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Gamma aminobutyric acid type A receptors (GABA A Rs) play a key role in the mammalian central nervous system (CNS) as drivers of neuroinhibitory circuits, which are commonly targeted for therapeutic purposes with potentiator drugs. However, due to their widespread expression and strong inhibitory action, systemic pharmaceutical potentiation of GABA A Rs inevitably causes adverse effects regardless of the drug selectivity. Therefore, therapeutic guidelines must often limit or exclude clinically available GABA A R potentiators, despite their high efficacy, good biodistribution, and favorable molecular properties. One solution to this problem is to use drugs with light-dependent activity (photopharmacology) in combination with on-demand, localized illumination. However, a suitable light-activated potentiator of GABA A Rs has been elusive so far for use in wildtype mammals. We have met this need by developing azocarnil, a diffusible GABAergic agonist-potentiator based on the anxiolytic drug abecarnil that is inactive in the dark and activated by visible violet light. Azocarnil can be rapidly deactivated with green light and by thermal relaxation in the dark. We demonstrate that it selectively inhibits neuronal currents in hippocampal neurons in vitro and in the dorsal horns of the spinal cord of mice, decreasing the mechanical sensitivity as a function of illumination without displaying systemic adverse effects. Azocarnil expands the in vivo photopharmacological toolkit with a novel chemical scaffold and achieves a milestone toward future phototherapeutic applications to safely treat muscle spasms, pain, anxiety, sleep disorders, and epilepsy.

GABAergic neurons and GABA A receptors (GABA A Rs) are critical elements of almost all neuronal circuits. Most GABA A Rs of the CNS are heteropentameric ion channels composed of two α, two β, and one γ subunits. These receptors serve as important drug targets for benzodiazepine (BDZ) site agonists, which potentiate the action of GABA at GABA A Rs. Most GABA A R classifications rely on the heterogeneity of the α subunit (α1-α6) included in the receptor complex. Heterogeneity of the γ subunits (γ1-γ3), which mediate synaptic clustering of GABA A Rs and contribute, together with α subunits, to the benzodiazepine (BDZ) binding site, has gained less attention, mainly because γ2 subunits greatly outnumber the other γ subunits in most brain regions. Here, we have investigated a potential role of non-γ2 GABA A Rs in neural circuits of the spinal dorsal horn, a key site of nociceptive processing. Female and male mice were studied. We demonstrate that besides γ2 subunits, γ1 subunits are significantly expressed in the spinal dorsal horn, especially in its superficial layers. Unlike global γ2 subunit deletion, which is lethal, spinal cord-specific loss of γ2 subunits was well tolerated. GABA A R clustering in the superficial dorsal horn remained largely unaffected and antihyperalgesic actions of HZ-166, a nonsedative BDZ site agonist, were partially retained. Our results thus suggest that the superficial dorsal horn harbors functionally relevant amounts of γ1 subunits that support the synaptic clustering of GABA A Rs in this site. They further suggest that γ1 containing GABA A Rs contribute to the spinal control of nociceptive information flow., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Neumann et al.)

Neuronal hyperexcitability is a key driver of persistent pain states including neuropathic pain. Leucine-rich, glioma inactivated 1 (LGI1), is a secreted protein known to regulate excitability within the nervous system and is the target of autoantibodies from neuropathic pain patients. Therapies that block or reduce antibody levels are effective at relieving pain in these patients, suggesting that LGI1 has an important role in clinical pain. Here we have investigated the role of LGI1 in regulating neuronal excitability and pain-related sensitivity by studying the consequences of genetic ablation in specific neuron populations using transgenic mouse models. LGI1 has been well studied at the level of the brain, but its actions in the spinal cord and peripheral nervous system (PNS) are poorly understood. We show that LGI1 is highly expressed in DRG and spinal cord dorsal horn neurons in both mouse and human. Using transgenic muse models, we genetically ablated LGI1, either specifically in nociceptors (LGI1fl/Nav1.8+), or in both DRG and spinal neurons (LGI1fl/Hoxb8+). On acute pain assays, we find that loss of LGI1 resulted in mild thermal and mechanical pain-related hypersensitivity when compared to littermate controls. In from LGI1fl/Hoxb8+ mice, we find loss of Kv1 currents and hyperexcitability of DRG neurons. LGI1fl/Hoxb8+ mice displayed a significant increase in nocifensive behaviours in the second phase of the formalin test (not observed in LGI1fl/Nav1.8+ mice) and extracellular recordings in LGI1fl/Hoxb8+ mice revealed hyperexcitability in spinal dorsal horn neurons, including enhanced wind-up. Using the spared nerve injury model, we find that LGI1 expression is dysregulated in the spinal cord. LGI1fl/Nav1.8+ mice showed no differences in nerve injury induced mechanical hypersensitivity, brush-evoked allodynia or spontaneous pain behaviour compared to controls. However, LGI1fl/Hoxb8+ mice showed a significant exacerbation of mechanical hypersensitivity and allodynia. Our findings point to effects of LGI1 at both the level of the DRG and spinal cord, including an important impact of spinal LGI1 on pathological pain. Overall, we find a novel role for LGI1 with relevance to clinical pain., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Aim of the Study: The aim of this study is to provide an analysis of the career trajectory of the recipients of a Swiss National MD-PhD grant thirty years after the creation of the Swiss interuniversity MD-PhD programme., Methods: The study surveyed 277 recipients of a Swiss National MD-PhD grant using an online questionnaire in April 2022. There were twenty questions about participants' demographics, the duration of their MD-PhD training, their career trajectory, current position, research and clinical activity, the impact of the support on the recipients' careers, and their satisfaction with various aspects of the grant., Results: The study showed that 141 out of the 277 grant recipients contacted returned the survey (51% response rate). The gender distribution of the participants was 33% women, 63% men, 4% unknown, which is almost the same as that of all grantees (35% women, 65% men). One hundred and fourteen (81%) respondents had completed their MD-PhD thesis and were graduates, while 27 (19%) were still MD-PhD students. The mean duration of the MD-PhD training was 4.27 years, with a slight upward trend over time. A large proportion of graduates, 81%, remained scientifically active after the grant, most of them in academic settings. Of the grantees who had completed their MD-PhD at least eight years before the survey, 55% had a paid research position with 40% combining research and clinical roles, and 15% doing research only. Seventy-six per cent remained clinically active, 54% occupied leadership positions, and 25% were professors. Most grantees believed that the grant had had a positive impact on their career trajectory. The main challenges included a delay in clinical training, a limited number of clinical positions with dedicated research time after the MD-PhD period, and sub-optimal recognition by hospital hierarchies., Conclusion: The data collected for this study confirm that the competitive Swiss National MD-PhD Grants Programme excels in supporting promising physician scientists who remain active in both research and clinical contexts in the long term. The individual grants are perceived as a distinction that acts as the basis for a successful career in academic medicine. Continued support and alternative funding sources, however, will be essential to ensure the programme's sustainability.

