A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types.

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We used transcriptomic and epigenomic cortical cell type taxonomies from mouse and human to define marker genes and putative enhancers and created a large toolkit of transgenic lines and enhancer AAVs for selective targeting of cortical cell populations. We report evaluation of fifteen new transgenic driver lines, two new reporter lines, and >800 different enhancer AAVs covering most subclasses of cortical cells. The tools reported here as well as the scaled process of tool creation and modification enable diverse experimental strategies towards understanding mammalian cortex and brain function.

The cellular basis of distinct thirst modalities.

Fluid intake is an essential innate behaviour that is mainly caused by two distinct types of thirst1-3. Increased blood osmolality induces osmotic thirst that drives animals to consume pure water. Conversely, the loss of body fluid induces hypovolaemic thirst, in which animals seek both water and minerals (salts) to recover blood volume. Circumventricular organs in the lamina terminalis are critical sites for sensing both types of thirst-inducing stimulus4-6. However, how different thirst modalities are encoded in the brain remains unknown. Here we employed stimulus-to-cell-type mapping using single-cell RNA sequencing to identify the cellular substrates that underlie distinct types of thirst. These studies revealed diverse types of excitatory and inhibitory neuron in each circumventricular organ structure. We show that unique combinations of these neuron types are activated under osmotic and hypovolaemic stresses. These results elucidate the cellular logic that underlies distinct thirst modalities. Furthermore, optogenetic gain of function in thirst-modality-specific cell types recapitulated water-specific and non-specific fluid appetite caused by the two distinct dipsogenic stimuli. Together, these results show that thirst is a multimodal physiological state, and that different thirst states are mediated by specific neuron types in the mammalian brain.

Complementary networks of cortical somatostatin interneurons enforce layer specific control.

The neocortex is functionally organized into layers. Layer four receives the densest bottom up sensory inputs, while layers 2/3 and 5 receive top down inputs that may convey predictive information. A subset of cortical somatostatin (SST) neurons, the Martinotti cells, gate top down input by inhibiting the apical dendrites of pyramidal cells in layers 2/3 and 5, but it is unknown whether an analogous inhibitory mechanism controls activity in layer 4. Using high precision circuit mapping, in vivo optogenetic perturbations, and single cell transcriptional profiling, we reveal complementary circuits in the mouse barrel cortex involving genetically distinct SST subtypes that specifically and reciprocally interconnect with excitatory cells in different layers: Martinotti cells connect with layers 2/3 and 5, whereas non-Martinotti cells connect with layer 4. By enforcing layer-specific inhibition, these parallel SST subnetworks could independently regulate the balance between bottom up and top down input.

Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion.

Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.

The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline.

Slits and their Roundabout (Robo) receptors mediate repulsive axon guidance at the Drosophila ventral midline and in the vertebrate spinal cord. Slit is cleaved to produce fragments with distinct signaling properties. In a screen for genes involved in Slit-Robo repulsion, we have identified the Adam family metalloprotease Kuzbanian (Kuz). Kuz does not regulate midline repulsion through cleavage of Slit, nor is Slit cleavage essential for repulsion. Instead, Kuz acts in neurons to regulate repulsion and Kuz can cleave the Robo extracellular domain in Drosophila cells. Genetic rescue experiments using an uncleavable form of Robo show that this receptor does not maintain normal repellent activity. Finally, Kuz activity is required for Robo to recruit its downstream signaling partner, Son of sevenless (Sos). These observations support the model that Kuz-directed cleavage is important for Robo receptor activation.

Axon growth and guidance: receptor regulation and signal transduction.

The development of precise connectivity patterns during the establishment of the nervous system depends on the regulated action of diverse, conserved families of guidance cues and their neuronal receptors. Determining how these signaling pathways function to regulate axon growth and guidance is fundamentally important to understanding wiring specificity in the nervous system and will undoubtedly shed light on many neural developmental disorders. Considerable progress has been made in defining the mechanisms that regulate the correct spatial and temporal distribution of guidance receptors and how these receptors in turn signal to the growth cone cytoskeleton to control steering decisions. This review focuses on recent advances in our understanding of the mechanisms mediating growth cone guidance with a particular emphasis on the control of guidance receptor regulation and signaling.