Virally based transsynaptic tracing technologies are powerful experimental tools for neuronal circuit mapping. The glycoprotein-deletion variant of the SAD-B19 vaccine strain rabies virus (RABV) has been the reagent of choice in monosynaptic tracing, since it permits the mapping of synaptic inputs to genetically marked neurons. Since its introduction, new helper viruses and reagents that facilitate complementation have enhanced the efficiency of SAD-B19(ΔG) transsynaptic transfer, but there has been little focus on improvements to the core RABV strain. Here we generate a new deletion mutant strain, CVS-N2c(ΔG), and examine its neuronal toxicity and efficiency in directing retrograde transsynaptic transfer. We find that by comparison with SAD-B19(ΔG), the CVS-N2c(ΔG) strain exhibits a reduction in neuronal toxicity and a marked enhancement in transsynaptic neuronal transfer. We conclude that the CVS-N2c(ΔG) strain provides a more effective means of mapping neuronal circuitry and of monitoring and manipulating neuronal activity in vivo in the mammalian CNS.

Pyramidal neuron dendrites integrate synaptic input from multiple partners. Different inputs converging on the same dendrite have distinct structural and functional features, but the molecular mechanisms organizing input-specific properties are poorly understood. We identify the orphan receptor GPR158 as a binding partner for the heparan sulfate proteoglycan (HSPG) glypican 4 (GPC4). GPC4 is enriched on hippocampal granule cell axons (mossy fibers), whereas postsynaptic GPR158 is restricted to the proximal segment of CA3 apical dendrites receiving mossy fiber input. GPR158-induced presynaptic differentiation in contacting axons requires cell-surface GPC4 and the co-receptor LAR. Loss of GPR158 increases mossy fiber synapse density but disrupts bouton morphology, impairs ultrastructural organization of active zone and postsynaptic density, and reduces synaptic strength of this connection, while adjacent inputs on the same dendrite are unaffected. Our work identifies an input-specific HSPG-GPR158 interaction that selectively organizes synaptic architecture and function of developing mossy fiber-CA3 synapses in the hippocampus.

Leucine-rich repeat (LRR) proteins have recently been identified as important regulators of synapse development and function, but for many LRR proteins the ligand-receptor interactions are not known. Here we identify the heparan sulfate (HS) proteoglycan glypican as a receptor for LRRTM4 using an unbiased proteomics-based approach. Glypican binds LRRTM4, but not LRRTM2, in an HS-dependent manner. Glypican 4 (GPC4) and LRRTM4 localize to the pre- and postsynaptic membranes of excitatory synapses, respectively. Consistent with a trans-synaptic interaction, LRRTM4 triggers GPC4 clustering in contacting axons and GPC4 induces clustering of LRRTM4 in contacting dendrites in an HS-dependent manner. LRRTM4 positively regulates excitatory synapse development in cultured neurons and in vivo, and the synaptogenic activity of LRRTM4 requires the presence of HS on the neuronal surface. Our results identify glypican as an LRRTM4 receptor and indicate that a trans-synaptic glypican-LRRTM4 interaction regulates excitatory synapse development.

Synaptic connectivity within adult circuits exhibits a remarkable degree of cellular and subcellular specificity. We report that the axon guidance receptor Robo2 plays a role in establishing synaptic specificity in hippocampal CA1. In vivo, Robo2 is present and required postsynaptically in CA1 pyramidal neurons (PNs) for the formation of excitatory (E) but not inhibitory (I) synapses, specifically in proximal but not distal dendritic compartments. In vitro approaches show that the synaptogenic activity of Robo2 involves a trans-synaptic interaction with presynaptic Neurexins, as well as binding to its canonical extracellular ligand Slit. In vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in awake behaving mice shows that preventing Robo2-dependent excitatory synapse formation cell autonomously during development alters place cell properties of adult CA1 PNs. Our results identify a trans-synaptic complex linking the establishment of synaptic specificity to circuit function.

Mossy cells in the hilus of the dentate gyrus constitute a major excitatory principal cell type in the mammalian hippocampus; however, it remains unknown how these cells behave in vivo. Here, we have used two-photon Ca2+ imaging to monitor the activity of mossy cells in awake, behaving mice. We find that mossy cells are significantly more active than dentate granule cells in vivo, exhibit spatial tuning during head-fixed spatial navigation, and undergo robust remapping of their spatial representations in response to contextual manipulation. Our results provide a functional characterization of mossy cells in the behaving animal and demonstrate their active participation in spatial coding and contextual representation.

Sharp-wave ripples are transient oscillatory events in the hippocampus that are associated with the reactivation of neuronal ensembles within specific circuits during memory formation. Fast-spiking, parvalbumin-expressing interneurons (FS-PV INs) are thought to provide fast integration in these oscillatory circuits by suppressing regenerative activity in their dendrites. Here, using fast 3D two-photon imaging and a caged glutamate, we challenge this classical view by demonstrating that FS-PV IN dendrites can generate propagating Ca(2+) spikes during sharp-wave ripples. The spikes originate from dendritic hot spots and are mediated dominantly by L-type Ca(2+) channels. Notably, Ca(2+) spikes were associated with intrinsically generated membrane potential oscillations. These oscillations required the activation of voltage-gated Na(+) channels, had the same frequency as the field potential oscillations associated with sharp-wave ripples, and controlled the phase of action potentials. Furthermore, our results demonstrate that the smallest functional unit that can generate ripple-frequency oscillations is a segment of a dendrite.

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.