Glycine receptors (GlyRs), together with GABA A receptors, mediate postsynaptic inhibition in most spinal cord and hindbrain neurons. In several CNS regions, GlyRs are also expressed in presynaptic terminals. Here, we analysed the effects of a phospho-deficient mutation (S346A) in GlyR α3 subunits on inhibitory synaptic transmission in superficial spinal dorsal horn neurons, where this subunit is abundantly expressed. Unexpectedly, we found that not only were the amplitudes of evoked glycinergic inhibitory postsynaptic currents (IPSCs) significantly larger in GlyRα3(S346A) mice than in mice expressing wild-type α3GlyRs (GlyRα3(WT) mice), but so were those of GABAergic IPSCs. Decreased frequencies of spontaneously occurring glycinergic and GABAergic miniature IPSCs (mIPSCs) with no accompanying change in mIPSC amplitudes suggested a change in presynaptic transmitter release. Paired-pulse experiments on glycinergic IPSCs revealed an increased paired-pulse ratio and a smaller coefficient of variation in GlyRα3(S346A) mice, which together indicate a reduction in transmitter release probability and an increase in the number of releasable vesicles. Paired-pulse ratios of GABAergic IPSCs recorded in the presence of strychnine were not different between genotypes, while the coefficient of variation was smaller in GlyRα3(S346A) mice, demonstrating that the decrease in release probability was readily reversible by GlyR blockade, while the difference in the size of the pool of releasable vesicles remained. Taken together, our results suggest that presynaptic α3 GlyRs regulate synaptic glycine and GABA release in superficial dorsal horn neurons, and that this effect is potentially regulated by their phosphorylation status. KEY POINTS: A serine-to-alanine point mutation was introduced into the glycine receptor α3 subunit of mice. This point mutation renders α3 glycine receptors resistant to protein kinase A mediated phosphorylation but has otherwise only small effects on receptor function. Patch-clamp recordings from neurons in mouse spinal cord slices revealed an unexpected increase in the amplitudes of both glycinergic and GABAergic evoked inhibitory postsynaptic currents (IPSCs). Miniature IPSCs, paired-pulse ratios and synaptic variation analyses indicate a change in synaptic glycine and GABA release. The results strongly suggest that α3 subunit-containing glycine receptors are expressed on presynaptic terminals of inhibitory dorsal horn neurons where they regulate transmitter release., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Unmyelinated non-peptidergic nociceptors (NP afferents) arborise in lamina II of the spinal cord and receive GABAergic axoaxonic synapses, which mediate presynaptic inhibition. However, until now the source of this axoaxonic synaptic input was not known. Here we provide evidence that it originates from a population of inhibitory calretinin-expressing interneurons (iCRs), which correspond to lamina II islet cells. The NP afferents can be assigned to 3 functionally distinct classes (NP1-3). NP1 afferents have been implicated in pathological pain states, while NP2 and NP3 afferents also function as pruritoceptors. Our findings suggest that all 3 of these afferent types innervate iCRs and receive axoaxonic synapses from them, providing feedback inhibition of NP input. The iCRs also form axodendritic synapses, and their targets include cells that are themselves innervated by the NP afferents, thus allowing for feedforward inhibition. The iCRs are therefore ideally placed to control the input from non-peptidergic nociceptors and pruritoceptors to other dorsal horn neurons, and thus represent a potential therapeutic target for the treatment of chronic pain and itch., (© 2023. The Author(s).)

Neurosteroids and benzodiazepines are modulators of the GABA A receptors, thereby causing anxiolysis. Furthermore, benzodiazepines such as midazolam are known to cause adverse side-effects on cognition upon administration. We previously found that midazolam at nanomolar concentrations (10 nM) blocked long-term potentiation (LTP). Here, we aim to study the effect of neurosteroids and their synthesis using XBD173, which is a synthetic compound that promotes neurosteroidogenesis by binding to the translocator protein 18 kDa (TSPO), since they might provide anxiolytic activity with a favourable side-effect profile. By means of electrophysiological measurements and the use of mice with targeted genetic mutations, we revealed that XBD173, a selective ligand of the translocator protein 18 kDa (TSPO), induced neurosteroidogenesis. In addition, the exogenous application of potentially synthesised neurosteroids (THDOC and allopregnanolone) did not depress hippocampal CA1-LTP, the cellular correlate of learning and memory. This phenomenon was observed at the same concentrations that neurosteroids conferred neuroprotection in a model of ischaemia-induced hippocampal excitotoxicity. In conclusion, our results indicate that TSPO ligands are promising candidates for post-ischaemic recovery exerting neuroprotection, in contrast to midazolam, without detrimental effects on synaptic plasticity.

Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-Maf EX ) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-Maf EX neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-Maf EX neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-Maf EX neuron activation. Our study identifies c-Maf EX neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Spinally projecting serotonergic neurons play a key role in controlling pain sensitivity and can either increase or decrease nociception depending on physiological context. It is currently unknown how serotonergic neurons mediate these opposing effects. Utilizing virus-based strategies and Tph2-Cre transgenic mice, we identified two anatomically separated populations of serotonergic hindbrain neurons located in the lateral paragigantocellularis (LPGi) and the medial hindbrain, which respectively innervate the superficial and deep spinal dorsal horn and have contrasting effects on sensory perception. Our tracing experiments revealed that serotonergic neurons of the LPGi were much more susceptible to transduction with spinally injected AAV2retro vectors than medial hindbrain serotonergic neurons. Taking advantage of this difference, we employed intersectional chemogenetic approaches to demonstrate that activation of the LPGi serotonergic projections decreases thermal sensitivity, whereas activation of medial serotonergic neurons increases sensitivity to mechanical von Frey stimulation. Together these results suggest that there are functionally distinct classes of serotonergic hindbrain neurons that differ in their anatomical location in the hindbrain, their postsynaptic targets in the spinal cord, and their impact on nociceptive sensitivity. The LPGi neurons that give rise to rather global and bilateral projections throughout the rostrocaudal extent of the spinal cord appear to be ideally poised to contribute to widespread systemic pain control., Competing Interests: RG, Md, KW, TÖ, RM, MR, HW, HZ No competing interests declared, (© 2023, Ganley et al.)

