Chemogenetic Tools for Causal Cellular and Neuronal Biology

Abstract

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

FIGURE 1.

DREADDs and their downstream intracellular signal transduction pathways.

FIGURE 2.

Cell type-specific or projection-specific transgene expression. A and B: Cre-dependent expression of DREADDs in neurons. A: schematic injection of Cre-dependent virus into brain via stereotactic delivery. B: selective DREADD expression FLEX switch. C: schematic of FLEX-switch used in Cre-dependent viruses and two step Cre-recombination events involving loxP and lox2272 heterotypic recombination sites for permanent inversion of DREADD transgenes. D and E: two-way intersectional viral strategies to express DREADDs in projection-specific neuronal subpopulations. D: axonally targeted CAV2-Cre injected into CeA for retrograde labeling and somatically targeted rAAV-FLEX-hM3Dq injected into PBN for expression only in CeA projecting PBN neurons. E: axonally targeted rAAV₅-Cre injected into NAc for retrograde labeling and somatically targeted rAAV-FLEX-KORDi injected into central subiculum for expression only in NAc projecting subiculum neurons. F: a 3-way intersectional strategy to express hM4Di in a molecularly defined neuron population with a defined axon projection. First, axon-targeted injection of a retroAAV expressing Flp recombinase in a Cre-recombinase-dependent manner (retroAAV-FLEX-FlpO) was made into spinal cord of Vgat-cre mice. Then somatic injection of Flp recombinase-inducible rAAV virus expressing hM4Di (rAAV-FD-hM4Di) targeted to RVM. In this configuration only the spinal cord projecting GABAergic neurons of RVM will be expressing hM4Di.

FIGURE 3.

Schematic of neural circuit epistasis for the PVH^TRH→ARC^AGRP feeding circuit. TRH activation using hM3Dq drives food intake which is abolished by concomitant hM4Di silencing of downstream AGRP neurons.

FIGURE 4.

Summary diagram depicting the experimental strategies to test the impact of synthetically activated memory engrams during fear learning and recall.

FIGURE 5.

Chemogenetic LGIC tools for activating and inhibiting neurons.

FIGURE 6.

Combination of pharmacological and functional Cys-loop modules to generate engineered chimeric ion channels for controlling ion flux. A: for a given PSAM (yellow)/PSEM (red) pair, combination with different IPDs allows multiple functional outcomes to be achieved ranging from neuronal activation, controlling calcium flux, or neuronal silencing. B: multiple orthogonal PSAM/PSEM pairs have been developed, which enables combinatorial generation of diverse chemogenetic ion channel tools.

FIGURE 7.

Efficacy, durability, and bidirectionality of PSAM/PSEM neuron perturbations. A: daily, reversible silencing of premotor neurons with PSAM-GlyR during rotorod motor learning compared with permanent neuron ablation (left). Subsequent training without PSEM injection led to efficient learning of the task. [From Esposito et al. (58), with permission from Nature Publishing Group.] B: bidirectional activation (Ex. PSAM) or silencing (Inh. PSAM) of D2R neurons in the striatum alters Von Frey (VF) threshold in spared nerve injury (SNI) pain model. [From Ren et al. (153), with permission from Nature Publishing Group.]

FIGURE 8.

Additional cell type-specific pharmacology methods. A: homodimerization (left) and heterodimerization (right). B: Cre recombinase fused to a destabilization domain (DD) can be stabilized by addition of the small molecule trimethoprim. C: targeting of the small molecule MK801 to specific cell populations by expressing porcine liver esterase (PLE) that can unmask CM-MK801. D: DART method for cell type-specific pharmacology in vivo. [From Shields et al. (164), with permission from American Association for the Advancement of Science.]