Enhancer viruses for combinatorial cell-subclass-specific labeling.

Rapid cell type identification by new genomic single-cell analysis methods has not been met with efficient experimental access to these cell types. To facilitate access to specific neural populations in mouse cortex, we collected chromatin accessibility data from individual cells and identified enhancers specific for cell subclasses and types. When cloned into recombinant adeno-associated viruses (AAVs) and delivered to the brain, these enhancers drive transgene expression in specific cortical cell subclasses. We extensively characterized several enhancer AAVs to show that they label different projection neuron subclasses as well as a homologous neuron subclass in human cortical slices. We also show how coupling enhancer viruses expressing recombinases to a newly generated transgenic mouse, Ai213, enables strong labeling of three different neuronal classes/subclasses in the brain of a single transgenic animal. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.

Optogenetic Activation of Normalization in Alert Macaque Visual Cortex.

Normalization has been proposed as a canonical computation that accounts for a variety of nonlinear neuronal response properties associated with sensory processing and higher cognitive functions. A key premise of normalization is that the excitability of a neuron is inversely proportional to the overall activity level of the network. We tested this by optogenetically activating excitatory neurons in alert macaque primary visual cortex and measuring changes in neuronal activity as a function of stimulation intensity, with or without variable-contrast visual stimulation. Optogenetic depolarization of excitatory neurons either facilitated or suppressed baseline activity, consistent with indirect recruitment of inhibitory networks. As predicted by the normalization model, neurons exhibited sub-additive responses to optogenetic and visual stimulation, which depended lawfully on stimulation intensity and luminance contrast. We conclude that the normalization computation persists even under the artificial conditions of optogenetic stimulation, underscoring the canonical nature of this form of neural computation.

Tracing inputs to inhibitory or excitatory neurons of mouse and cat visual cortex with a targeted rabies virus.

BACKGROUND: Cortical inhibition plays a critical role in controlling and modulating cortical excitation, and a more detailed understanding of the neuronal circuits contributing to each will provide more insight into their roles in complex cortical computations. Traditional neuronal tracers lack a means for easily distinguishing between circuits of inhibitory and excitatory neurons. To overcome this limitation, we have developed a technique for retrogradely labeling inputs to local clusters of inhibitory or excitatory neurons, but not both, using neurotropic adenoassociated and lentiviral vectors, cell-type-specific promoters, and a modified rabies virus. RESULTS: Applied to primary visual cortex (V1) in mouse, the cell-type-specific tracing technique labeled thousands of presynaptically connected neurons and revealed that the dominant source of input to inhibitory and excitatory neurons is local in origin. Neurons in other visual areas are also labeled; the percentage of these intercortical inputs to excitatory neurons is somewhat higher (~20%) than to inhibitory neurons (<10%), suggesting that intercortical connections have less direct control over inhibition. The inputs to inhibitory neurons were also traced in cat V1, and when aligned with the orientation preference map revealed for the first time that long-range inputs to inhibitory neurons are well tuned to orientation. CONCLUSIONS: These novel findings for inhibitory and excitatory circuits in the visual cortex demonstrate the efficacy of our new technique and its ability to work across species, including larger-brained mammals such as the cat. This paves the way for a better understanding of the roles of specific cell types in higher-order perceptual and cognitive processes.

Imaging light responses of retinal ganglion cells in the living mouse eye.

This study reports development of a novel method for high-resolution in vivo imaging of the function of individual mouse retinal ganglion cells (RGCs) that overcomes many limitations of available methods for recording RGC physiology. The technique combines insertion of a genetically encoded calcium indicator into RGCs with imaging of calcium responses over many days with FACILE (functional adaptive optics cellular imaging in the living eye). FACILE extends the most common method for RGC physiology, in vitro physiology, by allowing repeated imaging of the function of each cell over many sessions and by avoiding damage to the retina during removal from the eye. This makes it possible to track changes in the response of individual cells during morphological development or degeneration. FACILE also overcomes limitations of existing in vivo imaging methods, providing fine spatial and temporal detail, structure-function comparison, and simultaneous analysis of multiple cells.

Evoked axonal oxytocin release in the central amygdala attenuates fear response.

The hypothalamic neuropeptide oxytocin (OT), which controls childbirth and lactation, receives increasing attention for its effects on social behaviors, but how it reaches central brain regions is still unclear. Here we gained by recombinant viruses selective genetic access to hypothalamic OT neurons to study their connectivity and control their activity by optogenetic means. We found axons of hypothalamic OT neurons in the majority of forebrain regions, including the central amygdala (CeA), a structure critically involved in OT-mediated fear suppression. In vitro, exposure to blue light of channelrhodopsin-2-expressing OT axons activated a local GABAergic circuit that inhibited neurons in the output region of the CeA. Remarkably, in vivo, local blue-light-induced endogenous OT release robustly decreased freezing responses in fear-conditioned rats. Our results thus show widespread central projections of hypothalamic OT neurons and demonstrate that OT release from local axonal endings can specifically control region-associated behaviors.

New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits.

Glycoprotein-deleted (ΔG) rabies virus is a powerful tool for studies of neural circuit structure. Here, we describe the development and demonstrate the utility of new resources that allow experiments directly investigating relationships between the structure and function of neural circuits. New methods and reagents allowed efficient production of 12 novel ΔG rabies variants from plasmid DNA. These new rabies viruses express useful neuroscience tools, including the Ca(2+) indicator GCaMP3 for monitoring activity; Channelrhodopsin-2 for photoactivation; allatostatin receptor for inactivation by ligand application; and rtTA, ER(T2)CreER(T2), or FLPo, for control of gene expression. These new tools allow neurons targeted on the basis of their connectivity to have their function assayed or their activity or gene expression manipulated. Combining these tools with in vivo imaging and optogenetic methods and/or inducible gene expression in transgenic mice will facilitate experiments investigating neural circuit development, plasticity, and function that have not been possible with existing reagents.