Proper sensing of ambient temperature is of utmost importance for the survival of euthermic animals, including humans. While considerable progress has been made in our understanding of temperature sensors and transduction mechanisms, the higher-order neural circuits processing such information are still only incompletely understood. Using intersectional genetics in combination with circuit tracing and functional neuron manipulation, we identified Kcnip2-expressing inhibitory (Kcnip2 GlyT2 ) interneurons of the mouse spinal dorsal horn as critical elements of a neural circuit that tunes sensitivity to cold. Diphtheria toxin-mediated ablation of these neurons increased cold sensitivity without affecting responses to other somatosensory modalities, while their chemogenetic activation reduced cold and also heat sensitivity. We also show that Kcnip2 GlyT2 neurons become activated preferentially upon exposure to cold temperatures and subsequently inhibit spinal nociceptive output neurons that project to the lateral parabrachial nucleus. Our results thus identify a hitherto unknown spinal circuit that tunes cold sensitivity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cerebral ischemia is the leading cause for long-term disability and mortality in adults due to massive neuronal death. Currently, there is no pharmacological treatment available to limit progressive neuronal death after stroke. A major mechanism causing ischemia-induced neuronal death is the excessive release of glutamate and the associated overexcitation of neurons (excitotoxicity). Normally, GABA B receptors control neuronal excitability in the brain via prolonged inhibition. However, excitotoxic conditions rapidly downregulate GABA B receptors via a CaMKII-mediated mechanism and thereby diminish adequate inhibition that could counteract neuronal overexcitation and neuronal death. To prevent the deleterious downregulation of GABA B receptors, we developed a cell-penetrating synthetic peptide (R1-Pep) that inhibits the interaction of GABA B receptors with CaMKII. Administration of this peptide to cultured cortical neurons exposed to excitotoxic conditions restored cell surface expression and function of GABA B receptors. R1-Pep did not affect CaMKII expression or activity but prevented its T286 autophosphorylation that renders it autonomously and persistently active. Moreover, R1-Pep counteracted the aberrant downregulation of G protein-coupled inwardly rectifying K + channels and the upregulation of N-type voltage-gated Ca 2+ channels, the main effectors of GABA B receptors. The restoration of GABA B receptors activated the Akt survival pathway and inhibited excitotoxic neuronal death with a wide time window in cultured neurons. Restoration of GABA B receptors and neuroprotective activity of R1-Pep was verified by using brain slices prepared from mice after middle cerebral artery occlusion (MCAO). Treatment with R1-Pep restored normal GABA B receptor expression and GABA receptor-mediated K + channel currents. This reduced MCAO-induced neuronal excitability and inhibited neuronal death. These results support the hypothesis that restoration of GABA B receptor expression under excitatory conditions provides neuroprotection and might be the basis for the development of a selective intervention to inhibit progressive neuronal death after ischemic stroke., (© 2022 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

In mammals, the pontine noradrenergic system influences nearly every aspect of central nervous system function. A subpopulation of pontine noradrenergic neurons, called A5, are thought to be important in the cardiovascular response to physical stressors, yet their function is poorly defined. We hypothesized that activation of A5 neurons drives a sympathetically mediated increase in blood pressure (BP). To test this hypothesis, we conducted a comprehensive assessment of the cardiovascular effects of chemogenetic stimulation of A5 neurons in male and female adult rats using intersectional genetic and anatomical targeting approaches. Chemogenetic stimulation of A5 neurons in freely behaving rats elevated BP by 15 mmHg and increased cardiac baroreflex sensitivity with a negligible effect on resting HR. Importantly, A5 stimulation had no detectable effect on locomotor activity, metabolic rate, or respiration. Under anesthesia, stimulation of A5 neurons produced a marked elevation in visceral sympathetic nerve activity (SNA) and no change in skeletal muscle SNA, showing that A5 neurons preferentially stimulate visceral SNA. Interestingly, projection mapping indicates that A5 neurons target sympathetic preganglionic neurons throughout the spinal cord and parasympathetic preganglionic neurons throughout in the brainstem, as well as the nucleus of the solitary tract, and ventrolateral medulla. Moreover, in situ hybridization and immunohistochemistry indicate that a subpopulation of A5 neurons coreleases glutamate and monoamines. Collectively, this study suggests A5 neurons are a central modulator of autonomic function with a potentially important role in sympathetically driven redistribution of blood flow from the visceral circulation to critical organs and skeletal muscle.

Repeated application of noxious stimuli leads to a progressively increased pain perception; this temporal summation is enhanced in and predictive of clinical pain disorders. Its electrophysiological correlate is "wind-up," in which dorsal horn spinal neurons increase their response to repeated nociceptor stimulation. To understand the genetic basis of temporal summation, we undertook a GWAS of wind-up in healthy human volunteers and found significant association with SLC8A3 encoding sodium-calcium exchanger type 3 (NCX3). NCX3 was expressed in mouse dorsal horn neurons, and mice lacking NCX3 showed normal, acute pain but hypersensitivity to the second phase of the formalin test and chronic constriction injury. Dorsal horn neurons lacking NCX3 showed increased intracellular calcium following repetitive stimulation, slowed calcium clearance, and increased wind-up. Moreover, virally mediated enhanced spinal expression of NCX3 reduced central sensitization. Our study highlights Ca 2+ efflux as a pathway underlying temporal summation and persistent pain, which may be amenable to therapeutic targeting., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Midazolam amplifies synaptic inhibition via different γ-aminobutyric acid type A (GABAA) receptor subtypes defined by the presence of α1-, α2-, α3-, or α5-subunits in the channel complex. Midazolam blocks long-term potentiation and produces postoperative amnesia. The aims of this study were to identify the GABAA receptor subtypes targeted by midazolam responsible for affecting CA1 long-term potentiation and synaptic inhibition in neocortical neurons., Methods: The effects of midazolam on hippocampal CA1 long-term potentiation were studied in acutely prepared brain slices of male and female mice. Positive allosteric modulation on GABAA receptor-mediated miniature inhibitory postsynaptic currents was investigated in organotypic slice cultures of the mouse neocortex. In both experiments, wild-type mice and GABAA receptor knock-in mouse lines were compared in which α1-, α5-, α1/2/3-, α1/3/5- and α2/3/5-GABAA receptor subtypes had been rendered benzodiazepine-insensitive., Results: Midazolam (10 nM) completely blocked long-term potentiation (mean ± SD, midazolam, 98 ± 11%, n = 14/8 slices/mice vs. control 156 ± 19%, n = 20/12; P < 0.001). Experiments in slices of α1-, α5-, α1/2/3-, α1/3/5-, and α2/3/5-knock-in mice revealed a dominant role for the α1-GABAA receptor subtype in the long-term potentiation suppressing effect. In slices from wild-type mice, midazolam increased (mean ± SD) charge transfer of miniature synaptic events concentration-dependently (50 nM: 172 ± 71% [n = 10/6] vs. 500 nM: 236 ± 54% [n = 6/6]; P = 0.041). In α2/3/5-knock-in mice, charge transfer of miniature synaptic events did not further enhance when applying 500 nM midazolam (50 nM: 171 ± 62% [n = 8/6] vs. 500 nM: 175 ± 62% [n = 6/6]; P = 0.454), indicating two different binding affinities for midazolam to α2/3/5- and α1-subunits., Conclusions: These results demonstrate a predominant role of α1-GABAA receptors in the actions of midazolam at low nanomolar concentrations. At higher concentrations, midazolam also enhances other GABAA receptor subtypes. α1-GABAA receptors may already contribute at sedative doses to the phenomenon of postoperative amnesia that has been reported after midazolam administration., (Copyright © 2022, the American Society of Anesthesiologists. All Rights Reserved.)

