Show Summary Details

Page of

PRINTED FROM OXFORD HANDBOOKS ONLINE (www.oxfordhandbooks.com). © Oxford University Press, 2018. All Rights Reserved. Under the terms of the licence agreement, an individual user may print out a PDF of a single chapter of a title in Oxford Handbooks Online for personal use (for details see Privacy Policy and Legal Notice).

date: 15 December 2019

Chemo- and Optogenetic Strategies for the Elucidation of Pain Pathways

Abstract and Keywords

Pain is not a simple phenomenon and, beyond its conscious perception, involves circuitry that allows the brain to provide an affective context for nociception, which can influence mood and memory. In the past decade, neurobiological techniques have been developed that allow investigators to elucidate the importance of particular groups of neurons in different aspects of the pain response, something that may have important translational implications for the development of novel therapies. Chemo- and optogenetics represent two of the most important technical advances of recent times for gaining understanding of physiological circuitry underlying complex behaviors. The use of these techniques for teasing out the role of neurons and glia in nociceptive pathways is a rapidly growing area of research. The major findings of studies focused on understanding circuitry involved in different aspects of nociception and pain are highlighted. In addition, attention is drawn to the possibility of modification of chemo- and optogenetic techniques for use as potential therapies for treatment of chronic pain disorders in human patients.

Keywords: chemogenetics, optogenetics, nociception, pain, DREADD

The neuronal pathways that subserve the sensation of pain include sensory neurons of the peripheral nervous system (nociceptors) that are required to drive most pain states, as well as elements of the spinal cord and the brain. Pain can be elicited through the normal or abnormal activity of nerves at multiple levels of this pathway. Pain is not a simple phenomenon and, beyond its conscious perception, involves circuitry that allows the brain to provide an affective context for nociception, which can influence mood and memory. In the past decade, neurobiological techniques have been developed that allow investigators to elucidate the importance of particular groups of neurons in different aspects of the pain response, something that may have important translational implications for the development of novel therapies. Here, we review two of the major techniques that have been used in pain research: chemogenetics and optogenetics.

Chemogenetics

Chemogenetics refers to a suite of experimental techniques in which proteins are engineered to interact with normally inactive small-molecule ligands to excite or inhibit neuronal activity (reviewed by Roth, 2016). In effect, chemogenetics represents a molecular hoax in which cells are persuaded to respond to synthetic inputs that have been introduced by, and are under the control of, the investigator. A variety of protein classes have been chemogenetically engineered, including kinases, ligand-gated ion channels, and G protein–coupled receptors (GPCRs) (Roth, 2016). It is important to appreciate that the activity of neurons involved in the processing of pain is normally controlled by a diverse set of inputs, including chemical mediators released from damaged tissue, inflammatory mediators, neurotransmitters, and a variety of humoral agents or environmental factors, such as pressure and temperature. The effects produced are mediated by the interaction of these factors with receptors expressed by target neurons. Many of these receptors are ligand-gated ion channels or GPCRs. Activation of ligand-gated ion channels results in rapid changes in membrane potential, whereas GPCR activation may result in downstream G protein or β-arrestin signaling, leading to either increased or decreased neuronal excitability as well as numerous other consequences.

The types of receptors expressed by individual neurons are the result of the developmental program of the organism. The idea of producing GPCR-based chemogenetic tools goes back to the early 1990s and went through several iterations prior to arriving at the designer receptor exclusively activated by designer Drug (DREADD) platform initiated by Brian Roth, its current and now widely used manifestation. Earlier versions included allele-specific GPCRs (Strader et al., 1991) and receptors activated solely by synthetic ligands (RASSLs) (Coward et al., 1998). The more recent DREADD platform (Armbruster, Li, Pausch, Herlitze, & Roth, 2007) represents an experimental paradigm that is currently widely used to probe the role of different types of neurons in a large number of neurobiological contexts—including pain. Because DREADDs can be expressed in precisely defined populations of neurons, they enable the experimenter to exert à la carte control over the activity of particular nerve pathways, rather than the table d’hôte created by nature.

DREADD-Induced Neuronal Signaling

Structural and functional characteristics of GPCR signaling have been well described, although, even today, novel properties of GPCRs are still regularly discovered (Rosenbaum, Rasmussen, & Kobilka, 2009). GPCRs can interact with heterotrimeric G proteins (Gα, Gβ, and Gγ) as well as with β-arrestins in a ligand-dependent manner. DREADDs are engineered versions of GPCRs that have mutated binding sites so that they can no longer be activated by their normal endogenous ligand but, instead, can be activated by a synthetic small molecule. This “designer drug” is typically clozapine-N-oxide (CNO), originally thought to be a functionally “inactive” substance. The idea is that, once expressed in the desired population of neurons, DREADDs will remain inactive until CNO is introduced to activate them. This means that their active signaling is completely under the control of the experimenter.

Some caveats have recently been raised with respect to the idea that CNO is really a completely inactive substance, primarily because CNO can be metabolized to produce clozapine, an atypical antipsychotic drug that has high affinity for a number of important endogenous GPCRs. Moreover, the ability of clozapine to activate DREADDs turns out to be several orders of magnitude greater than CNO itself. Nevertheless, if the correct control experiments are included, it should still be possible to determine which effects are produced through the activation of DREADDs as opposed to some other off-target mechanism. Moreover, additional DREADD agonists such as “Compound 21,” which cannot be metabolized to clozapine, are now also available and may render the problems associated with the use of CNO moot (Chen et al., 2015).

