Next-Generation Tools to Study Autonomic Regulation In Vivo

Abstract

The recent development of tools to decipher the intricacies of neural networks has improved our understanding of brain function. Optogenetics allows one to assess the direct outcome of activating a genetically-distinct population of neurons. Neurons are tagged with light-sensitive channels followed by photo-activation with an appropriate wavelength of light to functionally activate or silence them, resulting in quantifiable changes in the periphery. Capturing and manipulating activated neuron ensembles, is a recently-designed technique to permanently label activated neurons responsible for a physiological function and manipulate them. On the other hand, neurons can be transfected with genetically-encoded Ca²⁺ indicators to capture the interplay between them that modulates autonomic end-points or somatic behavior. These techniques work with millisecond temporal precision. In addition, neurons can be manipulated chronically to simulate physiological aberrations by transfecting designer G-protein-coupled receptors exclusively activated by designer drugs. In this review, we elaborate on the fundamental concepts and applications of these techniques in research.

Keywords: Autonomic regulation; Calcium sensors; DREADD; Optogenetics.

Fig. 1

Optogenetics. The excitatory light-gated channel, channelrhodopsin, opens upon excitation with 475-nm light. This allows an influx of sodium ions that depolarizes the neuron. Halorhodopsin and archaerhodopsin silence the neuron by enabling hyperpolarization when they open in response to 575–600-nm light. Upon light activation, halorhodopsin allows chloride influx and archaerhodopsin pumps protons out of the cell, hyperpolarizing the neuron to silence it.

Fig. 2

Calcium imaging with GFP-based genetically encoded calcium indicators. Neuronal activation leads to an increase in intracellular Ca²⁺ which binds to calmodulin. Ca²⁺-calmodulin binds to M13, therefore re-orienting GFP subunits in the correct order. Now the GFP is detectable upon excitation with 473-nm light, indicating an activation-associated Ca²⁺ surge.

Fig. 3

DREADD: Designer receptor engineered for designer drugs. Receptors that mimic Gq protein-coupled (excitatory in neurons) and Gi protein-coupled (inhibitory in neurons) receptors can be selectively activated by the designer drug clozapine-n-oxide (CNO), leading to chronic activation or deactivation of a selected population of neurons.