In primary tauopathies, the deposition of tau neurofibrillary tangles and threads as well as neurodegenerative changes have been found within the brain and spinal cord. While degenerative changes have been intensively studied in the brain using structural magnetic resonance imaging (MRI), MRI studies investigating the spinal cord are still scarce. In the present study, we acquired ex vivo high resolution structural MRI of the cervical spinal cord of 8.5-9 month old hemizygous and homozygous P301L mice and non-transgenic littermates of both genders. We assessed the total cross-sectional area, and the gray and white matter anterior-posterior width and left-right width that are established imaging marker of spinal cord degeneration. We observed significant tissue-specific reductions in these parameters in female P301L mice that were stronger in homozygous than in hemizygous P301L mice, indicating both an effect of gender and transgene expression on cervical spinal cord atrophy. Moreover, atrophy was stronger in the gray matter than in the white matter. Immunohistochemical analysis revealed neurodegenerative and neuroinflammatory changes in the cervical spinal cord in both the gray and white matter of P301L mice. Collectively, our results provide evidence for cervical spinal cord atrophy that may directly contribute to the motor signs associated with tauopathy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sartoretti, Ganley, Ni, Freund, Zeilhofer and Klohs.)

Glycine receptors (GlyRs) are the primary mediators of fast inhibitory transmission in the mammalian spinal cord, where they modulate sensory and motor signaling. Mutations in GlyR genes as well as some other genes underlie the hereditary disorder hyperekplexia, characterized by episodic muscle stiffness and exaggerated startle responses. Here, we have investigated pain-related behavior and GlyR expression in the spinal cord of the GlyR deficient mutant mouse spastic ( spa ). In spastic mice, the GlyR number is reduced due to a β subunit gene ( Glrb ) mutation resulting in aberrant splicing of GlyRβ transcripts. Via direct physical interaction with the GlyR anchoring protein gephyrin, this subunit is crucially involved in the postsynaptic clustering of heteromeric GlyRs. We show that the mutation differentially affects aspects of the pain-related behavior of homozygous Glrb spa /Glrb spa mice. While response latencies to noxious heat were unchanged, chemically induced pain-related behavior revealed a reduction of the licking time and an increase in flinching in spastic homozygotes during both phases of the formalin test. Mechanically induced nocifensive behavior was reduced in spastic mice, although hind paw inflammation (by zymosan) resulted in allodynia comparable to wild-type mice. Immunohistochemical staining of the spinal cord revealed a massive reduction of dotted GlyRα subunit immunoreactivity in both ventral and dorsal horns, suggesting a reduction of clustered receptors at synaptic sites. Transcripts for all GlyRα subunit variants, however, were not reduced throughout the dorsal horn of spastic mice. These findings suggest that the loss of functional GlyRβ subunits and hence synaptically localized GlyRs compromises sensory processing differentially, depending on stimulus modality., Competing Interests: VE works as a consultant for Disc medicine, this was, however, completely independent of the work presented here. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Groemer, Triller, Zeilhofer, Becker, Eulenburg and Becker.)

The acute stress response mobilizes energy to meet situational demands and re-establish homeostasis. However, the underlying molecular cascades are unclear. Here, we use a brief swim exposure to trigger an acute stress response in mice, which transiently increases anxiety, without leading to lasting maladaptive changes. Using multiomic profiling, such as proteomics, phospho-proteomics, bulk mRNA-, single-nuclei mRNA-, small RNA-, and TRAP-sequencing, we characterize the acute stress-induced molecular events in the mouse hippocampus over time. Our results show the complexity and specificity of the response to acute stress, highlighting both the widespread changes in protein phosphorylation and gene transcription, and tightly regulated protein translation. The observed molecular events resolve efficiently within four hours after initiation of stress. We include an interactive app to explore the data, providing a molecular resource that can help us understand how acute stress impacts brain function in response to stress., (© 2022. The Author(s).)

Benzodiazepines are widely administered drugs to treat anxiety and insomnia. In addition to tolerance development and abuse liability, their chronic use may cause cognitive impairment and increase the risk for dementia. However, the mechanism by which benzodiazepines might contribute to persistent cognitive decline remains unknown. Here we report that diazepam, a widely prescribed benzodiazepine, impairs the structural plasticity of dendritic spines, causing cognitive impairment in mice. Diazepam induces these deficits via the mitochondrial 18 kDa translocator protein (TSPO), rather than classical γ-aminobutyric acid type A receptors, which alters microglial morphology, and phagocytosis of synaptic material. Collectively, our findings demonstrate a mechanism by which TSPO ligands alter synaptic plasticity and, as a consequence, cause cognitive impairment., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