In principle, DREADDs could be produced that couple to any G protein that is normally activated by a GPCR. However, to date, most DREADDs couple to heterotrimeric G proteins that contain αi, αo, αs, and αq subunits. Moreover, it should also be possible to produce DREADDs that exhibit signaling that is “biased” toward either G proteins or β-arrestin, something that is currently of great interest (Figure 1). Most widely used DREADDs are based on the structure of human muscarinic receptors, modified so that they have minimal sensitivity to their endogenous ligand, the neurotransmitter acetylcholine. The original DREADDs invented included three Gq-coupled receptors, each based on a different human muscarinic receptor: hM1Dq, hM3Dq, and hM5Dq—of which hM3Dq is currently the most frequently used. There are also three Gi/o-DREADDs that are based on human muscarinic receptors, hM2Di and hM4Di being the most commonly used. A DREADD coupled to Gs was created by swapping the intracellular regions of the turkey erythrocyte β-adrenergic receptor for equivalent regions of a rat M3 DREADD to create a rat Gs-coupled DREADD (Roth, 2016).

Chemo- and Optogenetic Strategies for the Elucidation of Pain Pathways

Figure 1. Chemogenetic receptors and their signaling properties. Different types of chemogenetic receptors (Rq, hM3Dq, GsD, hM4Di, and KORD) have been genetically engineered from muscarinic (blue and beige) or opioid (black) receptors. Each receptor is specifically activated by its ligand (CNO, clozapine-N-oxide; or Sal B, salvinorin B) to signal to downstream effectors (arrestin-2/arrestin-3 or G protein α subunits Gαq, Gαs, or Gαi/o).

Because DREADDs allow for both the inhibition and the activation of neurons, it would be useful to be able to achieve both of these outcomes in the same set of neurons in the same animal through the simultaneous expression of hM3Dq and hM4Di. However, the problem is that both of these are activated by the same synthetic ligand, CNO. To get around this problem, Vardy et al. produced a second Gi/o-activating DREADD based on the κ-opioid (KORD) receptor (Vardy et al., 2015). This receptor can be activated by the ligand salvinorin B (Sal B), an “inactive” metabolite of the κ-opioid receptor agonist salvinorin A (Figure 1). Hence, if hM3Dq and KORD are expressed in the same population of neurons, the activity of these cells can be increased or decreased depending on whether CNO or Sal B is administered.

Although activation of GPCRs can produce a wide array of signaling consequences, the most rapid manifestation of G protein activation observed in neurons is usually some change in excitability resulting from the downstream modulation of ion channels. For example, stimulation of Gi/o signaling in neurons often results in activation of G protein-coupled inwardly rectifying potassium channels (GIRKs) and inhibition of voltage-dependent calcium channels, both of which contribute to a reduction in neuronal excitability and neurotransmitter release (Armbruster et al., 2007). Hence, Gi/o-linked DREADDs are frequently used for producing rapid inhibition of target neurons. In contrast, activation of Gq signaling frequently results in modification of conductances that result in neuronal excitation. Consequently, activation of Gq-linked DREADDs can be used for rapidly activating specific neuronal pathways. These effects can also be achieved with other platforms (discussed in material that follows), such as optogenetics, or another approach using chemogenetically engineered ligand-gated ion channels (discussed further in this chapter). However, the DREADD platform has advantages in some situations, and these methods are considered complementary.

The Use of DREADDs in Pain Research

The use of DREADDs to dissect out the important neuronal pathways that contribute to pain physiology and pathology represents an attractive experimental paradigm that is now being employed by an increasing number of investigators. The peripheral component of the pain pathway consists of sensory neurons (some of which are nociceptors) whose cell bodies reside in the dorsal root ganglia (DRG) and trigeminal ganglia. These are pseudounipolar neurons that extend one process into the periphery, where they innervate the viscera or the skin, and a second process that carries information into the dorsal horn of the spinal cord. The activity of these neurons is normally controlled by different families of receptors, whose activation transduces stimuli such as heat, pressure, irritant chemical factors, inflammatory cytokines, kinins, prostanoids, and a variety of other stimuli into electrical information (Basbaum, Bautista, Scherrer, & Julius, 2009).

Other receptors, including those for μ-selective opioids and cannabinoids, are also expressed by nociceptors. Activation of this latter group of receptors results in G protein–mediated inhibition of neuronal activity and transmitter release in the dorsal horn, which is thought to be responsible for at least a portion of their analgesic effects. DRG neurons are phenotypically heterogeneous, and a current question of interest involves identifying the precise molecular and functional phenotypes of neuronal subsets that mediate different types of somatosensation—and this is something that may change over the course of a disease—with a view to targeting them with novel types of analgesic agents.

Theoretically, such questions are amenable to the use of DREADDs because their directed expression can be achieved through the use of promoters that are active in particular subtypes of DRG neurons. For example, the voltage-gated sodium channel, NaV1.8, is selectively expressed by a large number of DRG neurons, including most nociceptors (Shields et al., 2012). Hence, the use of NaV1.8-Cre mice, together with mouse lines that contain alleles allowing Cre-dependent expression of DREADDs, results in the expression of DREADDs restricted to the NaV1.8 population of nociceptors. Expression of DREADDs by DRG neurons can also be achieved through the injection of viral constructs, usually adeno-associated viruses (AAVs), which target particular neuronal subtypes.

Theoretically, activation of Gi/o DREADDs expressed by nociceptors following the injection of CNO should result in a reduction in the activity of target neurons and a change, usually a reduction, in pain behavior. Several reports have detailed the results of this type of experiment. Iyer et al. originally utilized a viral construct [AAV6-hSyn-HA-hM4D(Gi)-IRES-mCitrine] to achieve expression of hM4Di receptors in a population of small DRG neurons, presumably nociceptors, following injection of the virus into the sciatic nerve. Single injections of CNO into these mice produced a rapid increase in both mechanical and thermal pain thresholds over a period of at least 90 minutes (Iyer et al., 2016). Similar results have been obtained by other investigators using genetic strategies to express hM4Di in either NaV1.8 or DRG neurons expressing transient receptor potential channel vanilloid 1 (TRPV1).

