Thalamoseq

Deep sequencing of the cellular transcriptome has provided unprecedented access to the molecular diversity of neurons. However, it remains a challenge to make sense of the observed molecular diversity of neurons - including the challenge of relating molecular diversity to diversity in projection patterns, dendritic morphology, cellular biophysics, etc. To address this question a joint graduate student in our lab and the Hantman lab began a project to assess the molecular diversity of the mouse thalamus. We sought to use these approaches to both understand the logic by which the ~30 thalamic nuclei are organized as well as to provide molecular entry points that allow for precise manipulation of thalamic function. This developed into a project that drew upon diverse expertise within Janelia and in collaboration with Sacha Nelson at Brandeis. This effort produced a large dataset that describes molecular and anatomical diversity of major thalamic nuclei. The data that is publicly available at thalamoseq.janelia.org and the work is described in a recent paper in Nature Neuroscience.

This work was led by Adam Hantman's lab in collaboration with Sacha Nelson's lab and Quantitative Genomics in addition to the Neuroseq and MouseLight Janelia Project Teams, and Scientific Computing made the online resources for browsing the data.

flinchr

Optogenetics provides an exceptional and powerful toolkit for the exploration of brain function by allowing precise manipulation of individual cell types and circuits. Remarkably efficient tools exist for the stimulation of individual neurons discovered via mining of diverse genomes; however, while there are a number of tools that allow for inhibition - the result of both mining extant genomes and substantial directed mutagenesis - such tools are generally less robust and reliable in practice for many neuroscience applications. This suggests that alternate methods of diversifying the toolkit of available optogenetics reagents is required. While site directed mutagenesis has been a proven method we sought to exploit another method of diversification in evolution - recombination. Recombination is a process by which large coding regions of distinct genes may be combined to yield hybrid proteins of (potentially) novel function. In collaboration with Alla Karpova's lab several years ago we sought to begin to explore how to produce hybrid reagents through the modular combination of existing reagents and targeting domains. In one of first products of this collaboration we described the production of FLInChR (Full length inversion of channelrhodopsin) through the combination of a transmembrane domain on to the N-terminus of channelrhodopsin (ChR). This produced a topological inversion of ChR that, due to its pump-like properties, produced a dramatic alteration in function: it converted a neuronal activator into a potent neuronal silencer as described in our recent paper and summarized in a few places. Example data showing complete silencing of a relatively difficult to silence neuronal cell type (large layer 5 pyramidal cells in neocortex) is shown at right.

Through ongoing efforts we continue to explore this method of diversification of our toolkit for systems neuroscience and hope to produce additional useful resources. If you would like to try this inhibitor the plasmid is available at AddGene.

retro-AAV

Efficient retrograde access to projection neurons with the aim of delivering sensors and effectors constitutes an important future avenue for circuit dissection. Viral vectors are powerful gene delivery vehicles for the nervous system, but all available viral tools suffer from inefficient retrograde transport. Using directed molecular evolution together with our collaborators we engineered potent retrograde functionality into the capsid of AAV — a vector that already shows particular promise in neuroscience research. The newly evolved AAV2-retro variant permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde labeling reagents, and supports high-level payload expression adequate for functional circuit interrogation.

Plasmid is available at AddGene. We have set up a large-scale production of the following three basic tester preps at the Janelia facility: rAAV2-retro CAG-tdtomato, rAAV2-retro FLEX-tdtomato and rAAV2-retro Syn-Cre. These preps should be available within about a month. If you are unsure about the results you obtain with preps from any other sources, you will be able to request a free tester aliquot to use as a standard. To obtain a tester prep, please send a request to Janelia Virus Services or contact my lab.

Skilled movements & decisions

We have argued that the measurement of kinematic parameters of goal-directed movements is important for understanding the function of basal ganglia circuits that control purposive behavior. Recently, we have developed a behavioral task in which mice displace a spring-loaded 2D joystick past a hidden threshold to receive reward. Assuming a roughly constant transfer of work from the limbs to the joystick effort is related to the velocity of joystick movement. By using a continuous analog signal proportional to movement kinematics we can study how effort is traded off against reward, continuous neural representations of movement kinematics, as well as performing rapid (kHz), closed-loop feedback.

The system we developed has 3 key parts:

• a hall-effect sensor based, spring loaded joystick like this one;
• a mechanical system for stable head fixation. We like RIVETS;
• an embedded controller with fast, good A/D conversion to allow for rapid closed-loop feedback. We like the behavioral control system III.

You can contact us with specific questions or inquiries about the software we use to interact with the BCS if needed; however, there are many solutions available.