Manipulating Neural Circuits in Anesthesia Research

Abstract

The neural circuits underlying the distinct endpoints that define general anesthesia remain incompletely understood. It is becoming increasingly evident, however, that distinct pathways in the brain that mediate arousal and pain are involved in various endpoints of general anesthesia. To critically evaluate this growing body of literature, familiarity with modern tools and techniques used to study neural circuits is essential. This Readers' Toolbox article describes four such techniques: (1) electrical stimulation, (2) local pharmacology, (3) optogenetics, and (4) chemogenetics. Each technique is explained, including the advantages, disadvantages, and other issues that must be considered when interpreting experimental results. Examples are provided of studies that probe mechanisms of anesthesia using each technique. This information will aid researchers and clinicians alike in interpreting the literature and in evaluating the utility of these techniques in their own research programs.

Figure 1.. Four different techniques for manipulating neural activity.

Each of the techniques described in this Reader’s Toolbox excite or inhibit neural activity via their effects on membrane-bound proteins, including (A) voltage-gated proteins, (B) ion channels or other receptors (C) opsins, and (D) modified receptors that respond to an exogenous ligand instead of their endogenous ligand. Because of their different molecular mechanisms, these four techniques exhibit varying degrees of spatial range, temporal dynamics, and neuron-type specificity. Researchers use these techniques for manipulating neural activity in order to study the role of neural circuits in a range of physiological, behavioral, and cognitive states.

Figure 2.. Electrical stimulation method of circuit manipulation.

Electrical stimulation is used to manipulate neural activity near the stimulation electrode. (A) As the stimulus intensity increases, it affects neurons in a larger area of neural tissue, agnostic to the neuron type. (B) As an example, electrical stimulation of the ventral tegmental area (VTA) can be used to study its downstream effects on the prefrontal cortex (PFC) and nucleus accumbens (NAc). In this example, the strength and location of the electrical stimulation determines the extent of the effects on the downstream regions. Throughout the figures in this article, triangles indicate dopaminergic neurons, circles indicate non-dopaminergic neurons, red indicates activation of the neurons and black indicates no change in neuronal activity.

Figure 3.. Local pharmacological methods of circuit manipulation: microinjection and microdialysis.

Microinjection and microdialysis are used to manipulate neural activity near the cannula or probe tip by delivering drugs or other substances. In addition, microdialysis can be used to collect samples of substances from the brain for analysis. (A) Drugs and other receptor agonists or antagonists can be delivered to a small volume (typically <1 mm³) of brain tissue using microinjection. (B) Microdialysis is used to collect samples of drugs, neurotransmitters, or other small molecules from the brain to measure concentrations or other analyses. When the perfusate is isosmotic with the surrounding brain tissue, there is no net fluid delivery with microdialysis, in contrast with microinjection. (C) Microdialysis is also used to deliver drugs (often referred to as “reverse microdialysis”). In microdialysis and reverse microdialysis, diffusion of substances into or out of the probe tip depends on the pore size of the semi-permeable membrane.

Figure 4.. Optogenetic method of circuit manipulation.

Optogenetic stimulation is used to excite or inhibit targeted neurons that express the opsin, using an optic fiber. (A) In this example, light delivered through an optic fiber is used to excite only neurons that express the opsin, ChR2, in the path of the light. A given light pulse excites an action potential in neurons expressing the opsin (inset top), but not in neurons lacking opsin expression or out of the range of the delivered light (inset bottom). (B) The opsin is typically introduced to the neurons by injecting a virus containing the opsin gene (top). After about 3 weeks to allow for expression of the opsin, neuron bodies (bottom left) or axon terminals (bottom right) can be targeted with light from the optic fiber to determine which downstream targets are affected.

Figure 5.. Chemogenetic method of circuit manipulation.

Chemogenetic stimulation can be used to excite or inhibit neural activity by activating receptors on targeted neurons. (A) Only neurons expressing the chemogenetic receptor are affected by their respective exogenous ligand. In this case, the excitatory chemogenetic receptor, hM3Dq, is activated by its ligand, clozapine-N-oxide (CNO). (B) Similar to optogenetics, chemogenetic experiments begin with injection of a virus containing the gene for the chemogenetic receptor (top). After about 3 weeks to allow for expression of the receptors, transfected neurons can be targeted through systemic injection of CNO (bottom left) or local injection of CNO (bottom right; by microinjection). The method of CNO delivery depends on the experiment: systemic injection is ideal for experiments requiring activation of all transfected neurons, whereas local injection is ideal for experiments requiring activation of a small region of transfected neurons.