Some of these investigations have also started to examine the effects of DREADD activation in the context of models of disease-associated pain, with a view to understanding which populations of DRG neurons mediate different pain behaviors during the course of these diseases. For example, using a line of mice in which hM4Di was expressed in NaV1.8-positive DRG neurons, Miller et al. tested the effect of chemogenetic inhibition of nociceptors in the destabilization of the medial meniscus (DMM) model, a sophisticated surgical model of osteoarthritis, which produces a slowly developing chronic disease that closely resembles the human situation. Acute inhibition of NaV1.8-expressing neurons in mice following a single injection of CNO reduced knee hyperalgesia 4 weeks after DMM surgery and reduced mechanical allodynia 8 weeks after surgery. In contrast, CNO had no effect on pain-related behaviors 12 and 16 weeks after DMM surgery. Interestingly, morphine, a drug that activates GPCRs in the peripheral and central nervous systems, was still effectively reducing pain at both the earlier and the later stage of experimental osteoarthritis. These data suggest that osteoarthritis-associated pain attains a profile driven primarily by central sensitization or by Nav1.8-negative sensory neurons as the disease proceeds (Miller et al., 2017).

Jarayaj et al. used the same line of mice to examine pain behaviors in a high-fat diet (HFD) model of painful diabetic neuropathy (PDN). Electrophysiological studies showed that NaV1.8-positive DRG neurons taken from HFD-treated mice displayed a more excitable phenotype than their matched controls on a regular diet, and that this state of hyperexcitability could be inhibited through activation of hM4Di by CNO. In vivo acute activation of hM4Di produced rapid inhibition of mechanical allodynia in this model. HFD-treated mice also developed a number of other phenotypes that are normally associated with PDN in humans, primarily the dying back of their cutaneous innervation (Jayaraj et al., 2018).

Jayaraj et al. also examined how long-term activation of hM4Di, achieved through the continuous infusion of CNO using an osmotic minipump, affected the disease. The authors observed that this procedure not only prevented HFD-induced degeneration of cutaneous innervation but also, by starting the activation of hM4Di activation 10 weeks following the commencement of HFD, actually reversed the degeneration once it had been established. This “disease-modifying” effect has important implications for the treatment of diabetic neuropathy: The symptoms of the disease not only can be halted but also can be reversed with the appropriate treatment. Because activation of GPCR signaling has many downstream consequences, it is not clear how these long-term effects of hM4Di activation are mediated, that is, whether they are merely the result of the inhibition of neuronal hyperexcitability or some other signaling event. However, the former possibility is favored by the further observations of Jayaraj et al. that activation of hM3Dq receptors expressed in the NaV1.8 population increased excitability of these neurons and produced a phenotype in which the degeneration of cutaneous innervation observed with the HFD now occurred at earlier times in the treatment (Jayaraj et al., 2018).

Overall, these results appear to demonstrate the usefulness of DREADD-based manipulation of DRG neurons in investigations of pain. Nevertheless, a note of caution has been raised by Saloman et al., who expressed hM4Di in DRG neurons under the control of the TRPV1 promoter (Saloman et al., 2016). Whereas baseline heat thresholds in both male and female mice expressing this Gi-DREADD were normal, a single injection of CNO increased the heat threshold, an effect that returned to baseline 5 hours later. Consistent with these behavioral results, and the report by Jayaraj et al. (2018), CNO decreased action potential firing in isolated sensory neurons from hM4Di mice. Saloman et al. also observed, however, that expression of hM4Di receptors produced changes in the properties of voltage-gated Ca2+ and Na+ currents, as well as different signaling pathways, even in the absence of CNO (Saloman et al., 2016), which highlights potential limitations in the technology.

Further concerns were raised in a recent study published in Science (Gomez et al., 2017). Gomez and coworkers reported that the CNO metabolite clozapine, to which CNO is rapidly converted in vivo, shows high affinity for DREADD receptors and can activate them at much lower concentrations than CNO itself. It was proposed that clozapine was actually responsible for many of the DREADD-mediated effects of CNO administration to live animals. Moreover, as clozapine is an active antipsychotic drug that has high affinity for a number of important neurotransmitter receptors, these actions could also complicate the interpretation of CNO effects observed in vivo. Dr. Gold, the senior author of the Salomon study was so horrified by these results that in an online interview with The Scientist newspaper, he declared, “I think it’s a real concern. People continue to use these tools under the assumption they are not having off-target effects.” But, he said, his results “condemn” DREADDs as a strategy, and his laboratory has since dropped them. “We’re waving the red flag,” he added (Grens, 2017). Whether this is really good advice or whether it amounts to throwing the baby out with the proverbial bathwater is something that individual investigators will have to decide. Nevertheless, at the very least, they do underline the importance of performing the correct controls and also raise the possibility that, when expressed at high levels, DREADD receptors, like most GPCRs, might display some degree of constitutive (i.e., ligand-independent) activity.

In addition to peripheral nociceptors, an increasing number of investigators have begun to use DREADDs to dissect out the role of populations of neurons higher up the neuraxis in the physiology of pain perception. For example, Francois et al. examined the role in pain processing of enkephalin-expressing neurons in the dorsal horn of the spinal cord. In order to do this, they expressed hM4Di in these neurons. Administration of CNO to these mice produced a rapid pain phenotype in which mice began to flinch, bite, and lick their paws. As the enkephalinergic population of neurons contains both glutaminergic and GABAergic (mediated by γ-aminobutyric acid [GABA]) cells, the authors next restricted the expression of hMD4i to the GABAergic subtype using a vesicular GABA transporter (Vgat)–Cre and observed that CNO again produced a similar set of pain behaviors. On the other hand, inhibition or deletion of spinal excitatory neurons was antinociceptive, suggesting that the pain behavior that results from enkephalinergic interneuron inhibition is due to the GABAergic subpopulation (Francois et al., 2017). Hence, the use of chemogenetic methods allowed the investigators to conclude that GABAergic brainstem neurons regulated the release of the endogenous opioid enkephalin in the spinal cord to modulate inputs from sensory pain fibers. This conclusion has been supported by the work of Koga et al., who also expressed hM4Di in GABAergic inhibitory interneurons in the spinal dorsal horn and observed that CNO injections resulted in the rapid appearance of nocifensive behaviors (Koga et al., 2017).