Memory traces are believed to be ensembles of cells used to store memories. To visualize memory traces, we created a transgenic line that allows for the comparison between cells activated during encoding and expression of a memory. Mice re-exposed to a fear-inducing context froze more and had a greater percentage of reactivated cells in the dentate gyrus (DG) and CA3 than mice exposed to a novel context. Over time, these differences disappeared, in keeping with the observation that memories become generalized. Optogenetically silencing DG or CA3 cells that were recruited during encoding of a fear-inducing context prevented expression of the corresponding memory. Mice with reduced neurogenesis displayed less contextual memory and less reactivation in CA3 but, surprisingly, normal reactivation in the DG. These studies suggest that distinct memory traces are located in the DG and in CA3 but that the strength of the memory is related to reactivation in CA3.

During exploration, animals form an internal map of an environment by combining information about landmarks and the animal's movement, a process that depends on the hippocampus. The dentate gyrus (DG) is the first stage of the hippocampal circuit where self-motion ("where") and sensory cue information ("what") are integrated, but it remains unknown how DG neurons encode this information during cognitive map formation. Using two-photon calcium imaging in mice running on a treadmill along with online cue manipulation, we identify robust sensory cue responses in DG granule cells. Cue cell responses are stable, stimulus-specific, and accompanied by inhibition of nearby neurons. This demonstrates the existence of "cue cells" in addition to better characterized "place cells" in the DG. We hypothesize that the DG supports parallel channels of spatial and non-spatial information that contribute distinctly to downstream computations and affect roles of the DG in spatial navigation and episodic memory.

Viral vectors enable foreign proteins to be expressed in brains of non-genetic species, including non-human primates. However, viruses targeting specific neuron classes have proved elusive. Here we describe viral promoters and strategies for accessing GABAergic interneurons and their molecularly defined subsets in the rodent and primate. Using a set intersection approach, which relies on two co-active promoters, we can restrict heterologous protein expression to cortical and hippocampal somatostatin-positive and parvalbumin-positive interneurons. With an orthogonal set difference method, we can enrich for subclasses of neuropeptide-Y-positive GABAergic interneurons by effectively subtracting the expression pattern of one promoter from that of another. These methods harness the complexity of gene expression patterns in the brain and significantly expand the number of genetically tractable neuron classes across mammals.

Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation.

Pyramidal neurons express rich repertoires of leucine-rich repeat (LRR)-containing adhesion molecules with similar synaptogenic activity in culture. The in vivo relevance of this molecular diversity is unclear. We show that hippocampal CA1 pyramidal neurons express multiple synaptogenic LRR proteins that differentially distribute to the major excitatory inputs on their apical dendrites. At Schaffer collateral (SC) inputs, FLRT2, LRRTM1, and Slitrk1 are postsynaptically localized and differentially regulate synaptic structure and function. FLRT2 controls spine density, whereas LRRTM1 and Slitrk1 exert opposing effects on synaptic vesicle distribution at the active zone. All LRR proteins differentially affect synaptic transmission, and their combinatorial loss results in a cumulative phenotype. At temporoammonic (TA) inputs, LRRTM1 is absent; FLRT2 similarly controls functional synapse number, whereas Slitrk1 function diverges to regulate postsynaptic AMPA receptor density. Thus, LRR proteins differentially control synaptic architecture and function and act in input-specific combinations and a context-dependent manner to specify synaptic properties.

The substantia nigra pars reticulata (SNr) plays a key role in basal ganglia function. Projections from multiple basal ganglia nuclei converge at the SNr to regulate nigrothalamic output. The SNr is also characterized by abundant aminergic input, including dopaminergic dendrites and axons containing 5-hydroxytryptamine (5-HT) or histamine (HA). The functions of HA in the SNr include motor control via HA H3 receptors (H3Rs), although the mechanism remains far from elucidated. In Parkinson's disease, there is an increase in H3Rs and the density of HA-immunoreactive axons in the SN. We explored the role of H3Rs in the regulation of 5-HT release in SNr using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in rat midbrain slices. Immunohistochemistry identified a similar distribution for histaminergic and serotonergic processes in the SNr: immunoreactive varicosities were observed in the vicinity of dopaminergic dendrites. Electrically evoked 5-HT release was dependent on extracellular Ca2+ and prevented by NaV+-channel blockade. Extracellular 5-HT concentration was enhanced by inhibition of uptake transporters for 5-HT but not dopamine. Selective H3R agonists (R)-(-)-alpha-methyl-histamine or immepip inhibited evoked 5-HT release by up to 60%. This inhibition was prevented by the H3R antagonist thioperamide but not by the 5-HT1B receptor antagonist isamoltane. H3R inhibition of 5-HT release prevailed in the presence of GABA or glutamate receptor antagonists (ionotropic and metabotropic), suggesting minimal involvement of GABA or glutamate synapses. The potent regulation of 5-HT by H3Rs reported here not only elucidates HA function in the SNr but also raises the possibility of novel targets for basal ganglia therapies.

Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function.

The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and selectively localized to the axon initial segment (AIS), the subcellular compartment triggering action potentials. Ectopic expression of LRRC37B in mouse CPNs in vivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. Molecularly, LRRC37B binds to the secreted ligand FGF13A and to the voltage-gated sodium channel (Nav) β-subunit SCN1B. LRRC37B concentrates inhibitory effects of FGF13A on Nav channel function, thereby reducing excitability, specifically at the AIS level. Electrophysiological recordings in adult human cortical slices reveal lower neuronal excitability in human CPNs expressing LRRC37B. LRRC37B thus acts as a species-specific modifier of human neuron excitability, linking human genome and cell evolution, with important implications for human brain function and diseases.