Bryan A. Copits

Soft, stretchable, fully implantable miniaturized optoelectronic systems for wireless optogenetics

Optogenetics allows rapid, temporally specific control of neuronal activity by targeted expression and activation of light-sensitive proteins. Implementation typically requires remote light sources and fiber-optic delivery schemes that impose considerable physical constraints on natural behaviors. In this report we bypass these limitations using technologies that combine thin, mechanically soft neural interfaces with fully implantable, stretchable wireless radio power and control systems. The resulting devices achieve optogenetic modulation of the spinal cord and peripheral nervous system. This is demonstrated with two form factors; stretchable film appliqués that interface directly with peripheral nerves, and flexible filaments that insert into the narrow confines of the spinal epidural space. These soft, thin devices are minimally invasive, and histological tests suggest they can be used in chronic studies. We demonstrate the power of this technology by modulating peripheral and spinal pain circuitry, providing evidence for the potential widespread use of these devices in research and future clinical applications of optogenetics outside the brain.

Human sensory neurons: Membrane properties and sensitization by inflammatory mediators

Summary Naïve hDRG neurons cultured from young adults without chronic pain are highly chemosensitive to algogens and pruritogens, and can be sensitized by inflammatory mediators. Small‐ to medium‐size hDRG neurons exhibit AP waveforms similar to what has been described for rodent nociceptors. Cultured hDRG neurons represent a more direct way of studying analgesic translational efficacy and confirming target validation before initiating expensive and time‐consuming clinical trials. ABSTRACT Biological differences in sensory processing between human and model organisms may present significant obstacles to translational approaches in treating chronic pain. To better understand the physiology of human sensory neurons, we performed whole‐cell patch‐clamp recordings from 141 human dorsal root ganglion (hDRG) neurons from 5 young adult donors without chronic pain. Nearly all small‐diameter hDRG neurons (<50 &mgr;m) displayed an inflection on the descending slope of the action potential, a defining feature of rodent nociceptive neurons. A high proportion of hDRG neurons were responsive to the algogens allyl isothiocyanate (AITC) and ATP, as well as the pruritogens histamine and chloroquine. We show that a subset of hDRG neurons responded to the inflammatory compounds bradykinin and prostaglandin E2 with action potential discharge and show evidence of sensitization including lower rheobase. Compared to electrically evoked action potentials, chemically induced action potentials were triggered from less depolarized thresholds and showed distinct afterhyperpolarization kinetics. These data indicate that most small/medium hDRG neurons can be classified as nociceptors, that they respond directly to compounds that produce pain and itch, and that they can be activated and sensitized by inflammatory mediators. The use of hDRG neurons as preclinical vehicles for target validation is discussed.

Voluntary Exercise Training: Analysis of Mice in Uninjured, Inflammatory, and Nerve-Injured Pain States.

Both clinical and animal studies suggest that exercise may be an effective way to manage inflammatory and neuropathic pain conditions. However, existing animal studies commonly use forced exercise paradigms that incorporate varying degrees of stress, which itself can elicit analgesia, and thus may complicate the interpretation of the effects of exercise on pain. We investigated the analgesic potential of voluntary wheel running in the formalin model of acute inflammatory pain and the spared nerve injury model of neuropathic pain in mice. In uninjured, adult C57BL/6J mice, 1 to 4 weeks of exercise training did not alter nociceptive thresholds, lumbar dorsal root ganglia neuronal excitability, or hindpaw intraepidermal innervation. Further, exercise training failed to attenuate formalin-induced spontaneous pain. Lastly, 2 weeks of exercise training was ineffective in reversing spared nerve injury-induced mechanical hypersensitivity or in improving muscle wasting or hindpaw denervation. These findings indicate that in contrast to rodent forced exercise paradigms, short durations of voluntary wheel running do not improve pain-like symptoms in mouse models of acute inflammation and peripheral nerve injury.

Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures

Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization (OPO). Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 d, and they are amenable to electrophysiological recording, calcium imaging and viral gene transfer. The entire process of extraction and culturing can be completed in <7 h, and it can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research, and is an invaluable resource for improving translational research.

Divergent modulation of nociception by glutamatergic and GABAergic neuronal subpopulations in the periaqueductal gray

Abstract The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pronociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here, we demonstrate the different contributions of genetically defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception.