Reports such as these illustrate the increasing number of studies in which expression of DREADD receptors in the central nervous system has been used to dissect out the role of different subsets of neurons in pain physiology. This includes gastrin-releasing peptide neurons in the dorsal horn (Albisetti et al., 2019), dopaminergic neurons in the ventral tegmental area (Wakaizumi et al., 2016), hypothalamic orexinergic neurons (Zhou et al., 2018), and neurons expressing corticotropin-releasing factor (CRF) in the prefrontal cortex (Andreoli, Marketkar, & Dimitrov, 2017). Remarkably, these techniques even enable dissecting out negative affective aspects of pain, such as in a 2019 study by Corder et al., where a combination of time-lapse in vivo calcium imaging and neural activity manipulation in freely behaving mice identified a distinct set of neurons in the basolateral amygdala that encodes the negative affective valence of pain. Chemogenetic silencing of these neurons alleviated pain affective–motivational behaviors without altering the detection of noxious stimuli (Corder et al., 2019).

Overall, one cannot help but feel that the use of the DREADD platform in pain research is in its infancy. In many respects, DREADDs are ideal tools for probing the role of different neuronal populations in pain-related behaviors. One should also appreciate that GPCR signaling is extremely complex and includes phenomena such as biased agonism, which may hold the key to the production of next-generation analgesics. As it should be possible to produce DREADD-like molecules that reproduce different patterns of GPCR signaling, it is anticipated that molecular tools of this type will be extremely revealing when applied to pain research.

Chemogenetic Ligand-Gated Ion Channels

Activation of DREADDs is clearly a useful method for controlling neuronal activity. However, it will be appreciated that activation of a GPCR results in the downstream regulation of a large number of signaling pathways involving kinases, transcription factors, and other targets, in addition to the rapid regulation of ion channels. Hence, the effects of activating DREADDs, especially when this is done chronically, may be difficult to anticipate mechanistically. Nevertheless, activation of DREADDs has the advantage of indicating the possible effects of novel drugs designed to activate GPCRs, a common strategy in drug development. However, if investigators wish solely to manipulate neuronal excitability through regulation of ion channels, there are also chemogenetic strategies based on the modification of ligand-gated ion channels that perform this task more directly.

In its current iteration, chemogenetic control of ligand-gated ion channels utilizes the extracellular ligand-binding domain (LBD) of the α7 nicotinic acetylcholine receptor (nAChR) transplanted onto the ion translocating pore domain (IPD) of another member of the large Cys-loop receptor ion channel family. For example, attaching the α7 nAChR LBD to the pore domain of the serotonin receptor (5-HT3a) produces a channel (α7-5-HT3) with α7 nAChR pharmacology and 5-HT3a cation conductance properties (Eisele et al., 1993). Activation of these receptors produces rapid neuronal depolarization.

On the other hand, attaching the α7 nAChR LBD to the pore of the chloride-selective glycine receptor (GlyR), which renders an acetylcholine-responsive chloride channel (α7-GlyR), allows for rapid neuronal hyperpolarization. Of course, because these nicotinic receptor hybrids would normally be activated by endogenous acetylcholine, in practice what is used is a mutated α7 domain that can only be activated by a nonendogenous small molecule, just as DREADD receptors are activated by CNO/clozapine. Mutated LBDs are called pharmacologically selective actuator modules (PSAMs) (Figure 2). Many different versions of these can be produced depending on the mutation introduced into the LBD. The agonists that activate each PSAM are known as pharmacologically selective effector molecules (PSEMs). Just like DREADDs, PSAM/PSEM pairs can be used to investigate neuronal pathways underlying pain behaviors. Ren et al. used this strategy to investigate the role of GABAergic indirect spiny projection neurons (iSPNs) in the nucleus accumbens in mechanical allodynia in mice expressing excitatory PSAM (PSAM–5-HT3) and inhibitory PSAM (PSAM-GlyR) receptors. The administration of the appropriate PSEM to activate or inhibit these neurons, respectively, resulted in the effect of increasing or decreasing mechanical allodynia (Ren et al., 2016).

Although the PSAM/PSEM system represents an interesting variation of the chemogenetic approach, apart from the investigation discussed, it has not yet been widely employed in pain research. However, advances continue to be made for the utilization of this chemogenetic program, including the development of new ultrapotent PSEMs, some of which are currently available clinically used drugs. This development has encouraged the view that it may be possible to translate the PSAM/PSEM system for use in human subjects (Magnus et al., 2019).

Chemo- and Optogenetic Strategies for the Elucidation of Pain Pathways

Figure 2. Chemogenetic ligand-gated ion channels. Chimeric PSAM (pharmacologically selective actuator module)–based ligand-gated ion channels: PSAM–5-HT3 or PSAM-GlyR (glycine receptor) acutely modulate cation or chloride membrane conductances when activated by their cognate synthetic ligands (PSEMs, pharmacologically selective effector molecules). This results in rapid, reversible activation or inhibition of neuronal activity, respectively.

Optogenetics

Optogenetics refers to a set of techniques in which signaling proteins can be activated by light, as opposed to small chemical ligands, an approach initially proposed by Frances Crick and first achieved by Miesenböck in 2002 (see Miesenbock & Kevrekidis, 2005).

By analogy with chemogenetics, optogenetics allows the introduction of light-sensitive proteins called opsins into specific neuronal populations. Depending on the properties of the opsin selected, optogenetics can be used to increase or decrease neuronal activity in vivo. For example, channelrhodopsin is activated by blue light and triggers depolarization through the influx of cations, whereas halorhodopsin is activated by yellow light and hyperpolarizes neurons through anion influx (Zhang et al., 2011). We focus on two main uses of optogenetics in pain: in investigation of pathways involved in nociception and chronic pain and their potential for therapeutic use.