A growing body of experimental evidence shows that glycinergic inhibition plays vital roles in spinal pain processing. In spite of this, however, our knowledge about the morphology, neurochemical characteristics, and synaptic relations of glycinergic neurons in the spinal dorsal horn is very limited. The lack of this knowledge makes our understanding about the specific contribution of glycinergic neurons to spinal pain processing quite vague. Here we investigated the morphology and neurochemical characteristics of glycinergic neurons in laminae I-IV of the spinal dorsal horn using a GlyT2::CreERT2-tdTomato transgenic mouse line. Confirming previous reports, we show that glycinergic neurons are sparsely distributed in laminae I-II, but their densities are much higher in lamina III and especially in lamina IV. First in the literature, we provide experimental evidence indicating that in addition to neurons in which glycine colocalizes with GABA, there are glycinergic neurons in laminae I-II that do not express GABA and can thus be referred to as glycine-only neurons. According to the shape and size of cell bodies and dendritic morphology, we divided the tdTomato-labeled glycinergic neurons into three and six morphological groups in laminae I-II and laminae III-IV, respectively. We also demonstrate that most of the glycinergic neurons co-express neuronal nitric oxide synthase, parvalbumin, the receptor tyrosine kinase RET, and the retinoic acid-related orphan nuclear receptor β (RORβ), but there might be others that need further neurochemical characterization. The present findings may foster our understanding about the contribution of glycinergic inhibition to spinal pain processing., (© 2021 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Background and Purpose: Glycine receptors composed of α1 and β subunits are primarily found in the spinal cord and brainstem and are potentiated by ethanol (10-100 mM). However, much less is known about the presence, composition and ethanol sensitivity of these receptors in higher CNS regions. Here, we examined two regions of the brain reward system, the ventral tegmental area (VTA) and the prefrontal cortex (PFC), to determine their glycine receptor subunit composition and sensitivity to ethanol., Experimental Approach: We used Western blot, immunohistochemistry and electrophysiological techniques in three different models: wild-type C57BL/6, glycine receptor subunit α1 knock-in and glycine receptor subunit α2 knockout mice., Key Results: Similar levels of α and β receptor subunits were detected in both brain regions, and electrophysiological recordings demonstrated the presence of glycine-activated currents in both areas. Sensitivity of glycine receptors to glycine was lower in the PFC compared with VTA. Picrotoxin only partly blocked the glycine-activated current in the PFC and VTA, indicating that both regions express heteromeric αβ receptors. Glycine receptors in VTA neurons, but not in PFC neurons, were potentiated by ethanol., Conclusion and Implications: Glycine receptors in VTA neurons from WT and α2 KO mice were potentiated by ethanol, but not in neurons from the α1 KI mice, supporting the conclusion that α1 glycine receptors are predominantly expressed in the VTA. By contrast, glycine receptors in PFC neurons were not potentiated in any of the mouse models studied, suggesting the presence of α2/α3/α4, rather than α1 glycine receptor subunits., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Abstract: Glycinergic neurons and glycine receptors (GlyRs) exert a critical control over spinal nociception. Prostaglandin E2 (PGE2), a key inflammatory mediator produced in the spinal cord in response to peripheral inflammation, inhibits a certain subtype of GlyRs (α3GlyR) that is defined by the inclusion of α3 subunits and distinctly expressed in the lamina II of the spinal dorsal horn, ie, at the site where most nociceptive nerve fibers terminate. Previous work has shown that the hyperalgesic effect of spinal PGE2 is lost in mice lacking α3GlyRs and suggested that this phenotype results from the prevention of PGE2-evoked protein kinase A (PKA)-dependent phosphorylation and inhibition of α3GlyRs. However, direct proof for a contribution of this phosphorylation event to inflammatory hyperalgesia was still lacking. To address this knowledge gap, a phospho-deficient mouse line was generated that carries a serine to alanine point mutation at a strong consensus site for PKA-dependent phosphorylation in the long intracellular loop of the GlyR α3 subunit. These mice showed unaltered spinal expression of GlyR α3 subunits. In behavioral experiments, they showed no alterations in baseline nociception, but were protected from the hyperalgesic effects of intrathecally injected PGE2 and exhibited markedly reduced inflammatory hyperalgesia. These behavioral phenotypes closely recapitulate those found previously in GlyR α3-deficient mice. Our results thus firmly establish the crucial role of PKA-dependent phosphorylation of α3GlyRs in inflammatory hyperalgesia., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain.)

General consensus states that immunoglobulins are exclusively expressed by B lymphocytes to form the first line of defense against common pathogens. Here, we provide compelling evidence for the expression of two heavy chain immunoglobulin genes in subpopulations of neurons in the mouse brain and spinal cord. RNA isolated from excitatory and inhibitory neurons through ribosome affinity purification revealed Ighg3 and Ighm transcripts encoding for the constant (Fc), but not the variable regions of IgG3 and IgM. Because, in the absence of the variable immunoglobulin regions, these transcripts lack the canonical transcription initiation site used in lymphocytes, we screened for alternative 5' transcription start sites and identified a novel 5' exon adjacent to a proposed promoter element. Immunohistochemical, Western blot, and in silico analyses strongly support that these neuronal transcripts are translated into proteins containing four Immunoglobulin domains. Our data thus demonstrate the expression of two Fc-encoding genes Ighg3 and Ighm in spinal and supraspinal neurons of the murine CNS and suggest a hitherto unknown function of the encoded proteins., (© 2021 Scheurer et al.)

Diminished inhibitory control of spinal nociception is one of the major culprits of chronic pain states. Restoring proper synaptic inhibition is a well-established rational therapeutic approach explored by several pharmaceutical companies. A particular challenge arises from the need for site-specific intervention to avoid deleterious side effects such as sedation, addiction, or impaired motor control, which would arise from wide-range facilitation of inhibition. Specific targeting of glycinergic inhibition, which dominates in the spinal cord and parts of the hindbrain, may help reduce these side effects. Selective targeting of the α3 subtype of glycine receptors (GlyRs), which is highly enriched in the superficial layers of the spinal dorsal horn, a key site of nociceptive processing, may help to further narrow down pharmacological intervention on the nociceptive system and increase tolerability. This review provides an update on the physiological properties and functions of α3 subtype GlyRs and on the present state of related drug discovery programs.

The spinal dorsal horn harbors a sophisticated and heterogeneous network of excitatory and inhibitory neurons that process peripheral signals encoding different sensory modalities. Although it has long been recognized that this network is crucial both for the separation and the integration of sensory signals of different modalities, a systematic unbiased approach to the use of specific neuromodulatory systems is still missing. Here, we have used the translating ribosome affinity purification (TRAP) technique to map the translatomes of excitatory glutamatergic (vGluT2 + ) and inhibitory GABA and/or glycinergic (vGAT + or Gad67 + ) neurons of the mouse spinal cord. Our analyses demonstrate that inhibitory and excitatory neurons are not only set apart, as expected, by the expression of genes related to the production, release or re-uptake of their principal neurotransmitters and by genes encoding for transcription factors, but also by a differential engagement of neuromodulator, especially neuropeptide, signaling pathways. Subsequent multiplex in situ hybridization revealed eleven neuropeptide genes that are strongly enriched in excitatory dorsal horn neurons and display largely non-overlapping expression patterns closely adhering to the laminar and presumably also functional organization of the spinal cord grey matter.