Group II mGluRs suppress hyperexcitability in mouse and human nociceptors

Abstract We introduce a strategy for preclinical research wherein promising targets for analgesia are tested in rodent and subsequently validated in human sensory neurons. We evaluate group II metabotropic glutamate receptors, the activation of which is efficacious in rodent models of pain. Immunohistochemical analysis showed positive immunoreactivity for mGlu2 in rodent dorsal root ganglia (DRG), peripheral fibers in skin, and central labeling in the spinal dorsal horn. We also found mGlu2-positive immunoreactivity in human neonatal and adult DRG. RNA-seq analysis of mouse and human DRG revealed a comparative expression profile between species for group II mGluRs and for opioid receptors. In rodent sensory neurons under basal conditions, activation of group II mGluRs with a selective group II agonist produced no changes to membrane excitability. However, membrane hyperexcitability in sensory neurons exposed to the inflammatory mediator prostaglandin E2 (PGE2) was prevented by (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC). In human sensory neurons from donors without a history of chronic pain, we show that PGE2 produced hyperexcitability that was similarly blocked by group II mGluR activation. These results reveal a mechanism for peripheral analgesia likely shared by mice and humans and demonstrate a translational research strategy to improve preclinical validation of novel analgesics using cultured human sensory neurons.

Differential Regulation of Bladder Pain and Voiding Function by Sensory Afferent Populations Revealed by Selective Optogenetic Activation

Bladder-innervating primary sensory neurons mediate reflex-driven bladder function under normal conditions, and contribute to debilitating bladder pain and/or overactivity in pathological states. The goal of this study was to examine the respective roles of defined subtypes of afferent neurons in bladder sensation and function in vivo via direct optogenetic activation. To accomplish this goal, we generated transgenic lines that express a Channelrhodopsin-2-eYFP fusion protein (ChR2-eYFP) in two distinct populations of sensory neurons: TRPV1-lineage neurons (Trpv1Cre;Ai32, the majority of nociceptors) and Nav1.8+ neurons (Scn10aCre;Ai32, nociceptors and some mechanosensitive fibers). In spinal cord, eYFP+ fibers in Trpv1Cre;Ai32 mice were observed predominantly in dorsal horn (DH) laminae I-II, while in Scn10aCre;Ai32 mice they extended throughout the DH, including a dense projection to lamina X. Fiber density correlated with number of retrogradely-labeled eYFP+ dorsal root ganglion neurons (82.2% Scn10aCre;Ai32 vs. 62% Trpv1Cre;Ai32) and degree of DH excitatory synaptic transmission. Photostimulation of peripheral afferent terminals significantly increased visceromotor responses to noxious bladder distension (30–50 mmHg) in both transgenic lines, and to non-noxious distension (20 mmHg) in Scn10aCre;Ai32 mice. Depolarization of ChR2+ afferents in Scn10aCre;Ai32 mice produced low- and high-amplitude bladder contractions respectively in 53% and 27% of stimulation trials, and frequency of high-amplitude contractions increased to 60% after engagement of low threshold (LT) mechanoreceptors by bladder filling. In Trpv1Cre;Ai32 mice, low-amplitude contractions occurred in 27% of trials before bladder filling, which was pre-requisite for light-evoked high-amplitude contractions (observed in 53.3% of trials). Potential explanations for these observations include physiological differences in the thresholds of stimulated fibers and their connectivity to spinal circuits.

Macrophage-to-sensory neuron crosstalk mediated by Angiotensin II type-2 receptor elicits neuropathic pain

Peripheral nerve damage initiates a complex series of cellular and structural processes that culminate in chronic neuropathic pain. Our study defines local angiotensin signaling via activation of the Angiotensin II (Ang II) type-2 receptor (AT2R) on macrophages as the critical trigger of neuropathic pain. An AT2R-selective antagonist attenuates neuropathic, but not inflammatory pain hypersensitivity in mice, and requires the cell damage-sensing ion channel transient receptor potential family-A member-1 (TRPA1). Mechanical and cold pain hypersensitivity that are characteristic of neuropathic conditions can be attenuated by chemogenetic depletion of peripheral macrophages and AT2R-null hematopoietic cell transplantation. Our findings show no AT2R expression in mouse or human sensory neurons, rather AT2R expression and activation in macrophages triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on sensory neurons. Our study defines the precise neuro-immune crosstalk underlying nociceptor sensitization at the site of nerve injury. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic neuropathic pain, and therefore identifies multiple analgesic targets.