Molecular tools for precise manipulation of defined synaptic connections

Project Summary/Abstract Our project will address a major gap in existing neuroscience research tools, through the development of intersectional genetic tools for drug-controlled manipulation of specific synaptic connections between two genetically- or anatomically-defined neuronal populations. The tools we have engineered build from established cell

manipulation tools – DREADDs, PSAMs, and tetanus toxin – but critically, introduce trans-synaptic gating. Thus, the tools become activatable only at user-selected, genetically-defined cell-cell contact sites. We propose to develop three tool platforms in Aims 1-3, which enable drug-controlled synapse manipulation by three different mechanisms

and on three different timescales – ranging from seconds to minutes for antigen-gated PSAM ion channels to hours for antigen-gated trans-tetanus toxin. We have designed the antigen-gating of our tools to be both modular and programmable, so that either exogenous (e.g., surface GFP) or endogenous (e.g., tumor marker GD2) trans-synaptic

triggers can be used. To develop and optimize these tools, we will rely on the extensive protein engineering and directed evolution expertise of PI Alice Ting, who has previously developed proximity labeling enzymes and calcium integrators that are widely used in the neuroscience community. We will carefully validate the tools in two distinct mouse brain regions in the labs of PIs Xiaoke Chen and

Ivan Soltesz. Chen will evaluate the specificity and sensitivity of the proposed tools by assessing their ability to thoroughly and selectively silence connections from the paraventricular thalamus and prefrontal cortex onto two types of nucleus accumbens medium spiny neurons. We will use a combination of electrophysiological recording and

optogenetic pathway stimulation and calcium imaging on ex vivo brain slices to read out drug-gated inhibition of these circuits. We will then utilize these tools in vivo to study the circuit mechanisms of opioid withdrawal and

cocaine-induced behavioral sensitization. In the Soltesz lab, we plan to apply the tools to answer previously untestable questions about the roles of cell-type specific projections in regulating hippocampal function across scales from local microcircuits to long-range inputs. Our hippocampal experiments will use single cell electrophysiology, optogenetics,

in vivo imaging of behavior-associated neuronal activity, local field potential recordings and behavioral memory tests. Constant feedback between these ex vivo and in vivo studies and the tool engineering efforts in Aims 1-3 will ensure that our tools are optimized for maximal efficacy, specificity, and robustness.

Our team is highly interdisciplinary and diverse, combining the chemical biology and protein engineering expertise of Alice Ting with the systems and molecular neuroscience expertise of Xiaoke Chen and Ivan Soltesz. Soltesz is a pioneer in exploring the roles of hippocampal neuronal subpopulations in normal and pathological circuit

function, while Chen has uncovered novel circuit mchanisms underlying pain and addiction. Though our proposed aims are highly ambitious, they are feasible given the track record, expertise, and synergy of the team, as well as the strong preliminary data presented.