Surgical implantation of wireless, battery-free optoelectronic epidural implants for optogenetic manipulation of spinal cord circuits in mice.

The use of optogenetics to regulate neuronal activity has revolutionized the study of the neural circuitry underlying a number of complex behaviors in rodents. Advances have been particularly evident in the study of brain circuitry and related behaviors, while advances in the study of spinal circuitry have been less striking because of technical hurdles. We have developed and characterized a wireless and fully implantable optoelectronic device that enables optical manipulation of spinal cord circuitry in mice via a microscale light-emitting diode (µLED) placed in the epidural space (NeuroLux spinal optogenetic device). This protocol describes how to surgically implant the device into the epidural space and then analyze light-induced behavior upon µLED activation. We detail optimized optical parameters for in vivo stimulation and demonstrate typical behavioral effects of optogenetic activation of nociceptive spinal afferents using this device. This fully wireless spinal µLED system provides considerable versatility for behavioral assays compared with optogenetic approaches that require tethering of animals, and superior temporal and spatial resolution when compared with other methods used for circuit manipulation such as chemogenetics. The detailed surgical approach and improved functionality of these spinal optoelectronic devices substantially expand the utility of this approach for the study of spinal circuitry and behaviors related to mechanical and thermal sensation, pruriception and nociception. The surgical implantation procedure takes ~1 h. The time required for the study of behaviors that are modulated by the light-activated circuit is variable and will depend upon the nature of the study.

Myelinating Schwann cells ensheath multiple axons in the absence of E3 ligase component Fbxw7.

In the central nervous system (CNS), oligodendrocytes myelinate multiple axons; in the peripheral nervous system (PNS), Schwann cells (SCs) myelinate a single axon. Why are the myelinating potentials of these glia so fundamentally different? Here, we find that loss of Fbxw7, an E3 ubiquitin ligase component, enhances the myelinating potential of SCs. Fbxw7 mutant SCs make thicker myelin sheaths and sometimes appear to myelinate multiple axons in a fashion reminiscent of oligodendrocytes. Several Fbxw7 mutant phenotypes are due to dysregulation of mTOR; however, the remarkable ability of mutant SCs to ensheathe multiple axons is independent of mTOR signaling. This indicates distinct roles for Fbxw7 in SC biology including modes of axon interactions previously thought to fundamentally distinguish myelinating SCs from oligodendrocytes. Our data reveal unexpected plasticity in the myelinating potential of SCs, which may have important implications for our understanding of both PNS and CNS myelination and myelin repair.

Battery-free, fully implantable optofluidic cuff system for wireless optogenetic and pharmacological neuromodulation of peripheral nerves.

Studies of the peripheral nervous system rely on controlled manipulation of neuronal function with pharmacologic and/or optogenetic techniques. Traditional hardware for these purposes can cause notable damage to fragile nerve tissues, create irritation at the biotic/abiotic interface, and alter the natural behaviors of animals. Here, we present a wireless, battery-free device that integrates a microscale inorganic light-emitting diode and an ultralow-power microfluidic system with an electrochemical pumping mechanism in a soft platform that can be mounted onto target peripheral nerves for programmed delivery of light and/or pharmacological agents in freely moving animals. Biocompliant designs lead to minimal effects on overall nerve health and function, even with chronic use in vivo. The small size and light weight construction allow for deployment as fully implantable devices in mice. These features create opportunities for studies of the peripheral nervous system outside of the scope of those possible with existing technologies.

Macrophage angiotensin II type 2 receptor triggers neuropathic pain.

Peripheral nerve damage initiates a complex series of structural and cellular processes that culminate in chronic neuropathic pain. The recent success of a type 2 angiotensin II (Ang II) receptor (AT2R) antagonist in a phase II clinical trial for the treatment of postherpetic neuralgia suggests angiotensin signaling is involved in neuropathic pain. However, transcriptome analysis indicates a lack of AT2R gene (Agtr2) expression in human and rodent sensory ganglia, raising questions regarding the tissue/cell target underlying the analgesic effect of AT2R antagonism. We show that selective antagonism of AT2R attenuates neuropathic but not inflammatory mechanical and cold pain hypersensitivity behaviors in mice. Agtr2-expressing macrophages (MΦs) constitute the predominant immune cells that infiltrate the site of nerve injury. Interestingly, neuropathic mechanical and cold pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs and AT2R-null hematopoietic cell transplantation. Our study identifies AT2R on peripheral MΦs as a critical trigger for pain sensitization at the site of nerve injury, and therefore proposes a translatable peripheral mechanism underlying chronic neuropathic pain.

Deletion of Tsc2 in Nociceptors Reduces Target Innervation, Ion Channel Expression, and Sensitivity to Heat.

The mechanistic target of rapamycin complex 1 (mTORC1) is known to regulate cellular growth pathways, and its genetic activation is sufficient to enhance regenerative axon growth following injury to the central or peripheral nervous systems. However, excess mTORC1 activation may promote innervation defects, and mTORC1 activity mediates injury-induced hypersensitivity, reducing enthusiasm for the pathway as a therapeutic target. While mTORC1 activity is required for full expression of some pain modalities, the effects of pathway activation on nociceptor phenotypes and sensory behaviors are currently unknown. To address this, we genetically activated mTORC1 in mouse peripheral sensory neurons by conditional deletion of its negative regulator Tuberous Sclerosis Complex 2 (Tsc2). Consistent with the well-known role of mTORC1 in regulating cell size, soma size and axon diameter of C-nociceptors were increased in Tsc2-deleted mice. Glabrous skin and spinal cord innervation by C-fiber neurons were also disrupted. Transcriptional profiling of nociceptors enriched by fluorescence-associated cell sorting (FACS) revealed downregulation of multiple classes of ion channels as well as reduced expression of markers for peptidergic nociceptors in Tsc2-deleted mice. In addition to these changes in innervation and gene expression, Tsc2-deleted mice exhibited reduced noxious heat sensitivity and decreased injury-induced cold hypersensitivity, but normal baseline sensitivity to cold and mechanical stimuli. Together, these data show that excess mTORC1 activity in sensory neurons produces changes in gene expression, neuron morphology and sensory behavior.

Inflammation and nerve injury minimally affect mouse voluntary behaviors proposed as indicators of pain.

It has been suggested that the lack of rodent behavioral assays that represent the complexities of human pain contributes to the poor translational record of basic pain research findings. Clinically, chronic pain interferes with patient mobility and physical/social activities, and increases anxiety symptoms, in turn negatively impacting quality of life. To determine whether these behaviors are similarly influenced by putative pain manipulations in rodents, we systematically evaluated wheel running, locomotion, gait, social interaction, and anxiety-like behavior in models of inflammation and nerve injury in adult C57BL6/J male mice. We demonstrate that inflammation and nerve injury differentially affect voluntary behaviors while mice are hypersensitive to mechanical stimuli. Bilateral Complete Freund's Adjuvant (CFA)-induced inflammation transiently suppressed wheel running and locomotion and also induced gait deficits. In contrast, spared nerve injury (SNI) altered gait and impaired gross motor coordination. SNI-induced gait changes were not reversed by the analgesic PD123319, an angiotensin II type 2 receptor antagonist, and are therefore likely to be motor-related rather than pain-related. Neither CFA nor SNI significantly altered social interaction or elicited general anxiety-like behavior. Our findings suggest that in contrast to humans, mobility and physical/social activities are minimally altered, if at all, in mice following inflammation or nerve injury.

Fully implantable, battery-free wireless optoelectronic devices for spinal optogenetics.