Circuit Investigation

One of the first studies to employ optogenetics for probing of pain pathways in freely moving mice was published over 5 years ago by Philippe Séguéla and his colleagues at McGill University (Daou et al., 2013). In the study, a subset of primary afferent neurons expressing NaV1.8 were selectively targeted to express channelrhodopsin-2 (ChR2) channels. Blue light was used acutely to activate NaV1.8+ sensory neurons and was shown to elicit nocifensive behaviors. Selective activation of these afferents with light could induce central sensitization and conditioned place aversion in behavioral assays. Spinal cord isolated from these animals after receiving 10 minutes of blue light stimulation of afferent nerve fibers exhibited increased c-fos levels in the superficial dorsal horn, indicative of increased neuronal activity. More recently, another study delivered blue light via an epidural implant to the spinal dorsal horn of Nav1.8-ChR2+ mice and achieved similar results (Bonin et al., 2016). As expected, silencing of these same neurons optogenetically can reduce pain hypersensitivity in neuropathic and inflammatory pain states (Daou et al., 2016).

Since Séguéla’s studies, there have been dozens more employing optogenetics to study pain pathways in the periphery, brain, and spinal cord and to ask more specific questions regarding the specific subsets of neurons involved in different aspects of a pain response and in a variety of chronic pain conditions. For example, optogenetic activation of the central amygdala (CeA) causes an increase in visceral pain (Crock et al., 2012); optogenetic activation of inhibitory neurons in the anterior cingulate cortex (ACC) reduces mechanical hypersensitivity (Gu et al., 2015); and optoactivation of parvalbumin-positive neurons of the spinal dorsal horn evokes GABA release (Yang, Ma, Wang, Jiang, & Li, 2015).

The technique has also been used to resolve the repertoire of rapid protective responses to nociceptive stimuli at a millisecond timescale. Using a single pulse of light to activate nociceptors (TRPV1+ or substance P+ neurons) in freely behaving mice coupled with high-speed sampling video motion detection of mouse behaviors, it was possible to dissect the temporal relationship between a specific nociceptor input and its behavioral output (Browne et al., 2017). Such experiments would not be feasible without optogenetic stimulation.

An optical fiber-based nerve cuff has been designed for chronic stimulation of the peripheral nerve in freely behaving mice (Michoud et al., 2018). This is an improvement on the traditional polyethylene cuff that restricts the peripheral nerve in popular pain models, such as for chronic constriction injury (Mosconi & Kruger, 1996), where the injury is not very robust, and the animal is prone to recovery, thus complicating experimental results. The “optocuff” is made of soft, tubular material and is designed to be wrapped around the peripheral nerve. An optical fiber stemming from a miniaturized headstage that passes under the skin to the peripheral nerve is attached to the optocuff to deliver light for stimulation of the nerve. This allows for stricter control of peripheral neuron activity while the animal is freely behaving.

Optogenetics can also be combined with electrophysiology to allow interrogation of neuronal circuits both ex vivo and in vivo. For example, a wireless headstage system has been developed that can trigger neuronal activity optogenetically and then record and compress evoked neuronal signals from several channels at the same time in freely behaving ChR2 transgenic mice (Gagnon-Turcotte et al., 2017). Using this system, it is also possible to program optical stimulation patterns.

Neurons are not the only potential target of optogenetic manipulation. Recently, ChR2-expressing astrocytes of the spinal cord were stimulated optogenetically, leading to induction of pain hypersensitivity in vivo (Nam et al., 2016). In cultured cells and in spinal cord slices from these animals, optogenetic stimulation of astrocytes led to release of adenosine triphosphate (ATP), pro-inflammatory mediators, and disinhibition of projection neurons of the dorsal horn. This technique was very selective, as inward currents were only directed in astrocytes and not in dorsal horn neurons.

Moreover, optogenetics has been used to probe the two-way communication that exists between cells in the skin epidermis and cutaneous innervation. Two reports have examined this using either channelrhodopsin or archaerhodopsin to depolarize or hyperpolarize keratinocytes (Baumbauer et al., 2015; Moehring et al., 2018). These studies clearly indicated that changes in keratinocyte membrane potential altered the secretion of algogenic factors that regulated the excitability of cutaneous afferents, including nociceptors. In particular, they identified ATP-mediated (purinergic) signaling as controlling this interaction through the activation of P2X4 receptors expressed by nociceptors.

Another type of optogenetics, called OptoXR, has been developed that allows manipulation of receptor-initiated biochemical signaling pathways, but has yet to be used in pain studies (Airan, Thompson, Fenno, Bernstein, & Deisseroth, 2009). OptoXRs are opsin/GPCR chimeras; for example, opto-α1AR is a combination of rhodopsin and the α1-adrenergic receptor, and it can be used to transduce light to activate phospholipase C and increase intracellular inositol triphosphate (IP3) and diacylglycerol (DAG) levels. OptoXRs have been expressed in nucleus accumbens neurons to drive place preference behaviors in freely moving mice.

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system is an extremely popular technique for genome editing, and it has been proposed as a novel strategy for manipulating genes involved in chronic pain (Sun, Lutz, & Tao, 2016). The system consists of two main components: a guide RNA (gRNA), which recognizes the target DNA, and a Cas9 nuclease, which creates double-stranded breaks in the target sequence. Photoactivatable CRISPR systems have been developed that allow one to switch gene expression on or off with light. The system developed by Nihongaki et al. makes use of two fusion proteins where, in response to blue light, the proteins heterodimerize to move a transcriptional activator into position and initiate gene transcription (Nihongaki, Yamamoto, Kawano, Suzuki, & Sato, 2015).

Potential for Therapeutic Use

It goes without saying that all of the previously mentioned studies interrogating circuits involved in nociception are aimed toward the development of more effective therapies for treatment, some more directly aimed than others. A recent article demonstrated that a miniaturized bio-optoelectronic system in mice can be used for the modulation of sensory afferents that innervate the bladder to help treat conditions such as overactive bladder, urinary incontinence, and bladder pain syndrome (Mickle et al., 2019). A herpes simplex viral (HSV) vector containing a construct for expression of archaerhodopsin 3.0 (Arch) tagged with enhanced yellow fluorescent protein (eYFP) is injected directly into the bladder wall so that Arch is expressed in sensory neurons innervating the bladder. The closed-loop bio-optoelectronic system relies on coordination between a biophysical sensor for feedback control and a proximal light source for optogenetic stimulation. In practice, the device can detect dysfunctional bladder activity and normalize it by dampening sacral nerve activity. One can imagine that this sort of device would be extremely adaptable to humans with bladder pain disorders.