Abstract: Diminished synaptic inhibition in the spinal dorsal horn is a major contributor to pathological pain syndromes of neuropathic or inflammatory origin. Drugs that enhance the activity of dorsal horn α2/α3GABAARs normalize exaggerated nociceptive responses in rodents with neuropathic nerve lesions or peripheral inflammation but lack most of the typical side effects of less specific GABAergic drugs. It is however still unknown whether such drugs also reduce the clinically more relevant conscious perception of pain. Here, we investigated the effects of the α2/α3GABAAR subtype-selective modulator TPA023B on the tonic aversive component of pain in mice with peripheral inflammation or neuropathy. In neuropathic mice with a chronic constriction injury of the sciatic nerve, TPA023B not only reversed hyperalgesia to tactile and heat stimuli but also was highly effective in the conditioned place preference test. In the formalin test, TPA023B not only reduced licking of the injected paw but also reversed facial pain expression scores in the mouse grimace scale assay. Taken together, our results demonstrate that α2/α3GABAA receptor subtype-selective modulators not only reduce nociceptive withdrawal responses but also alleviate the tonic aversive components of chronic pain., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain.)

Anterolateral system (AS) neurons relay nociceptive information from the spinal cord to the brain, protecting the body from harm by evoking a variety of behaviours and autonomic responses. The developmental programs that guide the connectivity of AS neurons remain poorly understood. Spinofugal axons cross the spinal midline in response to Netrin-1 signalling through its receptor deleted in colorectal carcinoma (DCC); however, the relevance of this canonical pathway to AS neuron development has only been demonstrated recently. Here, we disrupted Netrin-1:DCC signalling developmentally in AS neurons and assessed the consequences on the path finding of the different classes of spinofugal neurons. Many lamina I AS neurons normally innervate the lateral parabrachial nucleus and periaqueductal gray on the contralateral side. The loss of DCC in the developing spinal cord resulted in increased frequency of ipsilateral projection of spinoparabrachial and spinoperiaqueductal gray neurons. Given that contralateral spinofugal projections are largely associated with somatotopic representation of the body, changes in the laterality of AS spinofugal projections may contribute to reduced precision in pain localization observed in mice and humans carrying Dcc mutations.

Background: The locus coeruleus (LC) is the principal source of noradrenaline (NA) in the central nervous system. Projection neurons in the ventral portion of the LC project to the spinal cord and are considered the main source of spinal NA. To understand the precise physiology of this pathway, it is important to have tools that allow specific genetic access to these descending projections. AAV2retro serotype vectors are a potential tool to transduce these neurons via their axon terminals in the spinal cord, and thereby limit the expression of genetic material to the spinal projections from the LC. Here, we assess the suitability of AAV2retro to target these neurons and investigate strategies to increase their labelling efficiency., Results: We show that the neurons in the LC that project to the spinal dorsal horn are largely resistant to transduction with AAV2retro serotype vectors. Compared to Cholera toxin B (CTb) tracing, AAV2retro.eGFP labelled far fewer neurons within the LC and surrounding regions, particularly within neurons that express tyrosine hydroxylase (TH), the rate-limiting enzyme for NA synthesis. We also show that the sensitivity for transduction of this projection can be increased using AAV2retro.eGFP.cre in ROSA26 tdTom reporter mice (23% increase), with a higher proportion of the newly revealed neurons expressing TH compared to those directly labelled with AAV2retro containing an eGFP expression sequence., Conclusion: These tracing studies identify limitations in AAV2retro-mediated retrograde transduction of a subset of projection neurons, specifically those that express NA and project to the spinal cord. This is likely to have implications for the study of NA-containing projections as well as other types of projection neuron in the central nervous system.

Neuronal circuits of the spinal dorsal horn integrate sensory information from the periphery with inhibitory and facilitating input from higher central nervous system areas. Most previous work focused on projections descending from the hindbrain. Less is known about inputs descending from the cerebral cortex. Here, we identified cholecystokinin (CCK) positive layer 5 pyramidal neurons of the primary somatosensory cortex (CCK + S1-corticospinal tract [CST] neurons) as a major source of input to the spinal dorsal horn. We combined intersectional genetics and virus-mediated gene transfer to characterize CCK + S1-CST neurons and to define their presynaptic input and postsynaptic target neurons. We found that S1-CST neurons constitute a heterogeneous population that can be subdivided into distinct molecular subgroups. Rabies-based retrograde tracing revealed monosynaptic input from layer 2/3 pyramidal neurons, from parvalbumin positive cortical interneurons, and from thalamic relay neurons in the ventral posterolateral nucleus. Wheat germ agglutinin-based anterograde tracing identified postsynaptic target neurons in dorsal horn laminae III and IV. About 60% of these neurons were inhibitory and about 60% of all spinal target neurons expressed the transcription factor c-Maf. The heterogeneous nature of both S1-CST neurons and their spinal targets suggest complex roles in the fine-tuning of sensory processing., (© The Author(s) 2020. Published by Oxford University Press.)