The advent of optogenetic tools has allowed unprecedented insights into the organization of neuronal networks. Although recently developed technologies have enabled implementation of optogenetics for studies of brain function in freely moving, untethered animals, wireless powering and device durability pose challenges in studies of spinal cord circuits where dynamic, multidimensional motions against hard and soft surrounding tissues can lead to device degradation. We demonstrate here a fully implantable optoelectronic device powered by near-field wireless communication technology, with a thin and flexible open architecture that provides excellent mechanical durability, robust sealing against biofluid penetration and fidelity in wireless activation, thereby allowing for long-term optical stimulation of the spinal cord without constraint on the natural behaviors of the animals. The system consists of a double-layer, rectangular-shaped magnetic coil antenna connected to a microscale inorganic light-emitting diode (µ-ILED) on a thin, flexible probe that can be implanted just above the dura of the mouse spinal cord for effective stimulation of light-sensitive proteins expressed in neurons in the dorsal horn. Wireless optogenetic activation of TRPV1-ChR2 afferents with spinal µ-ILEDs causes nocifensive behaviors and robust real-time place aversion with sustained operation in animals over periods of several weeks to months. The relatively low-cost electronics required for control of the systems, together with the biocompatibility and robust operation of these devices will allow broad application of optogenetics in future studies of spinal circuits, as well as various peripheral targets, in awake, freely moving and untethered animals, where existing approaches have limited utility.

Group II mGluRs suppress hyperexcitability in mouse and human nociceptors.

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.

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.

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.

ERK2 Alone Drives Inflammatory Pain But Cooperates with ERK1 in Sensory Neuron Survival.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are highly homologous yet distinct components of signal transduction pathways known to regulate cell survival and function. Recent evidence indicates an isoform-specific role for ERK2 in pain processing and peripheral sensitization. However, the function of ERK2 in primary sensory neurons has not been directly tested. To dissect the isoform-specific function of ERK2 in sensory neurons, we used mice with Cre-loxP-mediated deletion of ERK2 in Nav1.8(+) sensory neurons that are predominantly nociceptors. We find that ERK2, unlike ERK1, is required for peripheral sensitization and cold sensation. We also demonstrate that ERK2, but not ERK1, is required to preserve epidermal innervation in a subset of peptidergic neurons. Additionally, deletion of both ERK isoforms in Nav1.8(+) sensory neurons leads to neuron loss not observed with deletion of either isoform alone, demonstrating functional redundancy in the maintenance of sensory neuron survival. Thus, ERK1 and ERK2 exhibit both functionally distinct and redundant roles in sensory neurons. SIGNIFICANCE STATEMENT: ERK1/2 signaling affects sensory neuron function and survival. However, it was not clear whether ERK isoform-specific roles exist in these processes postnatally. Previous work from our laboratory suggested either functional redundancy of ERK isoforms or a predominant role for ERK2 in pain; however, the tools to discriminate between these possibilities were not available at the time. In the present study, we use new genetic knock-out lines to demonstrate that ERK2 in sensory neurons is necessary for development of inflammatory pain and for postnatal maintenance of peptidergic epidermal innervation. Interestingly, postnatal loss of both ERK isoforms leads to a profound loss of sensory neurons. Therefore, ERK1 and ERK2 display both functionally distinct and redundant roles in sensory neurons.

A simple and inexpensive method for determining cold sensitivity and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity. Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 °C to 5 °C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above. This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Enhanced nonpeptidergic intraepidermal fiber density and an expanded subset of chloroquine-responsive trigeminal neurons in a mouse model of dry skin itch.

UNLABELLED: Chronic pruritic conditions are often associated with dry skin and loss of epidermal barrier integrity. In this study, repeated application of acetone and ether followed by water (AEW) to the cheek skin of mice produced persistent scratching behavior with no increase in pain-related forelimb wiping, indicating the generation of itch without pain. Cheek skin immunohistochemistry showed a 64.5% increase in total epidermal innervation in AEW-treated mice compared to water-treated controls. This increase was independent of scratching, because mice prevented from scratching by Elizabethan collars showed similar hyperinnervation. To determine the effects of dry skin treatment on specific subsets of peripheral fibers, we examined Ret-positive, calcitonin gene-related peptide (CGRP)-positive, and glial cell line-derived neurotrophic factor family receptor α3 (GFRα3)-positive intraepidermal fiber density. AEW treatment increased Ret-positive fibers but not CGRP-positive or GFRα3-positive fibers, suggesting that a specific subset of nonpeptidergic fibers could contribute to dry skin itch. To test whether trigeminal ganglion neurons innervating the cheek exhibited altered excitability after AEW treatment, primary cultures of retrogradely labeled neurons were examined using whole-cell patch clamp electrophysiology. AEW treatment produced no differences in measures of excitability compared to water-treated controls. In contrast, a significantly higher proportion of trigeminal ganglion neurons was responsive to the nonhistaminergic pruritogen chloroquine after AEW treatment. We conclude that nonpeptidergic, Ret-positive fibers and chloroquine-sensitive neurons may contribute to dry skin pruritus. PERSPECTIVE: This study examines the underlying neurobiological mechanisms of persistent dry skin itch. Our results indicate that nonpeptidergic epidermal hyperinnervation and nonhistaminergic pruritic receptors are potential targets for chronic pruritus.