There is active interest in the use of optogenetics in patients. Circuit Therapeutics Incorporated is one of a few optogenetics companies that are squarely focused on the potential for optogenetics-based treatments for a host of neurological diseases, such as Parkinson disease and chronic pain. In addition, there is an ongoing Phase I/II clinical trial attempting to express ChR2 in the retina of patients with retinitis pigmentosa (https://clinicaltrials.gov/ct2/show/NCT02556736). The potential treatment is a gene therapy involving an injection of RST-001 to the eye.

With regard to pain research, a strategy involving delivery of a virus to a specific neuronal circuit is probably the most promising. The development of “minipromoters” (small promoters) may represent a very important advance in this regard as it allows the packaging of more genetic material into recombinant AAVs (de Leeuw et al., 2016). One must also consider the use of an effective light delivery system, and light-emitting diodes (LEDs) may be the most suitable for this. It is clear that significant progress is being made in both of these realms, and that an optogenetic method for treatment of chronic pain is most definitely possible.

Gene Therapy for Pain

In theory, one could silence the neurons in the basolateral amygdala involved in conferring unpleasantness of pain (Corder et al., 2019) or dampen the excitability of sensory neurons involved in peripheral neuropathies to treat different types of pain or the more troubling aspect of a particular patient’s pain. Because pain is really an individual “experience,” and different patients suffer different types of pain, a personalized approach to treatment is of particular interest. As mentioned, the use of chemo- or optogenetics as treatment strategies for pain would involve virally mediated expression of DREADDs or light-sensitive opsins in neurons of importance in specific pain pathways. This constitutes a form of gene therapy.

Gene therapy as an approach for treatment of chronic pain has been discussed previously (see review by Guedon et al., 2015); however, most studies using viral-gene transfer for chronic pain involve overexpression of pain-alleviating molecules directly, such as GABA (Chattopadhyay, Mata, & Fink, 2011) or opioids (Braz et al., 2001). While these are the most direct approaches, they suffer from lack of reversibility and the control possible with chemo- or optogenetic techniques as one cannot specify precisely which cells or when cells are activated or silenced. One could imagine that if GABA or opioid expression were too high following viral-gene transfer that these could have dangerous long-term side effects. In the case of DREADDs or opsins, these genes are not normally expressed in humans, so there is perhaps less risk. One could then carefully control delivery of ligands or light to control the effect on the particular patient. Of course, there is still a very high risk of complication with any gene therapy.

References

Airan, R. D., Thompson, K. R., Fenno, L. E., Bernstein, H., & Deisseroth, K. (2009). Temporally precise in vivo control of intracellular signalling. Nature, 458(7241), 1025–1029. doi:10.1038/nature07926Find this resource:

Albisetti, G. W., Pagani, M., Platonova, E., Hosli, L., Johannssen, H. C., Fritschy, J. M., … Zeilhofer, H. U. (2019). Dorsal horn gastrin-releasing peptide expressing neurons transmit spinal itch but not pain signals. The Journal of Neuroscience, 39(12), 2238–2250. doi:10.1523/JNEUROSCI.2559-18.2019Find this resource:

Andreoli, M., Marketkar, T., & Dimitrov, E. (2017). Contribution of amygdala CRF neurons to chronic pain. Experimental Neurology, 298(Pt. A), 1–12. doi:10.1016/j.expneurol.2017.08.010Find this resource:

Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S., & Roth, B. L. (2007). Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proceedings of the National Academy of Sciences of the United States of America, 104(12), 5163–5168. doi:10.1073/pnas.0700293104Find this resource:

Basbaum, A. I., Bautista, D. M., Scherrer, G., & Julius, D. (2009). Cellular and molecular mechanisms of pain. Cell, 139(2), 267–284. doi:10.1016/j.cell.2009.09.028Find this resource:

Baumbauer, K. M., DeBerry, J. J., Adelman, P. C., Miller, R. H., Hachisuka, J., Lee, K. H., … Albers, K. M. (2015). Keratinocytes can modulate and directly initiate nociceptive responses. Elife, 4. doi:10.7554/eLife.09674Find this resource:

Bonin, R. P., Wang, F., Desrochers-Couture, M., Ga Secka, A., Boulanger, M. E., Cote, D. C., & De Koninck, Y. (2016, March 9). Epidural optogenetics for controlled analgesia. Molecular Pain, 12. doi:10.1177/1744806916629051Find this resource:

Braz, J., Beaufour, C., Coutaux, A., Epstein, A. L., Cesselin, F., Hamon, M., & Pohl, M. (2001). Therapeutic efficacy in experimental polyarthritis of viral-driven enkephalin overproduction in sensory neurons. The Journal of Neuroscience, 21(20), 7881–7888.Find this resource:

Browne, L. E., Latremoliere, A., Lehnert, B. P., Grantham, A., Ward, C., Alexandre, C., … Woolf, C. J. (2017). Time-resolved fast mammalian behavior reveals the complexity of protective pain responses. Cell Reports, 20(1), 89–98. doi:10.1016/j.celrep.2017.06.024Find this resource:

Chattopadhyay, M., Mata, M., & Fink, D. J. (2011). Vector-mediated release of GABA attenuates pain-related behaviors and reduces Na(V)1.7 in DRG neurons. European Journal of Pain, 15(9), 913–920. doi:10.1016/j.ejpain.2011.03.007Find this resource:

Chen, X., Choo, H., Huang, X. P., Yang, X., Stone, O., Roth, B. L., & Jin, J. (2015). The first structure-activity relationship studies for designer receptors exclusively activated by designer drugs. ACS Chemical Neuroscience, 6(3), 476–484. doi:10.1021/cn500325vFind this resource:

Corder, G., Ahanonu, B., Grewe, B. F., Wang, D., Schnitzer, M. J., & Scherrer, G. (2019). An amygdalar neural ensemble that encodes the unpleasantness of pain. Science, 363(6424), 276–281. doi:10.1126/science.aap8586Find this resource:

Coward, P., Wada, H. G., Falk, M. S., Chan, S. D., Meng, F., Akil, H., & Conklin, B. R. (1998). Controlling signaling with a specifically designed Gi-coupled receptor. Proceedings of the National Academy of Sciences of the United States of America, 95(1), 352–357.Find this resource:

Crock, L. W., Kolber, B. J., Morgan, C. D., Sadler, K. E., Vogt, S. K., Bruchas, M. R., & Gereau, R. W. t. (2012). Central amygdala metabotropic glutamate receptor 5 in the modulation of visceral pain. The Journal of Neuroscience, 32(41), 14217–14226. doi:10.1523/JNEUROSCI.1473-12.2012Find this resource:

Daou, I., Beaudry, H., Ase, A. R., Wieskopf, J. S., Ribeiro-da-Silva, A., Mogil, J. S., & Seguela, P. (2016). Optogenetic silencing of Nav1.8-positive afferents alleviates inflammatory and neuropathic pain. eNeuro, 3(1), ENEURO.0140-15.2916. doi:10.1523/ENEURO.0140-15.2016Find this resource:

Daou, I., Tuttle, A. H., Longo, G., Wieskopf, J. S., Bonin, R. P., Ase, A. R., … Seguela, P. (2013). Remote optogenetic activation and sensitization of pain pathways in freely moving mice. The Journal of Neuroscience, 33(47), 18631–18640. doi:10.1523/JNEUROSCI.2424-13.2013Find this resource:

de Leeuw, C. N., Korecki, A. J., Berry, G. E., Hickmott, J. W., Lam, S. L., Lengyell, T. C., … Simpson, E. M. (2016). rAAV-compatible minipromoters for restricted expression in the brain and eye. Molecular Brain, 9(1), 52. doi:10.1186/s13041-016-0232-4Find this resource:

Eisele, J. L., Bertrand, S., Galzi, J. L., Devillers-Thiery, A., Changeux, J. P., & Bertrand, D. (1993). Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities. Nature, 366(6454), 479–483. doi:10.1038/366479a0Find this resource:

Francois, A., Low, S. A., Sypek, E. I., Christensen, A. J., Sotoudeh, C., Beier, K. T., … Scherrer, G. (2017). A brainstem-spinal cord inhibitory circuit for mechanical pain modulation by GABA and enkephalins. Neuron, 93(4), 822–839.e826. doi:10.1016/j.neuron.2017.01.008Find this resource:

Gagnon-Turcotte, G., LeChasseur, Y., Bories, C., Messaddeq, Y., De Koninck, Y., & Gosselin, B. (2017). A wireless headstage for combined optogenetics and multichannel electrophysiological recording. IEEE Transactions on Biomedical Circuits and Systems, 11(1), 1–14. doi:10.1109/TBCAS.2016.2547864Find this resource:

Gomez, J. L., Bonaventura, J., Lesniak, W., Mathews, W. B., Sysa-Shah, P., Rodriguez, L. A., … Michaelides, M. (2017). Chemogenetics revealed: DREADD occupancy and activation via converted clozapine. Science, 357(6350), 503–507. doi:10.1126/science.aan2475Find this resource:

Grens, K. (2017). Chemogenetics doesn’t work like many thought. The Scientist, August 4.Find this resource:

Gu, L., Uhelski, M. L., Anand, S., Romero-Ortega, M., Kim, Y. T., Fuchs, P. N., & Mohanty, S. K. (2015). Pain inhibition by optogenetic activation of specific anterior cingulate cortical neurons. PLoS One, 10(2), e0117746. doi:10.1371/journal.pone.0117746Find this resource:

Guedon, J. M., Wu, S., Zheng, X., Churchill, C. C., Glorioso, J. C., Liu, C. H., … Hao, S. (2015). Current gene therapy using viral vectors for chronic pain. Molecular Pain, 11, 27. doi:10.1186/s12990-015-0018-1Find this resource:

Iyer, S. M., Vesuna, S., Ramakrishnan, C., Huynh, K., Young, S., Berndt, A., … Delp, S. L. (2016). Optogenetic and chemogenetic strategies for sustained inhibition of pain. Scientific Reports, 6, 30570. doi:10.1038/srep30570Find this resource:

Jayaraj, N. D., Bhattacharyya, B. J., Belmadani, A. A., Ren, D., Rathwell, C. A., Hackelberg, S., … Menichella, D. M. (2018). Reducing CXCR4-mediated nociceptor hyperexcitability reverses painful diabetic neuropathy. The Journal of Clinical Investigation, 128(6), 2205–2225. doi:10.1172/JCI92117Find this resource:

Koga, K., Kanehisa, K., Kohro, Y., Shiratori-Hayashi, M., Tozaki-Saitoh, H., Inoue, K., … Tsuda, M. (2017). Chemogenetic silencing of GABAergic dorsal horn interneurons induces morphine-resistant spontaneous nocifensive behaviours. Scientific Reports, 7(1), 4739. doi:10.1038/s41598-017-04972-3Find this resource:

Magnus, C. J., Lee, P. H., Bonaventura, J., Zemla, R., Gomez, J. L., Ramirez, M. H., … Sternson, S. M. (2019). Ultrapotent chemogenetics for research and potential clinical applications. Science, 364(6436). doi:10.1126/science.aav5282Find this resource:

Michoud, F., Sottas, L., Browne, L. E., Asboth, L., Latremoliere, A., Sakuma, M., … Lacour, S. P. (2018). Optical cuff for optogenetic control of the peripheral nervous system. Journal of Neural Engineering, 15(1), 015002. doi:10.1088/1741-2552/aa9126Find this resource:

Mickle, A. D., Won, S. M., Noh, K. N., Yoon, J., Meacham, K. W., Xue, Y., … Rogers, J. A. (2019). A wireless closed-loop system for optogenetic peripheral neuromodulation. Nature, 565(7739), 361–365. doi:10.1038/s41586-018-0823-6Find this resource:

Miesenböck, G., & Kevrekidis, I. G. (2005). Optical imaging and control of genetically designated neurons in functioning circuits. Annual Review of Neuroscience, 28, 533–563. doi:10.1146/annurev.neuro.28.051804.101610Find this resource:

Miller, R. E., Ishihara, S., Bhattacharyya, B., Delaney, A., Menichella, D. M., Miller, R. J., & Malfait, A. M. (2017). Chemogenetic inhibition of pain neurons in a mouse model of osteoarthritis. Arthritis & Rheumatology (Hoboken, NJ), 69(7), 1429–1439. doi:10.1002/art.40118Find this resource:

Moehring, F., Cowie, A. M., Menzel, A. D., Weyer, A. D., Grzybowski, M., Arzua, T., … Stucky, C. L. (2018). Keratinocytes mediate innocuous and noxious touch via ATP-P2X4 signaling. Elife, 7. doi:10.7554/eLife.31684Find this resource:

Mosconi, T., & Kruger, L. (1996). Fixed-diameter polyethylene cuffs applied to the rat sciatic nerve induce a painful neuropathy: Ultrastructural morphometric analysis of axonal alterations. Pain, 64(1), 37–57.Find this resource:

Nam, Y., Kim, J. H., Kim, J. H., Jha, M. K., Jung, J. Y., Lee, M. G., … Suk, K. (2016). Reversible induction of pain hypersensitivity following optogenetic stimulation of spinal astrocytes. Cell Reports, 17(11), 3049–3061. doi:10.1016/j.celrep.2016.11.043Find this resource:

Nihongaki, Y., Yamamoto, S., Kawano, F., Suzuki, H., & Sato, M. (2015). CRISPR-Cas9-based photoactivatable transcription system. Chemistry and Biology, 22(2), 169–174. doi:10.1016/j.chembiol.2014.12.011Find this resource:

Ren, W., Centeno, M. V., Berger, S., Wu, Y., Na, X., Liu, X., … Surmeier, D. J. (2016). The indirect pathway of the nucleus accumbens shell amplifies neuropathic pain. Nature Neuroscience, 19(2), 220–222. doi:10.1038/nn.4199Find this resource:

Rosenbaum, D. M., Rasmussen, S. G., & Kobilka, B. K. (2009). The structure and function of G-protein-coupled receptors. Nature, 459(7245), 356–363. doi:10.1038/nature08144Find this resource:

Roth, B. L. (2016). DREADDs for neuroscientists. Neuron, 89(4), 683–694. doi:10.1016/j.neuron.2016.01.040Find this resource:

Saloman, J. L., Scheff, N. N., Snyder, L. M., Ross, S. E., Davis, B. M., & Gold, M. S. (2016). Gi-DREADD expression in peripheral nerves produces ligand-dependent analgesia, as well as ligand-independent functional changes in sensory neurons. The Journal of Neuroscience, 36(42), 10769–10781. doi:10.1523/JNEUROSCI.3480-15.2016Find this resource:

Shields, S. D., Ahn, H. S., Yang, Y., Han, C., Seal, R. P., Wood, J. N., … Dib-Hajj, S. D. (2012). Nav1.8 expression is not restricted to nociceptors in mouse peripheral nervous system. Pain, 153(10), 2017–2030. doi:10.1016/j.pain.2012.04.022Find this resource:

Strader, C. D., Gaffney, T., Sugg, E. E., Candelore, M. R., Keys, R., Patchett, A. A., & Dixon, R. A. (1991). Allele-specific activation of genetically engineered receptors. The Journal of Biological Chemistry, 266(1), 5–8.Find this resource:

Sun, L., Lutz, B. M., & Tao, Y. X. (2016). The CRISPR/Cas9 system for gene editing and its potential application in pain research. Translational Perioperative and Pain Medicine, 1(3), 22–33.Find this resource:

Vardy, E., Robinson, J. E., Li, C., Olsen, R. H. J., DiBerto, J. F., Giguere, P. M., … Roth, B. L. (2015). A new DREADD facilitates the multiplexed chemogenetic interrogation of behavior. Neuron, 86(4), 936–946. doi:10.1016/j.neuron.2015.03.065Find this resource:

Wakaizumi, K., Kondo, T., Hamada, Y., Narita, M., Kawabe, R., Narita, H., … Narita, M. (2016). Involvement of mesolimbic dopaminergic network in neuropathic pain relief by treadmill exercise: A study for specific neural control with Gi-DREADD in mice. Molecular Pain, Dec 1, 12. doi:10.1177/1744806916681567Find this resource:

Yang, K., Ma, R., Wang, Q., Jiang, P., & Li, Y. Q. (2015). Optoactivation of parvalbumin neurons in the spinal dorsal horn evokes GABA release that is regulated by presynaptic GABAB receptors. Neuroscience Letters, 594, 55–59. doi:10.1016/j.neulet.2015.03.050Find this resource:

Zhang, F., Vierock, J., Yizhar, O., Fenno, L. E., Tsunoda, S., Kianianmomeni, A., … Deisseroth, K. (2011). The microbial opsin family of optogenetic tools. Cell, 147(7), 1446–1457. doi:10.1016/j.cell.2011.12.004Find this resource:

Zhou, W., Cheung, K., Kyu, S., Wang, L., Guan, Z., Kurien, P. A., … Jan, L. Y. (2018). Activation of orexin system facilitates anesthesia emergence and pain control. Proceedings of the National Academy of Sciences of the United States of America, 115(45), E10740–E10747. doi:10.1073/pnas.1808622115Find this resource: