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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.

Summary 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.

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.

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 new 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 (~3s), the other by a slow endocytosis pathway (after 10s). The exocytosed vesicular proteins are preferentially recaptured in both pathways. RNAi knocking down 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, enabling the highly efficient endocytosis machinery at central synapses.

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-binding 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 (KA2-R/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 trafficking of intracellular kainate receptors following multimeric assembly.

When hippocampal synapses in culture are pharmacologically silenced for several days, synaptic strength increases. The structural correlate of this change in strength is an increase in the size of the synapses, with all synaptic components—active zone, postsynaptic density, and bouton—becoming larger. Further, the number of docked vesicles and the total number of vesicles per synapse increases, although the number of docked vesicles per area of active zone is unchanged. In parallel with these anatomical changes, the physiologically measured size of the readily releasable pool (RRP) and the release probability are increased. Ultrastructural analysis of individual synapses in which the RRP was previously measured reveals that, within measurement error, the same number of vesicles are docked as are estimated to be in the RRP.

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 nm 2 .