice.

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.

An Amacrine Cell Circuit for Signaling Steady Illumination in the Retina.

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.

Super-resolution two-photon microscopy via scanning patterned illumination.

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.

Hippocampal metaplasticity is required for the formation of temporal associative memories.

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 CA1 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.

Genetically targeted binary labeling of retinal neurons.

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.

Potentiating mGluR5 function with a positive allosteric modulator enhances adaptive learning.

Metabotropic glutamate receptor 5 (mGluR5) plays important roles in modulating neural activity and plasticity and has been associated with several neuropathological disorders. Previous work has shown that genetic ablation or pharmacological inhibition of mGluR5 disrupts fear extinction and spatial reversal learning, suggesting that mGluR5 signaling is required for different forms of adaptive learning. Here, we tested whether ADX47273, a selective positive allosteric modulator (PAM) of mGluR5, can enhance adaptive learning in mice. We found that systemic administration of the ADX47273 enhanced reversal learning in the Morris Water Maze, an adaptive task. In addition, we found that ADX47273 had no effect on single-session and multi-session extinction, but administration of ADX47273 after a single retrieval trial enhanced subsequent fear extinction learning. Together these results demonstrate a role for mGluR5 signaling in adaptive learning, and suggest that mGluR5 PAMs represent a viable strategy for treatment of maladaptive learning and for improving behavioral flexibility.

Organizational motifs for ground squirrel cone bipolar cells.

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 to morphology, immunocytochemistry, and function. The axons of the bipolar cell types stratify at different levels in the inner plexiform layer (IPL) and can interact with costratifying amacrine and ganglion cells. These interactions endow the ganglion cell types with unique functional properties. The wiring that underlies the interactions among bipolar, amacrine, and ganglion cells is poorly understood. It may be easier to elucidate this wiring if organizational rules can be established. We identify 13 types of cone bipolar cells in the ground squirrel, 11 of which contact contiguous cones, with the possible exception of short-wavelength-sensitive cones. Cells were identified by antibody labeling, tracer filling, and Golgi-like filling following transduction with an adeno-associated virus encoding for green fluorescent protein. The 11 bipolar cell types displayed two organizational patterns. In the first pattern, eight to 10 of the 11 types came in pairs with partially overlapping axonal stratification. Pairs shared morphological, immunocytochemical, and functional properties. The existence of similar pairs is a new motif that might have implications for how signals first diverge from a cone to bipolar cells and then reconverge onto a costratifying ganglion cell. The second pattern is a mirror symmetric organization about the middle of the IPL involving at least seven bipolar cell types. This anatomical symmetry may be associated with a functional symmetry in On and Off ganglion cell responses.

Synaptic vesicle exocytosis-endocytosis at central synapses: Fine-tuning at differential patterns of neuronal activity.

At nerve terminals, neurotransmitter release is mediated by exocytosis of synaptic vesicles at active zone. After exocytosis, vesicular components are efficiently retrieved by endocytosis. Tight coupling between synaptic vesicle exocytosis and endocytosis is critical for the maintenance of neurotransmission at central synapses. Recently, we have developed a new fluorescent pH reporter that permits us to examine exocytosis-endocytosis coupling at the level of individual synaptic vesicles at hippocampal synapses.1 To our surprise, we observed that the tight coupling of exocytosis and endocytosis broke down at very low stimulation frequencies, resulting in the generation of two endocytic vesicles per single exocytic fusion event. As stimulation frequency increased, exocytosis-endocytic coupling was restored with one endocytic vesicle generated for each vesicle exocytosed. Further studies revealed that the dissimilar patterns of exocytosis-endocytic coupling at different stimulation frequencies were mediated by two pathways of endocytosis that are orchestrated during differential patterns of neuronal activity.1 Here, we summarize our observations and further discuss their possible implications.

mGluR5 has a critical role in inhibitory learning.

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, we investigated the role of mGluR5 in the acquisition and reversal of an associative conditioned task and a spatial reference task. We found 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.

Two pathways of synaptic vesicle retrieval revealed by single-vesicle imaging.

Synaptic vesicle recycling is essential for maintaining efficient synaptic transmission. Detailed dissection of single-vesicle recycling still remains a major challenge. We have developed a fluorescent pH reporter that permits us to follow the fate of individual vesicles at hippocampal synapses after exocytosis. Here we show that, during low-frequency stimulation, single-vesicle fusion leads to two distinct vesicle internalizations, instead of one, as in general perception: one by a fast endocytosis pathway ( approximately 3 s), the other by a slow endocytosis pathway (after 10 s). The exocytosed vesicular proteins are preferentially recaptured in both pathways. RNAi knockdown of clathrin inhibits both pathways. As stimulation frequency increases, the number of endocytosed vesicles begins to match antecedent exocytosis. Meanwhile, the slow endocytosis is accelerated and becomes the predominant pathway. These results reveal that two pathways of endocytosis are orchestrated during neuronal activity, establishing a highly efficient endocytosis at central synapses.

Probing synaptic vesicle fusion by altering mechanical properties of the neuronal surface membrane.

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 approximately 3 times out of 4 and that the area of the fusion pore is approximately 4 nm(2).