Diminished synaptic inhibition in the superficial spinal dorsal horn contributes to exaggerated pain responses that accompany peripheral inflammation and neuropathy. α2GABA A receptors (α2GABA A R) constitute the most abundant GABA A R subtype at this site and are the targets of recently identified antihyperalgesic compounds. Surprisingly, hoxb8-α2 -/- mice that lack α2GABA A R from the spinal cord and peripheral sensory neurons exhibit unaltered sensitivity to acute painful stimuli and develop normal inflammatory and neuropathic hyperalgesia. Here, we provide a comprehensive analysis of GABAergic neurotransmission, of behavioral phenotypes and of possible compensatory mechanisms in hoxb8-α2 -/- mice. Our results confirm that hoxb8-α2 -/- mice show significantly diminished GABAergic inhibitory postsynaptic currents (IPSCs) in the superficial dorsal horn but no hyperalgesic phenotype. We also confirm that the potentiation of dorsal horn GABAergic IPSCs by the α2-preferring GABA A R modulator HZ-166 is reduced in hoxb8-α2 -/- mice and that hoxb8-α2 -/- mice are resistant to the analgesic effects of HZ-166. Tonic GABAergic currents, glycinergic IPSCs, and sensory afferent-evoked EPSCs did not show significant changes in hoxb8-α2 -/- mice rendering a compensatory up-regulation of other GABA A R subtypes or of glycine receptors unlikely. Although expression of serotonin and of the serotonin producing enzyme tryptophan hydroxylase (TPH2) was significantly increased in the dorsal horn of hoxb8-α2 -/- mice, ablation of serotonergic terminals from the lumbar spinal cord failed to unmask a nociceptive phenotype. Our results are consistent with an important contribution of α2GABA A R to spinal nociceptive control but their ablation early in development appears to activate yet-to-be identified compensatory mechanisms that protect hoxb8-α2 -/- mice from hyperalgesia., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Calorie-rich diets induce hyperphagia and promote obesity, although the underlying mechanisms remain poorly defined. We find that short-term high-fat-diet (HFD) feeding of mice activates prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC). PNOC ARC neurons represent a previously unrecognized GABAergic population of ARC neurons distinct from well-defined feeding regulatory AgRP or POMC neurons. PNOC ARC neurons arborize densely in the ARC and provide inhibitory synaptic input to nearby anorexigenic POMC neurons. Optogenetic activation of PNOC ARC neurons in the ARC and their projections to the bed nucleus of the stria terminalis promotes feeding. Selective ablation of these cells promotes the activation of POMC neurons upon HFD exposure, reduces feeding, and protects from obesity, but it does not affect food intake or body weight under normal chow consumption. We characterize PNOC ARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Astrocytes are essential contributors to neuronal function. As a consequence, disturbed astrocyte-neuron interactions are involved in the pathophysiology of several neurological disorders, with a strong impact on brain circuits and behavior. Here, we describe altered cortical physiology in a genetic mouse model of familial hemiplegic migraine type 2 (FHM2), with reduced expression of astrocytic Na + ,K + -ATPases. We used whole-cell electrophysiology, two-photon microscopy, and astrocyte gene rescue to demonstrate that an impairment in astrocytic glutamate uptake promotes NMDA spike generation in dendrites of cingulate cortex pyramidal neurons and enhances output firing of these neurons. Astrocyte compensation of the defective ATPase in the cingulate cortex rescued glutamate uptake, prevented abnormal NMDA spikes, and reduced sensitivity to cranial pain triggers. Together, our results demonstrate that impaired astrocyte function alters neuronal activity in the cingulate cortex and facilitates migraine-like cranial pain states in a mouse model of migraine., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of  GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs.

Arcuate nucleus agouti-related peptide (AgRP) neurons play a central role in feeding and are under complex regulation by both homeostatic hormonal and nutrient signals and hypothalamic neuronal pathways. Feeding may also be influenced by environmental cues, sensory inputs, and other behaviors, implying the involvement of higher brain regions. However, whether such pathways modulate feeding through direct synaptic control of AgRP neuron activity is unknown. Here, we show that nociceptin-expressing neurons in the anterior bed nuclei of the stria terminalis (aBNST) make direct GABAergic inputs onto AgRP neurons. We found that activation of these neurons inhibited AgRP neurons and feeding. The activity of these neurons increased upon food availability, and their ablation resulted in obesity. Furthermore, these neurons received afferent inputs from a range of upstream brain regions as well as hypothalamic nuclei. Therefore, aBNST GABAergic nociceptin neurons may act as a gateway to feeding behavior by connecting AgRP neurons to both homeostatic and nonhomeostatic neuronal inputs.

Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).

Optogenetics provide a potential alternative approach to the treatment of chronic pain, in which complex pathology often hampers efficacy of standard pharmacological approaches. Technological advancements in the development of thin, wireless, and mechanically flexible optoelectronic implants offer new routes to control the activity of subsets of neurons and nerve fibers in vivo . This study reports a novel and advanced design of battery-free, flexible, and lightweight devices equipped with one or two miniaturized LEDs, which can be individually controlled in real time. Two proof-of-concept experiments in mice demonstrate the feasibility of these devices. First, we show that blue-light devices implanted on top of the lumbar spinal cord can excite channelrhodopsin expressing nociceptors to induce place aversion. Second, we show that nocifensive withdrawal responses can be suppressed by green-light optogenetic (Archaerhodopsin-mediated) inhibition of action potential propagation along the sciatic nerve. One salient feature of these devices is that they can be operated via modern tablets and smartphones without bulky and complex lab instrumentation. In addition to the optical stimulation, the design enables the simultaneously wireless recording of the temperature in proximity of the stimulation area. As such, these devices are primed for translation to human patients with implications in the treatment of neurological and psychiatric conditions far beyond chronic pain syndromes.

Spinal transmission of pruritoceptive (itch) signals requires transneuronal signaling by gastrin-releasing peptide (GRP) produced by a subpopulation of dorsal horn excitatory interneurons. These neurons also express the glutamatergic marker vGluT2, raising the question of why glutamate alone is insufficient for spinal itch relay. Using optogenetics together with slice electrophysiology and mouse behavior, we demonstrate that baseline synaptic coupling between GRP and GRP receptor (GRPR) neurons is too weak for suprathreshold excitation. Only when we mimicked the endogenous firing of GRP neurons and stimulated them repetitively to fire bursts of action potentials did GRPR neurons depolarize progressively and become excitable by GRP neurons. GRPR but not glutamate receptor antagonism prevented this action. Provoking itch-like behavior by optogenetic activation of spinal GRP neurons required similar stimulation paradigms. These results establish a spinal gating mechanism for itch that requires sustained repetitive activity of presynaptic GRP neurons and postsynaptic GRP signaling to drive GRPR neuron output., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Genetically modified mice are widely used in studies on human and animal physiology and pharmacology, including pain research. The experimental design usually includes comparisons of genetically modified mice with wild-type littermates, requiring biopsy material for genotyping and methods for unequivocal identification of individual mice. Ethical standards and, in some countries, legislation require that both needs are reached with a single procedure. Clipping of the most distal phalanx of up to two toes per paw (toe clipping) is the favored procedure in most research fields, but it may be problematic in sensory physiology and pain research., Objectives: To systematically investigate whether toe-clipping influences later-in-life nociceptive sensitivity or the susceptibility to neuropathic or inflammatory hyperalgesia., Methods: We tested in male mice whether the clipping of 2 toes of a hind paw influences nociceptive sensitivities to noxious heat or cold, or to mechanical stimulation under baseline conditions, after peripheral nerve injury (chronic constriction of the sciatic nerve) or during peripheral inflammation induced by subcutaneous zymosan A injection. We tested not only for the presence of significant differences but also specifically addressed bioequivalence using the 2 one-sided t test procedure. We chose a threshold of 25% variation of the control value for nonequivalence, which is usually taken as a threshold for biological relevance in pain tests., Results: Using this value, we found that for all conditions (non-neuropathic and non-inflamed, neuropathic and inflamed), nociceptive sensitivities significantly fell within the equivalence bounds of the non-toe-clipped control mice., Conclusions: These results suggest that toe clipping does not have long-term effects on nociceptive sensitivities and does not alter the susceptibility of male mice to neuropathic or inflammatory hyperalgesia., Competing Interests: Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article., (Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain.)

