An adeno-associated viral labeling approach to visualize the meso- and microanatomy of mechanosensory afferents and autonomic innervation of the rat urinary bladder.

The urinary bladder is supplied by a rich network of sensory and autonomic axons, commonly visualized by immunolabeling for neural markers. This approach demonstrates overall network patterning but is less suited to understanding the structure of individual motor and sensory terminals within these complex plexuses. There is a further limitation visualizing the lightly myelinated (A-delta) class of sensory axons that provides the primary mechanosensory drive for initiation of voiding. Whereas most unmyelinated sensory axons can be revealed by immunolabeling for specific neuropeptides, to date no unique neural marker has been identified to immunohistochemically label myelinated visceral afferents. We aimed to establish a non-surgical method to visualize and map myelinated afferents in the bladder in rats. We found that in rats, the adeno-associated virus (AAV), AAV-PHP.S, which shows a high tropism for the peripheral nervous system, primarily transduced myelinated dorsal root ganglion neurons, enabling us to identify the structure and regional distribution of myelinated (mechanosensory) axon endings within the muscle and lamina propria of the bladder. We further identified the projection of myelinated afferents within the pelvic nerve and lumbosacral spinal cord. A minority of noradrenergic and cholinergic neurons in pelvic ganglia were transduced, enabling visualization and regional mapping of both autonomic and sensory axon endings within the bladder. Our study identified a sparse labeling approach for investigating myelinated sensory and autonomic axon endings within the bladder and provides new insights into the nerve-bladder interface.

Functional segregation within the pelvic nerve of male rats: a meso- and microscopic analysis.

The pelvic splanchnic nerves are essential for pelvic organ function and have been proposed as targets for neuromodulation. We have focused on the rodent homologue of these nerves, the pelvic nerves. Our goal was to define within the pelvic nerve the projections of organ-specific sensory axons labelled by microinjection of neural tracer (cholera toxin, subunit B) into the bladder, urethra or rectum. We also examined the location of peptidergic sensory axons within the pelvic nerves to determine whether they aggregated separately from sacral preganglionic and paravertebral sympathetic postganglionic axons travelling in the same nerve. To address these aims, microscopy was performed on the major pelvic ganglion (MPG) with attached pelvic nerves, microdissected from young adult male Sprague-Dawley rats (6-8 weeks old) and processed as whole mounts for fluorescence immunohistochemistry. The pelvic nerves were typically composed of five discrete fascicles. Each fascicle contained peptidergic sensory, cholinergic preganglionic and noradrenergic postganglionic axons. Sensory axons innervating the lower urinary tract (LUT) consistently projected in specific fascicles within the pelvic nerves, whereas sensory axons innervating the rectum projected in a complementary group of fascicles. These discrete aggregations of organ-specific sensory projections could be followed along the full length of the pelvic nerves. From the junction of the pelvic nerve with the MPG, sensory axons immunoreactive for calcitonin gene-related peptide (CGRP) showed several distinct patterns of projection: some projected directly to the cavernous nerve, others projected directly across the surface of the MPG to the accessory nerves and a third class entered the MPG, encircling specific cholinergic neurons projecting to the LUT. A subpopulation of preganglionic inputs to noradrenergic MPG neurons also showed CGRP immunoreactivity. Together, these studies reveal new molecular and structural features of the pelvic nerves and suggest functional targets of sensory nerves in the MPG. These anatomical data will facilitate the design of experimental bioengineering strategies to specifically modulate each axon class.

Neurite outgrowth in normal and injured primary sensory neurons reveals different regulation by nerve growth factor (NGF) and artemin.

Neurotrophic factors have been intensively studied as potential therapeutic agents for promoting neural regeneration and functional recovery after nerve injury. Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) that forms a signalling complex with GFRα3 and the tyrosine kinase Ret. Systemic administration of artemin in rodents is reported to facilitate regeneration of primary sensory neurons following axotomy, improve recovery of sensory function, and reduce sensory hypersensitivity that is a cause of pain. However, the biological mechanisms that underlie these effects are mostly unknown. This study has investigated the biological significance of the colocalisation of GFRα3 with TrkA (neurotrophin receptor for nerve growth factor [NGF]) in the peptidergic type of unmyelinated (C-fibre) sensory neurons in rat dorsal root ganglia (DRG). In vitro neurite outgrowth assays were used to study the effects of artemin and NGF by comparing DRG neurons that were previously uninjured, or were axotomised in vivo by transecting a visceral or somatic peripheral nerve. We found that artemin could facilitate neurite initiation but in comparison to NGF had low efficacy for facilitating neurite elongation and branching. This low efficacy was not increased when a preconditioning in vivo nerve injury was used to induce a pro-regenerative state. Neurite initiation was unaffected by artemin when PI3 kinase and Src family kinase signalling were blocked, but NGF had a reduced effect.

Co-cultures provide a new tool to probe communication between adult sensory neurons and urothelium.

PURPOSE: Recent evidence suggests that the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli and signals to nerve terminals and other cells in the bladder wall. The cellular and molecular basis of neuro-urothelial communication is not easily studied in the intact bladder. This led us to establish a method of co-culturing dorsal root ganglion sensory neurons and bladder urothelial cells. MATERIALS AND METHODS: Sensory neurons and urothelial cells obtained from dorsal root ganglia and bladders dissected from adult female Sprague-Dawley® rats were isolated by enzyme treatment and mechanical dissociation. They were plated together or separately on collagen coated substrate and cultured in keratinocyte medium for 48 to 72 hours. Retrograde tracer labeling was performed to identify bladder afferents used for functional testing. RESULTS: Neurite growth and complexity in neurons co-cultured with urothelial cells was increased relative to that in neuronal monocultures. The growth promoting effect of urothelial cells was reduced by the tyrosine kinase inhibitor K252a but upstream inhibition of nerve growth factor signaling with TrkA-Fc had no effect. Fura-2 calcium imaging of urothelial cells showed responses to adenosine triphosphate (100 µM) and activation of TRPV4 (4α-PDD, 10 µM) but not TRPV1 (capsaicin, 1 µM), TRPV3 (farnesyl pyrophosphate, 1 µM) or TRPA1 (mustard oil, 100 µM). In contrast, co-cultured neurons were activated by all agonists except farnesyl pyrophosphate. CONCLUSIONS: Co-culturing provides a new methodology for investigating neuro-urothelial interactions in animal models of urological conditions. Results suggest that neuronal properties are maintained in the presence of urothelium and neurite growth is potentiated by a nerve growth factor independent mechanism.

Selective recording of physiologically evoked neural activity in a mixed autonomic nerve using a minimally invasive array.

Real-time closed-loop control of neuromodulation devices requires long-term monitoring of neural activity in the peripheral nervous system. Although many signal extraction methods exist, few are both clinically viable and designed for extracting small signals from fragile peripheral visceral nerves. Here, we report that our minimally invasive recording and analysis technology extracts low to negative signal to noise ratio (SNR) neural activity from a visceral nerve with a high degree of specificity for fiber type and class. Complex activity was recorded from the rat pelvic nerve that was physiologically evoked during controlled bladder filling and voiding, in an extensively characterized in vivo model that provided an excellent test bed to validate our technology. Urethane-anesthetized male rats (n = 12) were implanted with a four-electrode planar array and the bladder instrumented for continuous-flow cystometry, which measures urodynamic function by recording bladder pressure changes during constant infusion of saline. We demonstrated that differential bipolar recordings and cross-correlation analyses extracts afferent and efferent activity, and discriminated between subpopulations of fibers based on conduction velocity. Integrated Aδ afferent fiber activity correlated with bladder pressure during voiding (r2: 0.66 ± 0.06) and was not affected by activating nociceptive afferents with intravesical capsaicin (r2: 0.59 ± 0.14, P = 0.54, and n = 3). Collectively, these results demonstrate our minimally invasive recording and analysis technology is selective in extracting mixed neural activity with low/negative SNR. Furthermore, integrated afferent activity reliably correlates with bladder pressure and is a promising first step in developing closed-loop technology for bladder control.

Corneal tissue-resident memory T cells form a unique immune compartment at the ocular surface.

The eye is considered immune privileged such that immune responses are dampened to protect vision. As the most anterior compartment of the eye, the cornea is exposed to pathogens and can mount immune responses that recruit effector T cells. However, presence of immune memory in the cornea is not defined. Here, we use intravital 2-photon microscopy to examine T cell responses in the cornea in mice. We show that recruitment of CD8+ T cells in response to ocular virus infection results in the formation of tissue-resident memory T (TRM) cells. Motile corneal TRM cells patrol the cornea and rapidly respond in situ to antigen rechallenge. CD103+ TRM cell generation requires antigen and transforming growth factor ß. In vivo imaging in humans also reveals highly motile cells that patrol the healthy cornea. Our study finds that TRM cells form in the cornea where they can provide local protective immunity.

Engineered AAVs for non-invasive gene delivery to rodent and non-human primate nervous systems.

Gene therapy offers great promise in addressing neuropathologies associated with the central and peripheral nervous systems (CNS and PNS). However, genetic access remains difficult, reflecting the critical need for the development of effective and non-invasive gene delivery vectors across species. To that end, we evolved adeno-associated virus serotype 9 (AAV9) capsid in mice and validated two capsids, AAV-MaCPNS1 and AAV-MaCPNS2, across rodent species (mice and rats) and non-human primate (NHP) species (marmosets and rhesus macaques). Intravenous administration of either AAV efficiently transduced the PNS in rodents and both the PNS and CNS in NHPs. Furthermore, we used AAV-MaCPNS1 in mice to systemically deliver the following: (1) the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia and (2) the neuronal actuator DREADD to dorsal root ganglia to mediate pain. This conclusively demonstrates the translatability of these two systemic AAVs across four species and their functional utility through proof-of-concept studies in mice.

Sex differences in c-Fos and EGR-1/Zif268 activity maps of rat sacral spinal cord following cystometry-induced micturition.

Storage and voiding of urine from the lower urinary tract (LUT) must be timed precisely to occur in appropriate behavioral contexts. A major part of the CNS circuit that coordinates this activity is found in the lumbosacral spinal cord. Immediate early gene (IEG) activity mapping has been widely used to investigate the lumbosacral LUT-related circuit, but most reports focus on the effects of noxious stimulation in anesthetized female rats. Here we use c-Fos and EGR-1 (Zif268) activity mapping of lumbosacral spinal cord to investigate cystometry-induced micturition in awake female and male rats. In females, after cystometry c-Fos neurons in spinal cord segments L5-S2 were concentrated in the sacral parasympathetic nucleus (SPN), dorsal horn laminae II-IV, and dorsal commissural nucleus (SDCom). Comparisons of cystometry and control groups in male and female revealed sex differences. Activity mapping suggested dorsal horn laminae II-IV was activated in females but showed net inhibition in males. However, inhibition in male rats was not detected by EGR-1 activity mapping, which showed low coexpression with c-Fos. A class of catecholamine neurons in SPN and SDCom neurons were also more strongly activated by micturition in females. In both sexes, most c-Fos neurons were identified as excitatory by their absence of Pax2 expression. In conclusion, IEG mapping in awake male and female rats has extended our understanding of the functional molecular anatomy of the LUT-related circuit in spinal cord. Using this approach, we have identified sex differences that were not detected by previous studies in anesthetized rats.

Computational modelling of nerve stimulation and recording with peripheral visceral neural interfaces.

Objective.Neuromodulation of visceral nerves is being intensively studied for treating a wide range of conditions, but effective translation requires increasing the efficacy and predictability of neural interface performance. Here we use computational models of rat visceral nerve to predict how neuroanatomical variability could affect both electrical stimulation and recording with an experimental planar neural interface.Approach.We developed a hybrid computational pipeline,VisceralNerveEnsembleRecording andStimulation (ViNERS), to couple finite-element modelling of extracellular electrical fields with biophysical simulations of individual axons. Anatomical properties of fascicles and axons in rat pelvic and vagus nerves were measured or obtained from public datasets. To validate ViNERS, we simulated pelvic nerve stimulation and recording with an experimental four-electrode planar array.Main results.Axon diameters measured from pelvic nerve were used to model a population of myelinated and unmyelinated axons and simulate recordings of electrically evoked single-unit field potentials (SUFPs). Across visceral nerve fascicles of increasing size, our simulations predicted an increase in stimulation threshold and a decrease in SUFP amplitude. Simulated threshold changes were dominated by changes in perineurium thickness, which correlates with fascicle diameter. We also demonstrated that ViNERS could simulate recordings of electrically-evoked compound action potentials (ECAPs) that were qualitatively similar to pelvic nerve recording made with the array used for simulation.Significance.We introduce ViNERS as a new open-source computational tool for modelling large-scale stimulation and recording from visceral nerves. ViNERS predicts how neuroanatomical variation in rat pelvic nerve affects stimulation and recording with an experimental planar electrode array. We show ViNERS can simulate ECAPS that capture features of our recordings, but our results suggest the underlying NEURON models need to be further refined and specifically adapted to accurately simulate visceral nerve axons.

Simulating bidirectional peripheral neural interfaces in EIDORS.

Bioelectronic neural interfaces that deliver adaptive therapeutic stimulation in an intelligent manner must be able to sense and stimulate activity within the same nerve. Existing minimally-invasive peripheral neural interfaces can provide a read-out of the aggregate level of activity via electrical recordings of nerve activity, but these recordings are limited in terms of their specificity. Computational simulations can provide fine-grained insight into the contributions of different neural populations to the extracellular recording, but integration of the signals from individual nerve fibers requires knowledge of spread of current in the complex (heterogenous, anisotropic) extracellular space. We have developed a model which uses the open-source EIDORS package for extracellular stimulation and recording in the pelvic nerve. The pelvic nerve is the primary source of autonomic innervation to the pelvic organs, and a prime target for electrical stimulation to treat a variety of voiding disorders. We simulated recordings of spontaneous and electrically-evoked activity using biophysical models for myelinated and unmyelinated axons. As expected, stimulus thresholds depended strongly on both fibre type and electrode-fibre distance. In conclusion, EIDORS can be used to accurately simulate extracellular recording in complex, heterogenous neural geometries.

Recording of Electrically Evoked Neural Activity and Bladder Pressure Responses in Awake Rats Chronically Implanted With a Pelvic Nerve Array.

Bioelectronic medical devices are well established and widely used in the treatment of urological dysfunction. Approved targets include the sacral S3 spinal root and posterior tibial nerve, but an alternate target is the group of pelvic splanchnic nerves, as these contain sacral visceral sensory and autonomic motor pathways that coordinate storage and voiding functions of the bladder. Here, we developed a device suitable for long-term use in an awake rat model to study electrical neuromodulation of the pelvic nerve (homolog of the human pelvic splanchnic nerves). In male Sprague-Dawley rats, custom planar four-electrode arrays were implanted over the distal end of the pelvic nerve, close to the major pelvic ganglion. Electrically evoked compound action potentials (ECAPs) were reliably detected under anesthesia and in chronically implanted, awake rats up to 8 weeks post-surgery. ECAP waveforms showed three peaks, with latencies that suggested electrical stimulation activated several subpopulations of myelinated A-fiber and unmyelinated C-fiber axons. Chronic implantation of the array did not impact on voiding evoked in awake rats by continuous cystometry, where void parameters were comparable to those published in naïve rats. Electrical stimulation with chronically implanted arrays also induced two classes of bladder pressure responses detected by continuous flow cystometry in awake rats: voiding contractions and non-voiding contractions. No evidence of tissue pathology produced by chronically implanted arrays was detected by immunohistochemical visualization of markers for neuronal injury or noxious spinal cord activation. These results demonstrate a rat pelvic nerve electrode array that can be used for preclinical development of closed loop neuromodulation devices targeting the pelvic nerve as a therapy for neuro-urological dysfunction.

17beta-estradiol activates estrogen receptor beta-signalling and inhibits transient receptor potential vanilloid receptor 1 activation by capsaicin in adult rat nociceptor neurons.

There is mounting evidence that estrogens act directly on the nervous system to affect the severity of pain. Estrogen receptors (ERs) are expressed by sensory neurons, and in trigeminal ganglia, 17beta-estradiol can indirectly enhance nociception by stimulating expression and release of prolactin, which increases phosphorylation of the nociceptor transducer transient receptor potential vanilloid receptor 1 (TRPV1). Here, we show that 17beta-estradiol acts directly on dorsal root ganglion (DRG) sensory neurons to reduce TRPV1 activation by capsaicin. Capsaicin-induced cobalt uptake and the maximum TRPV1 current induced by capsaicin were inhibited when isolated cultured DRGs neurons from adult female rats were exposed to 17beta-estradiol (10-100 nm) overnight. There was no effect of 17beta-estradiol on capsaicin potency, TRPV1 activation by protons (pH 6-4), and P2X currents induced by alpha,beta-methylene-ATP. Diarylpropionitrile (ERbeta agonist) also inhibited capsaicin-induced TRPV1 currents, whereas propylpyrazole triol (ERalpha agonist) and 17alpha-estradiol (inactive analog) were inactive, and 17beta-estradiol conjugated to BSA (membrane-impermeable agonist) caused a small increase. TRPV1 inhibition was antagonized by tamoxifen (1 microm), but ICI182870 (10 microm) was a potent agonist and mimicked 17beta-estradiol. We conclude that TRPV1 in DRG sensory neurons can be inhibited by a nonclassical estrogen-signalling pathway that is downstream of intracellular ERbeta. This affects the vanilloid binding site targeted by capsaicin but not the TRPV1 activation site targeted by protons. These actions could curtail the nociceptive transducer functions of TRPV1 and limit chemically induced nociceptor sensitization during inflammation. They are consistent with clinical reports that female pelvic pain can increase after reductions in circulating estrogens.

Axonal Injury Induces ATF3 in Specific Populations of Sacral Preganglionic Neurons in Male Rats.

Compared to other neurons of the central nervous system, autonomic preganglionic neurons are unusual because most of their axon lies in the periphery. These axons are vulnerable to injury during surgical procedures, yet in comparison to peripheral neurons and somatic motor neurons, the impact of injury on preganglionic neurons is poorly understood. Here, we have investigated the impact of axotomy on sacral preganglionic neurons, a functionally diverse group of neurons required for micturition, defecation, and sexual function. We have previously observed that after axotomy, the injury-related transcription factor activating transcription factor-3 (ATF3) is upregulated in only half of these neurons (Peddie and Keast, 2011: PMID: 21283532). In the current study, we have investigated if this response is constrained to particular subclasses of preganglionic neurons that have specific functions or signaling properties. Seven days after unilateral pelvic nerve transection, we quantified sacral preganglionic neurons expressing ATF3, many but not all of which co-expressed c-Jun. This response was independent of soma size. Subclasses of sacral preganglionic neurons expressed combinations of somatostatin, calbindin, and neurokinin-1 receptor, each of which showed a similar response to injury. We also found that in contrast to thoracolumbar preganglionic neurons, the heat shock protein-25 (Hsp25) was not detected in naive sacral preganglionic neurons but was upregulated in many of these neurons after axotomy; the majority of these Hsp25 neurons expressed ATF3. Together, these studies reveal the molecular complexity of sacral preganglionic neurons and their responses to injury. The simultaneous upregulation of Hsp25 and ATF3 may indicate a distinct mechanism of regenerative capacity after injury.

Acute and chronic changes in dorsal horn innervation by primary afferents and descending supraspinal pathways after spinal cord injury.

Sprouting of peptidergic nociceptive and descending supraspinal projections to the dorsal horn following spinal cord injury (SCI) has been proposed as a mechanism of neuropathic pain. To identify structural changes that could initiate or maintain SCI pain, we used a complete transection model in rats to examine how structural remodeling in the dorsal horn rostral to the lesion relates to distance from injury, laminar region, and duration of injury. The major classes of C-fiber primary afferents differed greatly in their susceptibility to structural and chemical changes and their ability to undergo plasticity. Peptidergic primary afferents showed a widespread loss throughout the dorsal horn of segments approaching the injury site. Some of this loss may have been due to decreased neuropeptide expression. The reduction in peptidergic fibers was transient, indicating compensatory sprouting and perhaps also increased neuropeptide expression within the cord. Nonpeptidergic afferents expressing GFRalpha1 were largely unaffected by SCI. In contrast, in GFRalpha2-expressing nonpeptidergic afferents SCI caused a permanent loss of dorsal horn innervation. Unexpectedly, GFRalpha2 was transiently induced throughout deeper laminae but this was not due to upregulation of GFRalpha2 in dorsal root ganglia. We also observed permanent sprouting of catecholamine terminals of supraspinal origin. This was restricted to the superficial laminae. Our results show that SCI caused a loss of sensory input as well as structural remodeling such that the balance of nociceptive inputs and descending modulation was permanently altered. These changes may contribute to mechanisms rostral to the site of SCI that trigger and maintain neuropathic pain.

Coexpression of prodynorphin and corticotrophin-releasing hormone in the rat central amygdala: evidence of two distinct endogenous opioid systems in the lateral division.

The lateral subdivision of the central nucleus of the amygdala (CeA) comprises two groups of gamma-aminobutyric acid (GABA) neurons that express corticotrophin-releasing hormone (CRH) and enkephalin. Regulation of the expression and release of these neuropeptides by glucocorticoids and other factors has been suggested to have a regulatory function on the diverse somatic, autonomic, and neuroendocrine responses that are coordinated by the CeA. Because another opioid peptide, dynorphin, has been reported to be also expressed by neurons in the lateral CeA, this study examined the neuronal expression of this kappa-opioid (KOP) receptor-preferring ligand by using immunohistochemistry for the precursor peptide prodynorphin. Prodynorphin neurons in the extended amygdala were observed mostly in the medial and central regions of the lateral CeA and the oval of the bed nucleus of the stria terminalis (BST). About one-third of the prodynorphin neurons in the CeA coexpressed CRH, whereas no coexpression with CRH was detected in the BST. Prodynorphin was not expressed by calbindin neurons in the medial part of the lateral CeA, and indirect evidence suggested that it was not expressed by enkephalin neurons. Coexpression of prodynorphin in extrahypothalamic CRH neurons in the CeA could provide an anatomical basis for regulation of the stress responses and other CRH-related functions by the brain dynorphin/KOP receptor system.

Induction of c-Fos and zif268 in the nociceptive amygdala parallel abstinence hyperalgesia in rats briefly exposed to morphine.

Opioid-induced analgesia can be followed by spontaneous pain in humans, and hyperalgesia in rodents. In this study, opioid-induced hyperalgesia was measured by the tail-flick test when acute abstinence was precipitated by administering naloxone to drug naive rats that had experienced morphine analgesia for only 30 min. In a further experiment, the drug treatment that previously caused opioid-induced hyperalgesia was found to increase neurons expressing nuclear c-Fos or zif268 proteins in extended amygdalar regions targeted by projections of the ascending spino-parabrachio-amygdaloid nociceptive pathway. Transcription factor induction, however, was not detected in multiple brain regions known to respond in parallel with the same extended amygdalar structures when (1) rats are exposed to interoceptive/physical stressors, or (2) naloxone is used to precipitate abstinence in opioid dependent rats. Surprisingly, in many regions c-Fos induction by morphine was reduced or blocked by naloxone, even though these subjects had also experienced the effects of morphine for 30 min prior to antagonist administration. It is suggested transcription factor induction during opioid hyperalgesia in non-dependent rats could support the induction or consolidation of neural plasticity in nociceptive amygdaloid circuitry previously suggested to function in bi-directional control of pain and expression of pain-related behaviors.

A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat.

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.

Characterization of neurons in the rat central nucleus of the amygdala: cellular physiology, morphology, and opioid sensitivity.

The central nucleus of the amygdala (CeA) orchestrates autonomic and other behavioral and physiological responses to conditioned stimuli that are aversive or elicit fear. As a related CeA function is the expression of hypoalgesia induced by conditioned stimuli or systemic morphine administration, we examined postsynaptic opioid modulation of neurons in each major CeA subdivision. Following electrophysiological recording, biocytin-filled neurons were precisely located in CeA regions identified by chemoarchitecture (enkephalin-immunoreactivity) and cytoarchitecture (DAPI nuclear staining) in fixed adult rat brain slices. This revealed a striking distribution of physiological types, as 92% of neurons in capsular CeA were classified as late-firing, whereas no neurons in the medial CeA were of this class. In contrast, 60% or more of neurons in the lateral and medial CeA were low-threshold bursting neurons. Mu-opioid receptor (MOPR) agonists induced postsynaptic inhibitory potassium currents in 61% of CeA cells, and this ratio was maintained in each subdivision and for each physiological class of neuron. However, MOPR agonists more frequently inhibited bipolar/fusiform cells than triangular or multipolar neurons. A subpopulation of MOPR-expressing neurons were also inhibited by delta opioid receptor agonists, whereas a separate population were inhibited kappa opioid receptors (KOPR). The MOPR agonist DAMGO inhibited 9/9 CeM neurons with projections to the parabrachial nucleus identified by retrograde tracer injection. These data support models of striatopallidal organization that have identified striatal-like and pallidal-like CeA regions. Opioids can directly inhibit output from each subdivision by activating postsynaptic MOPRs or KOPRs on distinct subpopulations of opioid-sensitive neurons.