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Most reports agree that aging negatively impacts pain processing and that the prevalence of chronic pain increases significantly with age. To improve current therapies, it is critical that aged animals be included in preclinical studies. Here we compared sensitivities to pain and itch-provoking stimuli in naïve and injured young and aged mice. Surprisingly, we found that in the absence of injury, aged male and female mice are significantly less responsive to mechanical stimuli and, in females, also to noxious thermal (heat) stimuli. In both older male and female mice, compared to younger (6 month-old mice), we also recorded reduced pruritogen-evoked scratching. On the other hand, after nerve injury, aged mice nevertheless developed significant mechanical hypersensitivity. Interestingly, however, and in contrast to young mice, aged mice developed both ipsilateral and contralateral post-injury mechanical allodynia. In a parallel immunohistochemical analysis of microglial and astrocyte markers, we found that the ipsilateral to contralateral ratio of nerve injury-induced expression decreased with age. That observation is consistent with our finding of contralateral hypersensitivity after nerve injury in the aged, but not the young mice. We conclude that aging has opposite effects on baseline vs post injury pain and itch processing. PERSPECTIVE: Aged male and female mice (22-24 months) are less sensitive to mechanical, thermal (heat) and itch-provoking stimuli than are younger mice (6 months).

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

AbstractNeuroimaging is a powerful tool to investigate potential associations between chronic pain and brain structure. However, the proliferation of studies across diverse chronic pain syndromes and heterogeneous results challenges data integration and interpretation. We conducted a preregistered anatomical likelihood estimate meta-analysis on structural magnetic imaging studies comparing patients with chronic pain and healthy controls. Specifically, we investigated a broad range of measures of brain structure as well as specific alterations in gray matter and cortical thickness. A total of 7849 abstracts of experiments published between January 1, 1990, and April 26, 2021, were identified from 8 databases and evaluated by 2 independent reviewers. Overall, 103 experiments with a total of 5075 participants met the preregistered inclusion criteria. After correction for multiple comparisons using the gold-standard family-wise error correction ( P < 0.05), no significant differences associated with chronic pain were found. However, exploratory analyses using threshold-free cluster enhancement revealed several spatially distributed clusters showing structural alterations in chronic pain. Most of the clusters coincided with regions implicated in nociceptive processing including the amygdala, thalamus, hippocampus, insula, anterior cingulate cortex, and inferior frontal gyrus. Taken together, these results suggest that chronic pain is associated with subtle, spatially distributed alterations of brain structure.

Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α2A-adrenergic receptor (α2AAR), seeking new α2AAR agonists chemotypes that lack the sedation conferred by known α2AAR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential Gi and Go signaling. Experimental structures of α2AAR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit '9087 [mean effective concentration (EC50) of 52 nanomolar] and two analogs, '7075 and PS75 (EC50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.

The 5-HT5A receptor (5-HT5AR), for which no selective agonists and a few antagonists exist, remains the least understood serotonin receptor. A single commercial antagonist, SB-699551, has been widely used to investigate the 5-HT5AR function in neurological disorders, including pain, but this molecule has substantial liabilities as a chemical probe. Accordingly, we sought to develop an internally controlled probe set. Docking over 6 million molecules against a 5-HT5AR homology model identified 5 mid-μM ligands, one of which was optimized to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT5AR with a more restricted off-target profile and decreased assay liabilities versus SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678. Surprisingly, analogs of UCSF678 that lost the 5-HT5AR activity revealed that 5-HT5AR engagement is nonessential for alleviating pain, contrary to studies with less-selective ligands. UCSF678 and analogs constitute a selective probe set with which to study the function of the 5-HT5AR.

TRPV1, a capsaicin- and heat-activated ion channel, is expressed by peripheral nociceptors and has been implicated in various inflammatory and neuropathic pain conditions. Although pharmacological modulation of TRPV1 has attracted therapeutic interest, many TRPV1 agonists and antagonists produce thermomodulatory side effects in animal models and human clinical trials, limiting their utility. These on-target effects may result from the perturbation of TRPV1 receptors on nociceptors, which transduce signals to central thermoregulatory circuits and release proinflammatory factors from their peripheral terminals, most notably the potent vasodilative neuropeptide, calcitonin gene-related peptide (CGRP). Alternatively, these body temperature effects may originate from the modulation of TRPV1 on vascular smooth muscle cells (vSMCs), where channel activation promotes arteriole constriction. Here, we ask which of these pathways is most responsible for the body temperature perturbations elicited by TRPV1 drugs in vivo. We address this question by selectively eliminating TRPV1 expression in sensory neurons or vSMCs and show that only the former abrogates agonist-induced hypothermia and antagonist-induced hyperthermia. Furthermore, lesioning the central projections of TRPV1-positive sensory nerve fibers also abrogates drug-mediated thermomodulation, whereas eliminating CGRP has no effect. Thus, TRPV1 drugs alter core body temperature by modulating sensory input to the central nervous system, rather than through peripheral actions on the vasculature. These findings suggest how mechanistically distinct TRPV1 antagonists may diminish inflammatory pain without affecting core body temperature.

The σ2 receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 μM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ1 receptor. Crystal structures of two ligands bound to the σ2 receptor confirmed the docked poses. To investigate the contribution of the σ2 receptor in pain, two potent σ2-selective ligands and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology.

A remarkable molecular and functional heterogeneity of the primary sensory neurons and dorsal horn interneurons transmits pain- and or itch-relevant information, but the molecular signature of the projection neurons that convey the messages to the brain is unclear. Here, using retro-TRAP (translating ribosome affinity purification) and RNA sequencing, we reveal extensive molecular diversity of spino- and trigeminoparabrachial projection neurons. Among the many genes identified, we highlight distinct subsets of Cck+ -, Nptx2+ -, Nmb+ -, and Crh+ -expressing projection neurons. By combining in situ hybridization of retrogradely labeled neurons with Fos-based assays, we also demonstrate significant functional heterogeneity, including both convergence and segregation of pain- and itch-provoking inputs into molecularly diverse subsets of NK1R- and non-NK1R-expressing projection neurons.

Febrile seizures (FSs) are the most common convulsion in infancy and childhood. Considering the limitations of current treatments, it is important to examine the mechanistic cause of FSs. Prompted by a genome-wide association study identifying TMEM16C (also known as ANO3) as a risk factor of FSs, we showed previously that loss of TMEM16C function causes hippocampal neuronal hyperexcitability [Feenstra et al., Nat. Genet. 46, 1274-1282 (2014)]. Our previous study further revealed a reduction in the number of warm-sensitive neurons that increase their action potential firing rate with rising temperature of the brain region harboring these hypothalamic neurons. Whereas central neuronal hyperexcitability has been implicated in FSs, it is unclear whether the maximal temperature reached during fever or the rate of body temperature rise affects FSs. Here we report that mutant rodent pups with TMEM16C eliminated from all or a subset of their central neurons serve as FS models with deficient thermoregulation. Tmem16c knockout (KO) rat pups at postnatal day 10 (P10) are more susceptible to hyperthermia-induced seizures. Moreover, they display a more rapid rise of body temperature upon heat exposure. In addition, conditional knockout (cKO) mouse pups (P11) with TMEM16C deletion from the brain display greater susceptibility of hyperthermia-induced seizures as well as deficiency in thermoregulation. We also found similar phenotypes in P11 cKO mouse pups with TMEM16C deletion from Ptgds-expressing cells, including temperature-sensitive neurons in the preoptic area (POA) of the anterior hypothalamus, the brain region that controls body temperature. These findings suggest that homeostatic thermoregulation plays an important role in FSs.

ABSTRACTDespite a robust literature associating IL-31 with pruritic inflammatory skin diseases, its influence on cutaneous inflammation and on the interplay between inflammatory and neurosensory pathways remain unmapped. Here, we examined the effects of IL-31 and its receptor IL31RA on both inflammation and pruritus in mouse models of dermatitis, including chronic topical house dust mite (HDM) exposure. Unexpectedly, Il31 deficiency increased cutaneous adaptive type 2 cytokine-producing cells and serum IgE. In addition, M2-like macrophages capable of fueling feedforward pro-inflammatory loops were selectively enriched in Il31ra-deficient skin. Thus, IL-31 is not strictly a pro-inflammatory cytokine, but rather an immunoregulatory factor that limits the magnitude of allergic skin inflammation. In contrast, Il31-deficient mice displayed a deficit in HDM-induced scratching. Itch reduction occurred despite intact – and in some cases increased – responsiveness of sensory neurons to other pruritogens released during HDM challenge, highlighting the non-redundant contribution of IL-31-receptive sensory afferents to pruritus in environmental allergen-induced dermatitis. When present, therefore, IL-31 uncouples circuits driven by sensory neurons and immune cells that converge in inflamed skin.

Pruritus is a common symptom of inflammatory skin conditions, including atopic dermatitis (AD). Although primary sensory neurons that transmit pruritic signals are well-cataloged, little is known about the neuronal alterations that occur as a result of skin disruption in AD. To address this question, we examined the molecular and behavioral consequences of challenging Grhl3PAR2/+ mice, which overexpress PAR2 in suprabasal keratinocytes, with serial topical application of the environmental allergen house dust mite (HDM). We monitored behavior and used RNA sequencing, qPCR, and in situ hybridization to evaluate gene expression in trigeminal ganglia (TG), before and after HDM. We found that neither Grhl3PAR2/+ nor wild-type (WT) mice exhibited spontaneous scratching, and pruritogen-induced acute scratching did not differ. In contrast, HDM exacerbated scratching in Grhl3PAR2/+ mice. Despite the absence of scratching in untreated Grhl3PAR2/+ mice, several TG genes in these mice were up-regulated compared to WT. HDM treatment of the Grhl3PAR2/+ mice enhanced up-regulation of this set of genes and induced additional genes, many within the subset of TG neurons that express TRPV1. The same set of genes was up-regulated in HDM-treated Grhl3PAR2/+ mice that did not scratch, but at lesser magnitude. Finally, we recorded comparable transcriptional changes in IL31Tg mice, demonstrating that a common genetic program is induced in two AD models. Taken together, we conclude that transcriptional changes that occur in primary sensory neurons in dermatitis-susceptible animals underlie a genetic priming that not only sensitizes the animal to chronic allergens but also contributes to pruritus in atopic skin disease.

ImportanceFunctional neuroimaging is a valuable tool for understanding how patients with chronic pain respond to painful stimuli. However, past studies have reported heterogenous results, highlighting opportunities for a quantitative meta-analysis to integrate existing data and delineate consistent associations across studies.ObjectiveTo identify differential brain responses to noxious stimuli in patients with chronic pain using functional magnetic resonance imaging (fMRI) while adhering to current best practices for neuroimaging meta-analyses.Data sourcesAll fMRI experiments published from January 1, 1990, to May 28, 2019, were identified in a literature search of PubMed/MEDLINE, EMBASE, Web of Science, Cochrane Library, PsycINFO, and SCOPUS.Study selectionExperiments comparing brain responses to noxious stimuli in fMRI between patients and controls were selected if they reported whole-brain results, included at least 10 patients and 10 healthy control participants, and used adequate statistical thresholding (voxel-height P

ABSTRACT The 5-HT 5A receptor (5-HT 5A R), for which no selective agonists and only a few antagonists exist, remains the least understood serotonin (5-HT) receptor. A single commercial antagonist (SB-699551) has been widely used to investigate central nervous system (CNS) 5-HT 5A R function in neurological disorders, including pain. However, because SB-699551 has affinity for many 5-HTRs, lacks inactive property-matched controls, and has assay interference concerns, it has liabilities as a chemical probe. To better illuminate 5-HT 5A R function, we developed a probe set through iterative rounds of molecular docking, pharmacological testing, and optimization. Docking over six million lead-like molecules against a 5-HT 5A R homology model identified five mid-μM ligand starting points with unique scaffolds. Over multiple rounds of structure-based design and testing, a new quinoline scaffold with high affinity and enhanced selectivity for the 5-HT 5A R was developed, leading to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT 5A R with a much more restricted off-target profile and decreased assay liabilities vs. SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678, which was also consistent with recent published 5-HTR structures. Surprisingly, property-matched analogs of UCSF678 that were either inactive across 5-HTRs or retained affinity for UCSF678’s off-targets revealed that 5-HT 5A R engagement is nonessential for alleviating pain in a mouse model, contrary to previous studies using less-selective ligands. Relative to SB-699551, these molecules constitute a well-characterized and more selective probe set with which to study the function of the 5-HT 5A receptor. Table of Contents Graphic

Estrogens are presumed to underlie, at least in part, the greater pain sensitivity and chronic pain prevalence that women experience compared to men. Although previous studies revealed populations of estrogen receptor-expressing neurons in primary afferents and in superficial dorsal horn neurons, there is little to no information as to the contribution of these neurons to the generation of acute and chronic pain. Here we molecularly characterized neurons in the mouse superficial spinal cord dorsal horn that express estrogen receptor α (ERα) and explored the behavioral consequences of their ablation. We found that spinal ERα-positive neurons are largely excitatory interneurons and many coexpress substance P, a marker for a discrete subset of nociceptive, excitatory interneurons. After viral, caspase-mediated ablation of spinal ERα-expressing cells, we observed a significant decrease in the first phase of the formalin test, but in male mice only. ERα-expressing neuron-ablation also reduced pruritogen-induced scratching in both male and female mice. There were no ablation-related changes in mechanical or heat withdrawal thresholds or in capsaicin-induced nocifensive behavior. In chronic pain models, we found no change in Complete Freund's adjuvant-induced thermal or mechanical hypersensitivity, or in partial sciatic nerve injury-induced mechanical allodynia. We conclude that ERα labels a subpopulation of excitatory interneurons that are specifically involved in chemically evoked persistent pain and pruritogen-induced itch.

Characterizing a reliable, pain-related neural signature is critical for translational applications. Many prior fMRI studies have examined acute nociceptive pain-related brain activation in healthy participants. However, synthesizing these data to identify convergent patterns of activation can be challenging due to the heterogeneity of experimental designs and samples. To address this challenge, we conducted a comprehensive meta-analysis of fMRI studies of stimulus-induced pain in healthy participants. Following pre-registration, two independent reviewers evaluated 4,927 abstracts returned from a search of 8 databases, with 222 fMRI experiments meeting inclusion criteria. We analyzed these experiments using Activation Likelihood Estimation with rigorous type I error control (voxel height p < 0.001, cluster p < 0.05 FWE-corrected) and found a convergent, largely bilateral pattern of pain-related activation in the secondary somatosensory cortex, insula, midcingulate cortex, and thalamus. Notably, these regions were consistently recruited regardless of stimulation technique, location of induction, and participant sex. These findings suggest a highly-conserved core set of pain-related brain areas, encouraging applications as a biomarker for novel therapeutics targeting acute nociceptive pain.

Paralleling the activation of dorsal horn microglia after peripheral nerve injury is a significant expansion and proliferation of macrophages around injured sensory neurons in dorsal root ganglia (DRG). Here we demonstrate a critical contribution of DRG macrophages, but not those at the nerve injury site, to both the initiation and maintenance of the mechanical hypersensitivity that characterizes the neuropathic pain phenotype. In contrast to the reported sexual dimorphism in the microglial contribution to neuropathic pain, depletion of DRG macrophages reduces nerve injury-induced mechanical hypersensitivity and expansion of DRG macrophages in both male and female mice. However, fewer macrophages are induced in the female mice and deletion of colony-stimulating factor 1 from sensory neurons, which prevents nerve injury-induced microglial activation and proliferation, only reduces macrophage expansion in male mice. Finally, we demonstrate molecular cross-talk between axotomized sensory neurons and macrophages, revealing potential peripheral DRG targets for neuropathic pain management.

Calcium binding proteins are expressed throughout the central and peripheral nervous system and disruption of their activity has major consequences in a wide array of cellular processes, including transmission of nociceptive signals that are processed at the level of the spinal cord. We previously reported that the calcium binding protein, hippocalcin-like 4 (Hpcal4), is heavily expressed in interneurons of the superficial dorsal horn, and that its expression is significantly downregulated in a TR4 mutant mouse model that exhibits major pain and itch deficits due to loss of a subpopulation of excitatory interneurons. That finding suggested that Hpcal4 may be a contributor to the behavioral phenotype of the TR4 mutant mouse. To address this question, here we investigated the behavioral consequences of global deletion of Hpcal4 in a battery of acute and persistent pain and itch tests. Unexpectedly, with the exception of a mild reduction in acute baseline thermal responses, Hpcal4-deficient mice exhibit no major deficits in pain or itch responses, under normal conditions or in the setting of tissue or nerve injury. Taken together, our results indicate that the neural calcium sensor Hpcal4 likely makes a limited contribution to pain and itch processing.

Today there are extensive maps of the molecular heterogeneity of primary afferents and dorsal horn interneurons, yet there is a dearth of molecular and functional information regarding the projection neurons that transmit pain and itch information to the brain. Additionally, most contemporary research into the spinal cord and medullary projection neurons focuses on neurons in the superficial dorsal horn; the contribution of deep dorsal horn and even ventral horn projection neurons to pain and itch processing is often overlooked. In the present review we integrate conclusions from classical as well as contemporary studies and provide a more balanced view of the diversity of projection neurons. A major question addressed is the extent to which labeled-lines are maintained in these different populations or whether the brain generates distinct pain and itch percepts by decoding complex convergent inputs that engage projection neurons.

Dysfunction of inhibitory circuits in the rostral anterior cingulate cortex underlies the affective (aversive), but not the sensory-discriminative features (hypersensitivity) of the pain experience. To restore inhibitory controls, we transplanted inhibitory interneuron progenitor cells into the rostral anterior cingulate cortex in a chemotherapy-induced neuropathic pain model. The transplants integrated, exerted a GABA-A mediated inhibition of host pyramidal cells and blocked gabapentin preference (i.e. relieved ongoing pain) in a conditioned place preference paradigm. Surprisingly, pain aversiveness persisted when the transplants populated both the rostral and posterior anterior cingulate cortex. We conclude that selective and long lasting inhibition of the rostral anterior cingulate cortex, in the mouse, has a profound pain relieving effect against nerve injury-induced neuropathic pain. However, the interplay between the rostral and posterior anterior cingulate cortices must be considered when examining circuits that influence ongoing pain and pain aversiveness.

Hyperexcitability of the anterior cingulate cortex (ACC) is thought to drive aversion associated with chronic neuropathic pain. Here, we studied the contribution of input from the mediodorsal thalamus (MD) to ACC, using sciatic nerve injury and chemotherapy-induced mouse models of neuropathic pain. Activating MD inputs elicited pain-related aversion in both models. Unexpectedly, excitatory responses of layer V ACC neurons to MD inputs were significantly weaker in pain models compared to controls. This caused the ratio between excitation and feedforward inhibition elicited by MD input to shift toward inhibition, specifically for subcortically projecting (SC) layer V neurons. Furthermore, direct inhibition of SC neurons reproduced the pain-related aversion elicited by activating MD inputs. Finally, both the ability to elicit pain-related aversion and the decrease in excitation were specific to MD inputs; activating basolateral amygdala inputs produced opposite effects. Thus, chronic pain-related aversion may reflect activity changes in specific pathways, rather than generalized ACC hyperactivity.

Reelin (Reln) and Disabled-1 (Dab1) participate in the Reln-signaling pathway and when either is deleted, mutant mice have the same spinally mediated behavioral abnormalities, increased sensitivity to noxious heat and a profound loss in mechanical sensitivity. Both Reln and Dab1 are highly expressed in dorsal horn areas that receive and convey nociceptive information, Laminae I-II, lateral Lamina V, and the lateral spinal nucleus (LSN). Lamina I contains both projection neurons and interneurons that express Neurokinin-1 receptors (NK1Rs) and they transmit information about noxious heat both within the dorsal horn and to the brain. Here, we ask whether the increased heat nociception in Reln and dab1 mutants is due to incorrectly positioned dorsal horn neurons that express NK1Rs. We found more NK1R-expressing neurons in Reln-/- and dab1-/- Laminae I-II than in their respective wild-type mice, and some NK1R neurons co-expressed Dab1 and the transcription factor Lmx1b, confirming their excitatory phenotype. Importantly, heat stimulation in dab1-/- mice induced Fos in incorrectly positioned NK1R neurons in Laminae I-II. Next, we asked whether these ectopically placed and noxious-heat responsive NK1R neurons participated in pain behavior. Ablation of the superficial NK1Rs with an intrathecal injection of a substance P analog conjugated to the toxin saporin (SSP-SAP) eliminated the thermal hypersensitivity of dab1-/- mice, without altering their mechanical insensitivity. These results suggest that ectopically positioned NK1R-expressing neurons underlie the heat hyperalgesia of Reelin-signaling pathway mutants, but do not contribute to their profound mechanical insensitivity.

Excitatory interneurons account for the majority of neurons in the superficial dorsal horn, but despite their presumed contribution to pain and itch, there is still limited information about their organisation and function. We recently identified 2 populations of excitatory interneuron defined by expression of gastrin-releasing peptide (GRP) or substance P (SP). Here, we demonstrate that these cells show major differences in their morphological, electrophysiological, and pharmacological properties. Based on their somatodendritic morphology and firing patterns, we propose that the SP cells correspond to radial cells, which generally show delayed firing. By contrast, most GRP cells show transient or single-spike firing, and many are likely to correspond to the so-called transient central cells. Unlike the SP cells, few of the GRP cells had long propriospinal projections, suggesting that they are involved primarily in local processing. The 2 populations also differed in responses to neuromodulators, with most SP cells, but few GRP cells, responding to noradrenaline and 5-HT; the converse was true for responses to the μ-opioid agonist DAMGO. Although a recent study suggested that GRP cells are innervated by nociceptors and are strongly activated by noxious stimuli, we found that very few GRP cells receive direct synaptic input from TRPV1-expressing afferents, and that they seldom phosphorylate extracellular signal-regulated kinases in response to noxious stimuli. These findings indicate that the SP and GRP cells differentially process somatosensory information.

“Somehow scientists still pursue the same questions, if now on higher levels of theoretical abstraction rooted in deeper layers of empirical evidence… To paraphrase an old philosophy joke, science is more like it is today than it has ever been. In other words, science remains as challenging as ever to human inquiry. And the need to communicate its progress… remains as essential now as then.” — Tom Siegfried, Science News 2021 In fact, essential questions about pain have not changed since IASP’s creation in Issaquah: what causes it and how can we treat it? Are we any closer to answering these questions, or have we just widened the gap between bench and bedside? The technology used to answer questions about pain mechanisms has certainly changed, whether the focus is on sensory neurons, spinal cord circuitry, descending controls or cortical pain processing. In this paper, we will describe how transgenics, transcriptomics, optogenetics, calcium imaging, fMRI, neuroimmunology and in silico drug development have transformed the way we examine the complexity of pain processing. But does it all, as our founders hoped, help people with pain? Are voltage-gated Na channels the new holy grail for analgesic development, is there a pain biomarker, can we completely replace opioids, will proteomic analyses identify novel targets, is there a “pain matrix,” and can it be targeted? Do the answers lie in our tangible discoveries, or in the seemingly intangible? Our founders could barely imagine what we know now, yet their questions remain. [ABSTRACT FROM AUTHOR]

BDNF is a critical contributor to neuronal growth, development, learning, and memory. Although extensively studied in the brain, BDNF is also expressed by primary afferent sensory neurons in the peripheral nervous system. Unfortunately, anatomical and functional studies of primary afferent-derived BDNF have been limited by the availability of appropriate molecular tools. Here, we used targeted, inducible molecular approaches to characterize the expression pattern of primary afferent BDNF and the extent to which it contributes to a variety of pain and itch behaviors. Using a BDNF-LacZ reporter mouse, we found that BDNF is expressed primarily by myelinated primary afferents and has limited overlap with the major peptidergic and non-peptidergic subclasses of nociceptors and pruritoceptors. We also observed extensive neuronal, but not glial, expression in the spinal cord dorsal horn. In addition, because BDNF null mice are not viable and even Cre-mediated deletion of BDNF from sensory neurons could have developmental consequences, here we deleted BDNF selectively from sensory neurons, in the adult, using an advillin-Cre-ER line crossed to floxed BDNF mice. We found that BDNF deletion in the adult altered few itch or acute and chronic pain behaviors, beyond sexually dimorphic phenotypes in the tail immersion, histamine, and formalin tests. Based on the anatomical distribution of sensory neuron-derived BDNF and its limited contribution to pain and itch processing, we suggest that future studies of primary afferent-derived BDNF should examine behaviors evoked by activation of myelinated primary afferents.

Introduction:Gabapentin regulates pain processing by direct action on primary afferent nociceptors and dorsal horn nociresponsive neurons. Through an action at supraspinal levels, gabapentin also engages descending noradrenergic inhibitory controls that indirectly regulate spinal cord pain processing. Although direct injection of gabapentin into the anterior cingulate cortex provides pain relief independent of descending inhibitory controls, it remains unclear whether that effect is representative of what occurs when gabapentin interacts at multiple brain loci, eg, after intracerebroventricular (i.c.v.) injection. Methods:We administered gabapentin i.c.v. in a mouse model of chemotherapy (paclitaxel)-induced neuropathic pain. To distinguish spinal from supraspinally processed features of the pain experience, we examined mechanical hypersensitivity and assessed relief of pain aversiveness using an analgesia-induced conditioned place preference paradigm. Results:Paclitaxel-treated mice showed a preference for a 100-μg i.c.v. gabapentin-paired chamber that was accompanied by reduced mechanical allodynia, indicative of concurrent engagement of descending controls. As expected, the same dose in uninjured mice did not induce place preference, demonstrating that gabapentin, unlike morphine, is not inherently rewarding. Furthermore, a lower dose of supraspinal gabapentin (30 μg), which did not reverse mechanical allodynia, did not induce conditioned place preference. Finally, concurrent injections of i.c.v. gabapentin (100 μg) and intrathecal yohimbine, an α2-receptor antagonist, blocked preference for the gabapentin-paired chamber. Conclusion:We conclude that pain relief, namely a reduction of pain aversiveness, induced by supraspinal gabapentin administered by an i.c.v. route is secondary to its activation of descending noradrenergic inhibitory controls that block transmission of the "pain" message from the spinal cord to the brain.

Although chronic pain is one of the most important medical problems facing society, there has been very limited progress in the development of novel therapies for this condition. Here, we discuss high-impact research priorities to reduce the number of people transitioning from acute to chronic intractable pain.

Cellular interactions between delta and mu opioid receptors (DORs and MORs), including heteromerization, are thought to regulate opioid analgesia. However, the identity of the nociceptive neurons in which such interactions could occur in vivo remains elusive. Here we show that DOR-MOR co-expression is limited to small populations of excitatory interneurons and projection neurons in the spinal cord dorsal horn and unexpectedly predominates in ventral horn motor circuits. Similarly, DOR-MOR co-expression is rare in parabrachial, amygdalar, and cortical brain regions processing nociceptive information. We further demonstrate that in the discrete DOR-MOR co-expressing nociceptive neurons, the two receptors internalize and function independently. Finally, conditional knockout experiments revealed that DORs selectively regulate mechanical pain by controlling the excitability of somatostatin-positive dorsal horn interneurons. Collectively, our results illuminate the functional organization of DORs and MORs in CNS pain circuits and reappraise the importance of DOR-MOR cellular interactions for developing novel opioid analgesics.

Transplanting embryonic precursors of GABAergic neurons from the medial ganglionic eminence (MGE) into adult mouse spinal cord ameliorates mechanical and thermal hypersensitivity in peripheral nerve injury models of neuropathic pain. Although Fos and transneuronal tracing studies strongly suggest that integration of MGE-derived neurons into host spinal cord circuits underlies recovery of function, the extent to which there is synaptic integration of the transplanted cells has not been established. Here, we used electron microscopic immunocytochemistry to assess directly integration of GFP-expressing MGE-derived neuronal precursors into dorsal horn circuitry in intact, adult mice with short- (5-6 weeks) or long-term (4-6 months) transplants. We detected GFP with pre-embedding avidin-biotin-peroxidase and GABA with post-embedding immunogold labeling. At short and long times post-transplant, we found host-derived synapses on GFP-immunoreactive MGE cells bodies and dendrites. The proportion of dendrites with synaptic input increased from 50% to 80% by 6 months. In all mice, MGE-derived terminals formed synapses with GFP-negative (host) cell bodies and dendrites and, unexpectedly, with some GFP-positive (i.e., MGE-derived) dendrites, possibly reflecting autoapses or cross talk among transplanted neurons. We also observed axoaxonic appositions between MGE and host terminals. Immunogold labeling for GABA confirmed that the transplanted cells were GABAergic and that some transplanted cells received an inhibitory GABAergic input. We conclude that transplanted MGE neurons retain their GABAergic phenotype and integrate dynamically into host-transplant synaptic circuits. Taken together with our previous electrophysiological analyses, we conclude that MGE cells are not GABA pumps, but alleviate pain and itch through synaptic release of GABA.

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion-derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.

BackgroundDespite recent insights into the pathophysiology of acute and chronic itch, chronic itch remains an often intractable condition. Among major contributors to chronic itch is dysfunction of spinal cord gamma aminobutyric acidergic (GABAergic) inhibitory controls.ObjectivesWe sought to test the hypothesis that selective GABA agonists as well as cell transplant-derived GABA are antipruritic against acute itch and in a transgenic mouse model of atopic dermatitis produced by overexpression of the TH2 cell-associated cytokine, IL-31 (IL-31Tg mice).MethodsWe injected wild-type and IL-31Tg mice with combinations of GABA-A (muscimol) or GABA-B (baclofen) receptor agonists 15 to 20 minutes prior to injection of various pruritogens (histamine, chloroquine, or endothelin-1) and recorded spontaneous scratching before and after drug administration. We also tested the antipruritic properties of intraspinal transplantation of precursors of GABAergic interneurons in the IL-31Tg mice.ResultsSystemic muscimol or baclofen are antipruritic against both histamine-dependent and -independent pruritogens, but the therapeutic window using either ligand alone was very small. In contrast, combined subthreshold doses of baclofen and muscimol produced a significant synergistic antipruritic effect, with no sedation. Finally, transplant-mediated long-term enhancement of GABAergic signaling not only reduced spontaneous scratching in the IL-31Tg mice but also dramatically resolved the associated skin lesions.ConclusionsAlthough additional research is clearly needed, existing approved GABA agonists should be considered in the management of chronic itch, notably atopic dermatitis.

Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X2 and/or P2X3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.

Cell transplantation offers an attractive alternative to pharmacotherapy for the management of a host of clinical conditions. Most importantly, the transplanted cells provide a continuous, local delivery of therapeutic compounds, which avoids many of the adverse side effects associated with systemically administered drugs. Here, we describe the broad therapeutic utility of transplanting precursors of cortical inhibitory interneurons derived from the embryonic medial ganglionic eminence (MGE), in a variety of chronic pain and itch models in the mouse. Despite the cortical environment in which the MGE cells normally develop, these cells survive transplantation and will even integrate into the circuitry of an adult host spinal cord. When transplanted into the spinal cord, the cells significantly reduce the hyperexcitability that characterizes both chronic neuropathic pain and itch conditions. This MGE cell-based strategy differs considerably from traditional pharmacological treatments as the approach is potentially disease modifying (i.e., the therapy targets the underlying etiology of the pain and itch pathophysiology).

Cell transplantation offers an attractive alternative to pharmacotherapy for the management of a host of clinical conditions. Most importantly, the transplanted cells provide a continuous, local delivery of therapeutic compounds, which avoids many of the adverse side effects associated with systemically administered drugs. Here, we describe the broad therapeutic utility of transplanting precursors of cortical inhibitory interneurons derived from the embryonic medial ganglionic eminence (MGE), in a variety of chronic pain and itch models in the mouse. Despite the cortical environment in which the MGE cells normally develop, these cells survive transplantation and will even integrate into the circuitry of an adult host spinal cord. When transplanted into the spinal cord, the cells significantly reduce the hyperexcitability that characterizes both chronic neuropathic pain and itch conditions. This MGE cell-based strategy differs considerably from traditional pharmacological treatments as the approach is potentially disease modifying (i.e., the therapy targets the underlying etiology of the pain and itch pathophysiology).

Spinal cord transplants of embryonic cortical GABAergic progenitor cells derived from the medial ganglionic eminence (MGE) can reverse mechanical hypersensitivity in the mouse models of peripheral nerve injury- and paclitaxel-induced neuropathic pain. Here, we used electrophysiology, immunohistochemistry, and electron microscopy to examine the extent to which MGE cells integrate into host circuitry and recapitulate endogenous inhibitory circuits. Whether the transplants were performed before or after nerve injury, the MGE cells developed into mature neurons and exhibited firing patterns characteristic of subpopulations of cortical and spinal cord inhibitory interneurons. Conversely, the transplanted cells preserved cortical morphological and neurochemical properties. We also observed a robust anatomical and functional synaptic integration of the transplanted cells into host circuitry in both injured and uninjured animals. The MGE cells were activated by primary afferents, including TRPV1-expressing nociceptors, and formed GABAergic, bicuculline-sensitive, synapses onto host neurons. Unexpectedly, MGE cells transplanted before injury prevented the development of mechanical hypersensitivity. Together, our findings provide direct confirmation of an extensive, functional synaptic integration of MGE cells into host spinal cord circuits. This integration underlies normalization of the dorsal horn inhibitory tone after injury and may be responsible for the prophylactic effect of preinjury transplants.Significance statementSpinal cord transplants of embryonic cortical GABAergic interneuron progenitors from the medial ganglionic eminence (MGE), can overcome the mechanical hypersensitivity produced in different neuropathic pain models in adult mice. Here, we examined the properties of transplanted MGE cells and the extent to which they integrate into spinal cord circuitry. Using electrophysiology, immunohistochemistry, and electron microscopy, we demonstrate that MGE cells, whether transplanted before or after nerve injury, develop into inhibitory neurons, are activated by nociceptive primary afferents, and form GABA-A-mediated inhibitory synapses with the host. Unexpectedly, cells transplanted into naive spinal cord prevented the development of nerve-injury-induced mechanical hypersensitivity. These results illustrate the remarkable plasticity of adult spinal cord and the potential of cell-based therapies against neuropathic pain.

Experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis produced by immunization with myelin oligodendrocyte glycoprotein (MOG) and adjuvants, results from profound T-cell mediated CNS demyelination. EAE is characterized by progressive, ascending motor dysfunction and symptoms of ongoing pain and hypersensitivity, in some cases preceding or concomitant with the motor deficits. In this regard, the EAE model mimics major features of multiple sclerosis, where a central neuropathic pain state is common. Although the latter condition is presumed to arise from a CNS loss of inhibitory controls secondary to the demyelination, dysfunction of sensory neurons may also contribute. Based on our previous studies that demonstrated the utility of monitoring expression of activating transcription factor 3 (ATF3), a sensitive marker of injured sensory neurons, here we followed both ATF3 and CD4+ T cells invasion of sensory ganglia (as well as the CNS) at different stages of the EAE model. We found that ATF3 is induced in peripheral sensory ganglia and brainstem well before the appearance of motor deficits. Unexpectedly, the ATF3 induction always preceded T cell infiltration, typically in adjacent, but non-overlapping regions. Surprisingly, control administration of the pertussis toxin and/or Complete Freund's adjuvants, without MOG, induced ATF3 in sensory neurons. In contrast, T cell infiltration only occurred with MOG. Taken together, our results suggest that the clinical manifestations in the EAE result not only from central demyelination but also from neuronal stress and subsequent pathophysiology of sensory neurons.

There is growing concern about lack of scientific rigor and transparent reporting across many preclinical fields of biological research. Poor experimental design and lack of transparent reporting can result in conscious or unconscious experimental bias, producing results that are not replicable. The Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION) public-private partnership with the U.S. Food and Drug Administration sponsored a consensus meeting of the Preclinical Pain Research Consortium for Investigating Safety and Efficacy (PPRECISE) Working Group. International participants from universities, funding agencies, government agencies, industry, and a patient advocacy organization attended. Reduction of publication bias, increasing the ability of others to faithfully repeat experimental methods, and increased transparency of data reporting were specifically discussed. Parameters deemed essential to increase confidence in the published literature were clear, specific reporting of an a priori hypothesis and definition of primary outcome measure. Power calculations and whether measurement of minimal meaningful effect size to determine these should be a core component of the preclinical research effort provoked considerable discussion, with many but not all agreeing. Greater transparency of reporting should be driven by scientists, journal editors, reviewers, and grant funders. The conduct of high-quality science that is fully reported should not preclude novelty and innovation in preclinical pain research, and indeed, any efforts that curtail such innovation would be misguided. We believe that to achieve the goal of finding effective new treatments for patients with pain, the pain field needs to deal with these challenging issues.

Among many mechanisms implicated in the development of neuropathic pain after nerve damage is a profound dysfunction of GABAergic inhibitory controls, manifested by ongoing pain, mechanical hypersensitivity, and thermal hyperalgesia. In some respects, neuropathic pain can be considered a "disease" of the nervous system, with features in common with trauma-induced seizures. Indeed, first-line management involves anticonvulsant therapy. An alternative to pharmacotherapy for neuropathic pain is an approach that reestablishes the inhibitory tone that is lost after nerve damage. To this end, we have transplanted embryonic cortical GABAergic precursor neurons into the spinal cord of nerve-injured mice. Using a combination of light and electron microscopic analyses, and also in vitro electrophysiological recordings from spinal cord slice preparations, we demonstrated remarkable integration of the transplants into the host, adult spinal cord. Most importantly, transplants produced a complete reversal of the hypersensitivity in a sciatic nerve injury model and in a paclitaxel-generated chemotherapy model of neuropathic pain. In related studies, we demonstrated that medial ganglionic eminence cell transplants are also effective in a chronic neuropathic itch model in which there is a significant loss of dorsal horn inhibitory interneurons. Most importantly, in contrast to systemic or intrathecal pharmacological therapies, adverse side effects are minimized when the inhibitory control, namely, γ-aminobutyric acid release, occurs in a spinal cord circuit. These studies suggest that therapy targeted at repairing the GABAergic dysfunction is a viable and novel alternative to the management of neuropathic pain and itch, particularly those that are or become refractory to traditional pharmacotherapy.

Neuropathic pain is a chronic debilitating disease that results from nerve damage, persists long after the injury has subsided, and is characterized by spontaneous pain and mechanical hypersensitivity. Although loss of inhibitory tone in the dorsal horn of the spinal cord is a major contributor to neuropathic pain, the molecular and cellular mechanisms underlying this disinhibition are unclear. Here, we combined pharmacogenetic activation and selective ablation approaches in mice to define the contribution of spinal cord parvalbumin (PV)-expressing inhibitory interneurons in naive and neuropathic pain conditions. Ablating PV neurons in naive mice produce neuropathic pain-like mechanical allodynia via disinhibition of PKCγ excitatory interneurons. Conversely, activating PV neurons in nerve-injured mice alleviates mechanical hypersensitivity. These findings indicate that PV interneurons are modality-specific filters that gate mechanical but not thermal inputs to the dorsal horn and that increasing PV interneuron activity can ameliorate the mechanical hypersensitivity that develops following nerve injury.

One approach to analgesia is to block pain at the site of origin or along the peripheral pathway by selectively ablating pain-transmitting neurons or nerve terminals directly. The heat/capsaicin receptor (TRPV1) expressed by nociceptive neurons is a compelling target for selective interventional analgesia because it leaves somatosensory and proprioceptive neurons intact. Resiniferatoxin (RTX), like capsaicin, is a TRPV1 agonist but has greater potency. We combine RTX-mediated inactivation with the precision of computed tomography (CT)-guided delivery to ablate peripheral pain fibers in swine. Under CT guidance, RTX was delivered unilaterally around the lumbar dorsal root ganglia (DRG), and vehicle only was administered to the contralateral side. During a 4-week observation period, animals demonstrated delayed or absent withdrawal responses to infrared laser heat stimuli delivered to sensory dermatomes corresponding to DRG receiving RTX treatment. Motor function was unimpaired as assessed by disability scoring and gait analysis. In treated DRG, TRPV1 mRNA expression was reduced, as were nociceptive neuronal perikarya in ganglia and their nerve terminals in the ipsilateral dorsal horn. CT guidance to precisely deliver RTX to sites of peripheral pain transmission in swine may be an approach that could be tailored to block an array of clinical pain conditions in patients.

Decreased spinal cord GABAergic inhibition is a major contributor to the persistent neuropathic pain that can follow peripheral nerve injury. Recently, we reported that restoring spinal cord GABAergic signaling by intraspinal transplantation of cortical precursors of GABAergic interneurons from the embryonic medial ganglionic eminence (MGE) can reverse the mechanical hypersensitivity (allodynia) that characterizes a neuropathic pain model in the mouse. We show that MGE cell transplants are also effective against both the mechanical allodynia and the heat hyperalgesia produced in a paclitaxel-induced chemotherapy model of neuropathic pain. To test the necessity of GABA release by the transplants, we also studied the utility of transplanting MGE cells from mice with a deletion of VGAT, the vesicular GABA transporter. Transplants from these mice, in which GABA is synthesized but cannot be stored or released, had no effect on mechanical hypersensitivity or heat hyperalgesia in the paclitaxel model. Taken together, these results demonstrate the therapeutic potential of GABAergic precursor cell transplantation in diverse neuropathic pain models and support our contention that restoration of inhibitory controls through release of GABA from the transplants is their mode of action.

There is continuing controversy relating to the primary afferent neurotransmitter that conveys itch signals to the spinal cord. Here, we investigated the DRG and spinal cord expression of the putative primary afferent-derived "itch" neurotransmitter, gastrin-releasing peptide (GRP). Using ISH, qPCR, and immunohistochemistry, we conclude that GRP is expressed abundantly in spinal cord, but not in DRG neurons. Titration of the most commonly used GRP antiserum in tissues from wild-type and GRP mutant mice indicates that the antiserum is only selective for GRP at high dilutions. Paralleling these observations, we found that a GRPeGFP transgenic reporter mouse has abundant expression in superficial dorsal horn neurons, but not in the DRG. In contrast to previous studies, neither dorsal rhizotomy nor an intrathecal injection of capsaicin, which completely eliminated spinal cord TRPV1-immunoreactive terminals, altered dorsal horn GRP immunoreactivity. Unexpectedly, however, peripheral nerve injury induced significant GRP expression in a heterogeneous population of DRG neurons. Finally, dual labeling and retrograde tracing studies showed that GRP-expressing neurons of the superficial dorsal horn are predominantly interneurons, that a small number coexpress protein kinase C gamma (PKCγ), but that none coexpress the GRP receptor (GRPR). Our studies support the view that pruritogens engage spinal cord "itch" circuits via excitatory superficial dorsal horn interneurons that express GRP and that likely target GRPR-expressing interneurons. The fact that peripheral nerve injury induced de novo GRP expression in DRG neurons points to a novel contribution of this peptide to pruritoceptive processing in neuropathic itch conditions.

The transmission of pruritoceptive (itch) messages involves specific neural circuits within the spinal cord that are distinct from those that transmit pain messages. These itch-specific circuits are tonically regulated by inhibitory interneurons in the dorsal horn. Consistent with these findings, it has previously been reported that loss of GABAergic interneurons in mice harboring a deletion of the transcription factor Bhlhb5 generates a severe, nonremitting condition of chronic itch. Here, we tested the hypothesis that the neuropathic itch in BHLHB5-deficient animals can be treated by restoring inhibitory controls through spinal cord transplantation and integration of precursors of cortical inhibitory interneurons derived from the embryonic medial ganglionic eminence. We specifically targeted the transplants to segments of the spinal cord innervated by areas of the body that were most severely affected. BHLHB5-deficient mice that received transplants demonstrated a substantial reduction of excessive scratching and dramatic resolution of skin lesions. In contrast, the scratching persisted and skin lesions worsened over time in sham-treated mice. Together, these results indicate that cell-mediated restoration of inhibitory controls has potential as a powerful, cell-based therapy for neuropathic itch that not only ameliorates symptoms of chronic itch, but also may modify disease.

Many neurologic and psychiatric disorders are marked by imbalances between neural excitation and inhibition. In the cerebral cortex, inhibition is mediated largely by GABAergic (γ-aminobutyric acid-secreting) interneurons, a cell type that originates in the embryonic ventral telencephalon and populates the cortex through long-distance tangential migration. Remarkably, when transplanted from embryos or in vitro culture preparations, immature interneurons disperse and integrate into host brain circuits, both in the cerebral cortex and in other regions of the central nervous system. These features make interneuron transplantation a powerful tool for the study of neurodevelopmental processes such as cell specification, cell death, and cortical plasticity. Moreover, interneuron transplantation provides a novel strategy for modifying neural circuits in rodent models of epilepsy, Parkinson's disease, mood disorders, and chronic pain.