Removal of synaptically-released glutamate by astrocytes is necessary to spatially and temporally limit neuronal activation. Recent evidence suggests that astrocytes may have specialized functions in specific circuits, but the extent and significance of such specialization are unclear. By performing direct patch-clamp recordings and two-photon glutamate imaging, we report that in the somatosensory cortex, glutamate uptake by astrocytes is slower during sustained synaptic stimulation when compared to lower stimulation frequencies. Conversely, glutamate uptake capacity is increased in the frontal cortex during higher frequency synaptic stimulation, thereby limiting extracellular buildup of glutamate and NMDA receptor activation in layer 5 pyramidal neurons. This efficient glutamate clearance relies on Na + /K + -ATPase function and both GLT-1 and non-GLT-1 transporters. Thus, by enhancing their glutamate uptake capacity, astrocytes in the frontal cortex may prevent excessive neuronal excitation during intense synaptic activity. These results may explain why diseases associated with network hyperexcitability differentially affect individual brain areas., Competing Interests: The authors declare no competing interests.

Gastrin-releasing peptide (GRP) is a spinal itch transmitter expressed by a small population of dorsal horn interneurons (GRP neurons). The contribution of these neurons to spinal itch relay is still only incompletely understood, and their potential contribution to pain-related behaviors remains controversial. Here, we have addressed this question in a series of experiments performed in GRP::cre and GRP::eGFP transgenic male mice. We combined behavioral tests with neuronal circuit tracing, morphology, chemogenetics, optogenetics, and electrophysiology to obtain a more comprehensive picture. We found that GRP neurons form a rather homogeneous population of central cell-like excitatory neurons located in lamina II of the superficial dorsal horn. Multicolor high-resolution confocal microscopy and optogenetic experiments demonstrated that GRP neurons receive direct input from MrgprA3-positive pruritoceptors. Anterograde HSV-based neuronal tracing initiated from GRP neurons revealed ascending polysynaptic projections to distinct areas and nuclei in the brainstem, midbrain, thalamus, and the somatosensory cortex. Spinally restricted ablation of GRP neurons reduced itch-related behaviors to different pruritogens, whereas their chemogenetic excitation elicited itch-like behaviors and facilitated responses to several pruritogens. By contrast, responses to painful stimuli remained unaltered. These data confirm a critical role of dorsal horn GRP neurons in spinal itch transmission but do not support a role in pain. SIGNIFICANCE STATEMENT Dorsal horn gastrin-releasing peptide neurons serve a well-established function in the spinal transmission of pruritic (itch) signals. A potential role in the transmission of nociceptive (pain) signals has remained controversial. Our results provide further support for a critical role of dorsal horn gastrin-releasing peptide neurons in itch circuits, but we failed to find evidence supporting a role in pain., (Copyright © 2019 the authors 0270-6474/19/392238-13$15.00/0.)

Background: Deleterious variants in the voltage-gated sodium channel type 2 (Na v 1.2) lead to a broad spectrum of phenotypes ranging from benign familial neonatal-infantile epilepsy (BFNIE), severe developmental and epileptic encephalopathy (DEE) and intellectual disability (ID) to autism spectrum disorders (ASD). Yet, the underlying mechanisms are still incompletely understood., Methods: To further elucidate the genotype-phenotype correlation of SCN2A variants we investigated the functional effects of six variants representing the phenotypic spectrum by whole-cell patch-clamp studies in transfected HEK293T cells and in-silico structural modeling., Results: The two variants p.L1342P and p.E1803G detected in patients with early onset epileptic encephalopathy (EE) showed profound and complex changes in channel gating, whereas the BFNIE variant p.L1563V exhibited only a small gain of channel function. The three variants identified in ID patients without seizures, p.R937C, p.L611Vfs*35 and p.W1716*, did not produce measurable currents. Homology modeling of the missense variants predicted structural impairments consistent with the electrophysiological findings., Conclusions: Our findings support the hypothesis that complete loss-of-function variants lead to ID without seizures, small gain-of-function variants cause BFNIE and EE variants exhibit variable but profound Na v 1.2 gating changes. Moreover, structural modeling was able to predict the severity of the variant impact, supporting a potential role of structural modeling as a prognostic tool. Our study on the functional consequences of SCN2A variants causing the distinct phenotypes of EE, BFNIE and ID contributes to the elucidation of mechanisms underlying the broad phenotypic variability reported for SCN2A variants.

Benzodiazepines (BDZ), which potentiate the action of GABA at four subtypes of GABA A receptors (α1, α2, α3, and α5GABA A Rs), are highly effective against anxiety disorders, but also cause severe side effects greatly limiting their clinical application. Both, preclinical studies in genetically engineered mice, and preclinical and clinical trials with subtype-selective compounds indicate that undesired effects can in principle be avoided by targeting specific GABA A R subtypes. While there is general consensus that activity at α1GABA A Rs should be avoided, controversy exists as to whether α2 or α3GABA A Rs need to be targeted for anxiolysis. While previous experiments in GABA A R point-mutated mice demonstrated a critical role of α2GABA A Rs, studies solely relying on pharmacological approaches suggested a dominant contribution of α3GABA A Rs. As most α1GABA A R-sparing BDZ site agonists discriminate little between α2 and α3GABA A Rs, these claims rest almost exclusively on a single compound, TP003, that has been reported to be a selective α3GABA A R modulator. Here, we have revisited the in vitro pharmacological profile of TP003 and, in addition, tested TP003 in GABA A R triple point-mutated mice, in which only either α1, α2, or α3GABA A Rs were left BDZ sensitive. These experiments revealed that TP003 behaves as a partial, rather non-selective BDZ site agonist in vitro that acts in vivo through α1, α2, and α3GABA A Rs (α5GABA A R-mediated effects were not tested). With respect to anxiolysis, our results support a critical contribution of α2GABA A Rs, but not of α3GABA A Rs. TP003 should therefore not be considered an α3GABA A R selective agent. Previously published studies using TP003 should be interpreted with caution., (Copyright © 2018 Elsevier Ltd. All rights reserved.)