Gi-DREADD Expression in Peripheral Nerves Produces Ligand-Dependent Analgesia, as well as Ligand-Independent Functional Changes in Sensory Neurons.

Designer receptors exclusively activated by designer drugs (DREADDs) are an advanced experimental tool that could potentially provide a novel approach to pain management. In particular, expression of an inhibitory (Gi-coupled) DREADD in nociceptors might enable ligand-dependent analgesia. To test this possibility, TRPV1-cre mice were used to restrict expression of Gi-DREADDs to predominantly C-fibers. Whereas baseline heat thresholds in both male and female mice expressing Gi-DREADD were normal, 1 mg/kg clozapine-N-oxide (CNO) produced a significant 3 h increase in heat threshold that returned to baseline by 5 h after injection. Consistent with these behavioral results, CNO decreased action potential firing in isolated sensory neurons from Gi-DREADD mice. Unexpectedly, however, the expression of Gi-DREADD in sensory neurons caused significant changes in voltage-gated Ca2+ and Na+ currents in the absence of CNO, as well as an increase in Na+ channel (NaV1.7) expression. Furthermore, CNO-independent excitatory and inhibitory second-messenger signaling was also altered in these mice, which was associated with a decrease in the analgesic effect of endogenous inhibitory G-protein-coupled receptor activation. These results highlight the potential of this exciting technology, but also its limitations, and that it is essential to identify the underlying mechanisms for any observed behavioral phenotypes. SIGNIFICANCE STATEMENT: DREADD technology is a powerful tool enabling manipulation of activity and/or transmitter release from targeted cell populations. The purpose of this study was to determine whether inhibitory DREADDs in nociceptive afferents could be used to produce analgesia, and if so, how. DREADD activation produced a ligand-dependent analgesia to heat in vivo and a decrease in neuronal firing at the single-cell level. However, we observed that expression of Gi-DREADD also causes ligand-independent changes in ion channel activity and second-messenger signaling. These findings highlight both the potential and the limitations of this exciting technology as well as the necessity to identify the mechanisms underlying any observed phenotype.

Bhlhb5::flpo allele uncovers a requirement for Bhlhb5 for the development of the dorsal cochlear nucleus.

Auditory information is initially processed in the cochlear nuclei before being relayed to the brain. The cochlear nuclei are subdivided into dorsal, anterior ventral, and posterior ventral domains, each containing several subtypes of neurons that are thought to play discrete roles in the processing of sound. However, the ontogeny of these neurons is poorly understood, and this gap in knowledge hampers efforts to understand the basic neural circuitry of this nucleus. Here, we reveal that Bhlhb5 is expressed in both excitatory (unipolar brush cells) and inhibitory neurons (cartwheel cells) of the DCN during development. To gain genetic access to Bhlhb5-expressing neurons in the DCN, we generated a Bhlhb5::flpo knockin allele. Using an intersectional genetic strategy, we labeled cartwheel cells, thereby providing proof of concept that subpopulations of Bhlhb5-expressing neurons can be genetically targeted. Moreover, fate-mapping experiments using this allele revealed that Bhlhb5 is required for the proper development of the DCN, since mice lacking Bhlhb5 showed a dramatically diminished number of neurons, including unipolar brush and cartwheel cells. Intriguingly, the Bhlhb5::flpo allele also genetically labels numerous other regions of the nervous system that process sensory input, including the dorsal horn, the retina, and the nucleus of the lateral olfactory tract, hinting at a more general role for Bhlhb5 in the development of neurons that mediate sensory integration.

Generation of a NK1R-CreER knockin mouse strain to study cells involved in Neurokinin 1 Receptor signaling.

The Neurokinin 1 Receptor (NK1R), which binds Substance P, is expressed in discrete populations of neurons throughout the nervous system, where it has numerous roles including the modulation of pain and affective behaviors. Here, we report the generation of a NK1R-CreER knockin allele, in which CreERT2 replaces the coding sequence of the TACR1 gene (encoding NK1R) in order to gain genetic access to these cells. We find that the NK1R-CreER allele mediates recombination in many regions of the nervous system that are important in pain and anxiety including the amygdala, hypothalamus, frontal cortex, raphe nucleus, and dorsal horn of the spinal cord. Other cell types that are labeled by this allele include amacrine cells in the retina and fibroblasts in the skin. Thus, the NK1R-CreER mouse line is a valuable new tool for conditional gene manipulation enabling the visualization and manipulation of cells that express NK1R.

Generation of a KOR-Cre knockin mouse strain to study cells involved in kappa opioid signaling.

The kappa opioid receptor (KOR) has numerous important roles in the nervous system including the modulation of mood, reward, pain, and itch. In addition, KOR is expressed in many non-neuronal tissues. However, the specific cell types that express KOR are poorly characterized. Here, we report the development of a KOR-Cre knockin allele, which provides genetic access to cells that express KOR. In this mouse, Cre recombinase (Cre) replaces the initial coding sequence of the Opkr1 gene (encoding the kappa opioid receptor). We demonstrate that the KOR-Cre allele mediates recombination by embryonic day 14.5 (E14.5). Within the brain, KOR-Cre shows expression in numerous areas including the cerebral cortex, nucleus accumbens and striatum. In addition, this allele is expressed in epithelium and throughout many regions of the body including the heart, lung, and liver. Finally, we reveal that KOR-Cre mediates recombination of a subset of bipolar and amacrine cells in the retina. Thus, the KOR-Cre mouse line is a valuable new tool for conditional gene manipulation to enable the study of KOR.

Itch and its inhibition by counter stimuli.

Recent studies have made significant progress in the knowledge of how itch sensation is processed, especially the molecular identity of neurons involved in itch signaling, both in the dorsal root ganglion and spinal cord. Despite these advances, the organization of these neurons in dorsal spinal cord circuits and how they interact with other somatosensory modalities, such as pain or temperature, remain relatively unexplored. Recent work from our lab and others has begun to shed light on these questions and will be the focus of this chapter. Here we describe the discovery of B5-I neurons, a population of inhibitory interneurons that function to inhibit itch, and review the evidence that these neurons mediate the inhibition of itch by counter stimuli. These studies are helping to solve the long-standing question of why itch makes us scratch.

Kappa Opioid Receptor Distribution and Function in Primary Afferents.

Primary afferents are known to be inhibited by kappa opioid receptor (KOR) signaling. However, the specific types of somatosensory neurons that express KOR remain unclear. Here, using a newly developed KOR-cre knockin allele, viral tracing, single-cell RT-PCR, and ex vivo recordings, we show that KOR is expressed in several populations of primary afferents: a subset of peptidergic sensory neurons, as well as low-threshold mechanoreceptors that form lanceolate or circumferential endings around hair follicles. We find that KOR acts centrally to inhibit excitatory neurotransmission from KOR-cre afferents in laminae I and III, and this effect is likely due to KOR-mediated inhibition of Ca2+ influx, which we observed in sensory neurons from both mouse and human. In the periphery, KOR signaling inhibits neurogenic inflammation and nociceptor sensitization by inflammatory mediators. Finally, peripherally restricted KOR agonists selectively reduce pain and itch behaviors, as well as mechanical hypersensitivity associated with a surgical incision. These experiments provide a rationale for the use of peripherally restricted KOR agonists for therapeutic treatment.

Automated acoustic detection of mouse scratching.

Itch is an aversive somatic sense that elicits the desire to scratch. In animal models of itch, scratching behavior is frequently used as a proxy for itch, and this behavior is typically assessed through visual quantification. However, manual scoring of videos has numerous limitations, underscoring the need for an automated approach. Here, we propose a novel automated method for acoustic detection of mouse scratching. Using this approach, we show that chloroquine-induced scratching behavior in C57BL/6 mice can be quantified with reasonable accuracy (85% sensitivity, 75% positive predictive value). This report is the first method to apply supervised learning techniques to automate acoustic scratch detection.

An Unexpected Role for TRPV4 in Serotonin-Mediated Itch.

Previous studies have revealed that TRPV1 and TRPA1 function downstream of many itch receptors, where they mediate inward current to trigger action potentials in primary afferents. Although other TRP channels, such as TRPV4, are expressed in primary afferents, whether or not they play an analogous role in itch was previously unknown. Now, Akiyama et al. provide evidence that TRPV4 is a key mediator of serotonin-induced itch. This finding is important because it uncovers an unanticipated role for TRPV4 in itch, thereby identifying a novel therapeutic target.

Semi-intact ex vivo approach to investigate spinal somatosensory circuits.

The somatosensory input that gives rise to the perceptions of pain, itch, cold and heat are initially integrated in the superficial dorsal horn of the spinal cord. Here, we describe a new approach to investigate these neural circuits in mouse. This semi-intact somatosensory preparation enables recording from spinal output neurons, while precisely controlling somatosensory input, and simultaneously manipulating specific populations of spinal interneurons. Our findings suggest that spinal interneurons show distinct temporal and spatial tuning properties. We also show that modality selectivity - mechanical, heat and cold - can be assessed in both retrogradely labeled spinoparabrachial projection neurons and genetically labeled spinal interneurons. Finally, we demonstrate that interneuron connectivity can be determined via optogenetic activation of specific interneuron subtypes. This new approach may facilitate key conceptual advances in our understanding of the spinal somatosensory circuits in health and disease.

Dynorphin acts as a neuromodulator to inhibit itch in the dorsal horn of the spinal cord.

Menthol and other counterstimuli relieve itch, resulting in an antipruritic state that persists for minutes to hours. However, the neural basis for this effect is unclear, and the underlying neuromodulatory mechanisms are unknown. Previous studies revealed that Bhlhb5(-/-) mice, which lack a specific population of spinal inhibitory interneurons (B5-I neurons), develop pathological itch. Here we characterize B5-I neurons and show that they belong to a neurochemically distinct subset. We provide cause-and-effect evidence that B5-I neurons inhibit itch and show that dynorphin, which is released from B5-I neurons, is a key neuromodulator of pruritus. Finally, we show that B5-I neurons are innervated by menthol-, capsaicin-, and mustard oil-responsive sensory neurons and are required for the inhibition of itch by menthol. These findings provide a cellular basis for the inhibition of itch by chemical counterstimuli and suggest that kappa opioids may be a broadly effective therapy for pathological itch.