Research Article

Deconstructing behavioral neuropharmacology with cellular specificity

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Science  07 Apr 2017:
Vol. 356, Issue 6333, eaaj2161
DOI: 10.1126/science.aaj2161

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A tailored look at behavioral pharmacology

It is important to understand how animal behavior is mediated by molecular, cellular, and circuit components of the brain. However, it has been difficult to link the activity of specific molecules in defined cells to behavioral roles. Shields et al. developed an approach to deconstruct behavioral neuropharmacology with cellular specificity. The technique, termed DART (drugs acutely restricted by tethering), uses enzymatic capture to restrict standard drugs to the surface of genetically specified cells without prior modification of the native pharmacological target. The method provides cell-type specificity, endogenous-protein specificity, acute onset, and utility in behaving animals. This enables the activity of specific molecules in defined circuit elements to be causally linked to behavior.

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Structured Abstract

INTRODUCTION

Animal behavior is mediated by molecular, cellular, and circuit components of the brain. However, because many proteins are broadly expressed, it has been difficult to link the activity of specific proteins in defined cells to behavioral roles. This challenge has particular relevance to neuropsychiatric disorders, which have largely been understood in relation to clinically effective drugs that act on known molecular targets. Such knowledge has been difficult to extend to circuit-level insight because cell type specificity has not been possible with traditional pharmacology.

RATIONALE

Here, we combined the speed and molecular specificity of pharmacology with the cell type specificity of genetic tools. DART (drugs acutely restricted by tethering) is a technique that uses a bacterial enzyme called HaloTag to capture and tether drugs to the surface of defined cells. A key feature is that the method does not require prior modification or overexpression of the native pharmacological target because HaloTag is expressed as a separate protein. Drug capture proceeds rapidly over seconds to minutes, producing a factor of ~100 enrichment of the drug at the surface of HaloTag-expressing cells. The method provides a unique feature set: (i) Cell type specificity arises from expression of HaloTag under control of a cell type–specific promoter; (ii) molecular specificity is inherited from the drug that is tethered; and (iii) acute onset upon delivery of the DART ligand is similar to that of traditional pharmacology, thus averting compensatory phenomena.

RESULTS

We first developed a DART that antagonizes the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR), a broadly expressed postsynaptic glutamate receptor. We validated the speed, cell type specificity, and molecular specificity of the method in cultured neuronal assays, in coronal slices of mouse dorsal striatum, and in behaving mice. We then applied the technique to a mouse model of Parkinson’s disease (PD). In PD, AMPARs are subject to dysregulated long-term potentiation (LTP) and long-term depression (LTD) in distinct cell types of the striatum, a brain region critical for movement. AMPAR antagonists have been studied extensively in animal models of PD and in human clinical trials for the disorder. However, it has been difficult to link AMPAR activity in defined cells to motor deficits. We found that motor deficits were causally attributed to AMPARs in indirect spiny projection neurons (iSPNs) and to excess phasic firing of tonically active interneurons (TANs) of the striatum. Together, iSPNs and TANs (i.e., D2 cells) drove akinesia, whereas movement execution deficits reflected the ratio of AMPARs in D2 versus D1 cells. Finally, we designed a muscarinic antagonist DART in one iteration, demonstrating applicability of the method to diverse targets.

CONCLUSION

Neuropsychiatric disorders have been examined with acute manipulations featuring either circuit or molecular specificity. DART combines these features, enabling interrogation of specific proteins in defined cells. The approach may provide a platform whereby the mechanism of action for widely prescribed drugs can be examined with cellular specificity in animal models of several disorders. Such studies could inform new translational strategies by advancing nonobvious drug combinations, or by providing a road map for the design of bivalent therapeutics based on the “message-address” concept of Schwyzer.

Deconstructing behavioral neuropharmacology.

Drugs that manipulate specific molecules in the brain (e.g., green but not purple postsynaptic receptor) have shaped our understanding of neuropathologies. The DART technique uses HaloTag (red) to capture and spatially restrict drugs to the surface of genetically defined cells. Behavioral effects of drugs can thus be deconstructed into the individual and combinatorial contributions produced by defined cell types.

Illustration: Julia Kuhl

Abstract

Behavior has molecular, cellular, and circuit determinants. However, because many proteins are broadly expressed, their acute manipulation within defined cells has been difficult. Here, we combined the speed and molecular specificity of pharmacology with the cell type specificity of genetic tools. DART (drugs acutely restricted by tethering) is a technique that rapidly localizes drugs to the surface of defined cells, without prior modification of the native target. We first developed an AMPAR antagonist DART, with validation in cultured neuronal assays, in slices of mouse dorsal striatum, and in behaving mice. In parkinsonian animals, motor deficits were causally attributed to AMPARs in indirect spiny projection neurons (iSPNs) and to excess phasic firing of tonically active interneurons (TANs). Together, iSPNs and TANs (i.e., D2 cells) drove akinesia, whereas movement execution deficits reflected the ratio of AMPARs in D2 versus D1 cells. Finally, we designed a muscarinic antagonist DART in one iteration, demonstrating applicability of the method to diverse targets.

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