Metabolic regulator LKB1 is crucial for Schwann cell-mediated axon maintenance.

Schwann cells (SCs) promote axonal integrity independently of myelination by poorly understood mechanisms. Current models suggest that SC metabolism is critical for this support function and that SC metabolic deficits may lead to axonal demise. The LKB1-AMP-activated protein kinase (AMPK) kinase pathway targets several downstream effectors, including mammalian target of rapamycin (mTOR), and is a key metabolic regulator implicated in metabolic diseases. We found through molecular, structural and behavioral characterization of SC-specific mutant mice that LKB1 activity is central to axon stability, whereas AMPK and mTOR in SCs are largely dispensable. The degeneration of axons in LKB1 mutants was most dramatic in unmyelinated small sensory fibers, whereas motor axons were comparatively spared. LKB1 deletion in SCs led to abnormalities in nerve energy and lipid homeostasis and to increased lactate release. The latter acts in a compensatory manner to support distressed axons. LKB1 signaling is essential for SC-mediated axon support, a function that may be dysregulated in diabetic neuropathy.

A dynamic set point for thermal adaptation requires phospholipase C-mediated regulation of TRPM8 in vivo.

The ability to sense and respond to thermal stimuli at varied environmental temperatures is essential for survival in seasonal areas. In this study, we show that mice respond similarly to ramping changes in temperature from a wide range of baseline temperatures. Further investigation suggests that this ability to adapt to different ambient environments is based on rapid adjustments made to a dynamic temperature set point. The adjustment of this set point requires transient receptor potential cation channel, subfamily member 8 (TRPM8), but not transient receptor potential cation channel, subfamily A, member 1 (TRPA1), and is regulated by phospholipase C (PLC) activity. Overall, our findings suggest that temperature response thresholds in mice are dynamic, and that this ability to adapt to environmental temperature seems to mirror the in vitro findings that PLC-mediated hydrolysis of phosphoinositol 4,5-bisphosphate modulates TRPM8 activity and thereby regulates the response thresholds to cold stimuli.

Dopamine-dependent compensation maintains motor behavior in mice with developmental ablation of dopaminergic neurons.

The loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and consequent depletion of striatal dopamine are known to underlie the motor deficits observed in Parkinson's disease (PD). Adaptive changes in dopaminergic terminals and in postsynaptic striatal neurons can compensate for significant losses of striatal dopamine, resulting in preservation of motor behavior. In addition, compensatory changes independent of striatal dopamine have been proposed based on PD therapies that modulate nondopaminergic circuits within the basal ganglia. We used a genetic strategy to selectively destroy dopaminergic neurons in mice during development to determine the necessity of these neurons for the maintenance of normal motor behavior in adult and aged mice. We find that loss of 90% of SNc dopaminergic neurons and consequent depletion of >95% of striatal dopamine does not result in changes in motor behavior in young-adult or aged mice as evaluated by an extensive array of motor behavior tests. Treatment of aged mutant mice with the dopamine receptor antagonist haloperidol precipitated motor behavior deficits in aged mutant mice, indicating that <5% of striatal dopamine is sufficient to maintain motor function in these mice. We also found that mutant mice exhibit an exaggerated response to l-DOPA compared with control mice, suggesting that preservation of motor function involves sensitization of striatal dopamine receptors. Our results indicate that congenital loss of dopaminergic neurons induces remarkable adaptions in the nigrostriatal system where limited amounts of dopamine in the dorsal striatum can maintain normal motor function.

