Table 1.

Optogenetics, CANE, GECI, and DREADDs.

Method	Advantages	Limitations	Application in autonomic system
Optogenetics	Simulates neuronal function in vivo by depolarizing or hyperpolarizing neurons with pulses of light Targets specific nuclei by harnessing genetic difference using the Cre-Lox system and stereotaxic injection Manipulates neurons in vivo and in vitro High spatio-temporal resolution in vivo	Involves lowering an optical fiber close to the nucleus of interest at the risk of displacing brain tissue Restricted to acute manipulation; chronic manipulation is not possible Prolonged light-pulses into the brain tissue can cause cell damage Involves 5–6-week wait for light-gated opsins to be expressed on the cell membrane	Photo-activating catecholaminergic neurons in the RVLM of mice with channelrhodopsin increases blood pressure and has adverse autonomic consequences leading to sleep apnea [19] Archaerhodopsin-induced photo-inhibition of left stellate ganglion in dogs using a wireless LED suppresses nerve activity, thus suppressing cardiac ventricular arrhythmias [41] Photo-activating neurons in the locus ceruleus with channelrhodopsin inhibits parasympathetic transmission to cardiac vagal neurons in the brainstem, leading to tachycardia [42]
CANE	Labels causative neurons activated by an autonomic function Compared to other techniques, this method has the lowest non-specific labelling Permanently transfects any gene of interest into neurons, like channelrhodopsin, cre-recombinase, DREADD, GCaMP, and GFP Trans-synaptically labels input circuits using pseudorabies virus to package EnvA and gene of interest High spatio-temporal resolution	Involves time-bound stereotaxic injection to ensure EnvA virus labels associated neurons Some autonomic and behavioral processes are sensitive to pre-surgery anesthesia which leads to cFos activation in neurons, causing non-specific labelling Must wait for weeks before the gene of interest is expressed in neurons	Can be used to identify neurons responsible for treatment-induced autonomic activity. This information can be harnessed to optogenetically activate neural pathways. It can also be used to map input circuits and knockout genes of interest
GECI	Visualizes neuronal activity that regulates or responds to physiological changes in vivo Allows simultaneous 2-color imaging of neurons and astrocytes It is possible to quantitate neuronal activity detected by the system GECI expression is long-lasting, allowing more than one experimental session with a transfected mouse GECI sensors can be expressed in vivo using transgenic mice, thus avoiding stereotaxic injection of virus and confining its expression to the area of interest High spatio-temporal resolution	Involves invasive stereotaxic surgery to install the detecting probe The GECI must be selected carefully in order to address a hypothesis correctly Can only provide information on neuronal activation in a selected nucleus but not the whole brain	GCaMP3 sensors indicate how vagal sensory neurons respond to enteric mechanoreceptors and chemoreceptors [43] GCaMP6 sensors expressed in heat-sensitive neurons in the ventromedial preoptic area detect changes in activity in response to temperature which results in an autonomic response [44] GCaMP3 sensors indicate that somatostatin GABAergic neurons in the dorsal motor nucleus of the vagus regulate parasympathetic gastric activity [45]
DREADD	Allows chronic long-term control of neurons Persistent neuronal manipulation is possible without damaging the cell. Activated exclusively by intraperitoneal injection of the designer drug, clozapine-n-oxide Transgenic mice that express DREADD receptors using the Cre-Lox mechanism eliminate the need for invasive stereotaxic surgery	CNO rapidly metabolizes to clozapine, an FDA-approved antipsychotic Clozapine binds to the designer receptor with greater affinity than CNO Therefore, experiments must be designed with appropriate controls, to eliminate any interference caused by clozapine itself	Activating excitatory hM3Dq DREADD in oxytocinergic neurons in the PVN rescues chronic intermittent hypoxia-hypercapnia-induced hypertension [37] Using hM3Dq DREADD to activate inhibitory neurons in the arcuate nucleus lowers sympathetic activity [38] hM3Dq DREADD-induced activation of astrocytes in mice leads to autonomic changes such as increases in heart rate, blood pressure, and saliva formation [46]