A novel behavioral assay for measuring cold sensation in mice.

Behavioral models of cold responses are important tools for exploring the molecular mechanisms of cold sensation. To complement the currently cold behavioral assays and allow further studies of these mechanisms, we have developed a new technique to measure the cold response threshold, the cold plantar assay. In this assay, animals are acclimated on a glass plate and a cold stimulus is applied to the hindpaw through the glass using a pellet of compressed dry ice. The latency to withdrawal from the cooled glass is used as a measure of the cold response threshold of the rodents, and the dry ice pellet provides a ramping cold stimulus on the glass that allows the correlation of withdrawal latency values to rough estimates of the cold response threshold temperature. The assay is highly sensitive to manipulations including morphine-induced analgesia, Complete Freund's Adjuvant-induced inflammatory allodynia, and Spinal Nerve Ligation-induced neuropathic allodynia.

Schwann cell mitochondrial metabolism supports long-term axonal survival and peripheral nerve function.

Mitochondrial dysfunction is a common cause of peripheral neuropathies. While the role of neuron and axonal mitochondria in peripheral nerve disease is well appreciated, whether Schwann cell (SC) mitochondrial deficits contribute to peripheral neuropathies is unclear. Here, we examine how SC mitochondrial dysfunction affects axonal survival and contributes to the decline of peripheral nerve function by generating mice with SC-specific mitochondrial deficits. These mice (Tfam-SCKOs) were produced through the tissue-specific deletion of the mitochondrial transcription factor A gene (Tfam), which is essential for mitochondrial DNA (mtDNA) transcription and maintenance. Tfam-SCKOs were viable, but as they aged, they developed a progressive peripheral neuropathy characterized by nerve conduction abnormalities as well as extensive muscle denervation. Morphological examination of Tfam-SCKO nerves revealed early preferential loss of small unmyelinated fibers followed by prominent demyelination and degeneration of larger-caliber axons. Tfam-SCKOs displayed sensory and motor deficits consistent with this pathology. Remarkably, the severe mtDNA depletion and respiratory chain abnormalities in Tfam-SCKO mice did not affect SC proliferation or survival. Mitochondrial function in SCs is therefore essential for maintenance of axonal survival and normal peripheral nerve function, suggesting that SC mitochondrial dysfunction contributes to human peripheral neuropathies.

RET signaling is required for survival and normal function of nonpeptidergic nociceptors.

Small unmyelinated sensory neurons classified as nociceptors are divided into two subpopulations based on phenotypic differences, including expression of neurotrophic factor receptors. Approximately half of unmyelinated nociceptors express the NGF receptor TrkA, and half express the GDNF family ligand (GFL) receptor Ret. The function of NGF/TrkA signaling in the TrkA population of nociceptors has been extensively studied, and NGF/TrkA signaling is a well established mediator of pain. The GFLs are analgesic in models of neuropathic pain emphasizing the importance of understanding the physiological function of GFL/Ret signaling in nociceptors. However, perinatal lethality of Ret-null mice has precluded the study of the physiological role of GFL/Ret signaling in the survival, maintenance, and function of nociceptors in viable mice. We deleted Ret exclusively in nociceptors by crossing nociceptor-specific Na(v)1.8 Cre and Ret conditional mice to produce Ret-Na(v)1.8 conditional knock-out (CKO) mice. Loss of Ret exclusively in nociceptors results in a reduction in nociceptor number and size, indicating that Ret signaling is important for the survival and trophic support of these cells. Ret-Na(v)1.8 CKO mice exhibit reduced epidermal innervation but normal central projections. In addition, Ret-Na(v)1.8 CKO mice have increased sensitivity to cold and increased formalin-induced pain, demonstrating that Ret signaling modulates the function of nociceptors in vivo. Enhanced inflammation-induced pain may be mediated by decreased prostatic acid phosphatase (PAP), as PAP levels are markedly reduced in Ret-Na(v)1.8 CKO mice. The results of this study identify the physiological role of endogenous Ret signaling in the survival and function of nociceptors.