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SummaryNeurons projecting from the rostral ventromedial medulla (RVM) to the spinal dorsal horn are critical elements of endogenous pain control systems. Here, we describe a GABA/glycinergic pathway that predominantly innervates the superficial dorsal horn. Anatomical and optogenetic tracing of these neurons from a single unilateral site of the lumbar spinal cord indicated that these neurons give rise to a dense bilateral innervation of the spinal cord along its entire rostrocaudal axis. Chemogenetic activation of these neurons caused a bilateral and widespread reduction in heat, cold, and mechanical sensitivity, while their silencing with tetanus toxin induced allodynia and spontaneous pain-like aversive behaviors. Consistent with a continuous role in the prevention of spontaneous pain, many descending RVM GABAergic neurons were found to be tonically active. This pathway may therefore be relevant for widespread conditioned analgesia, while its dysfunction may underlie chronic widespread pain syndromes.

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 (Kcnip2GlyT2) 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 Kcnip2GlyT2 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., Neuron, 111 (1), ISSN:0896-6273, ISSN:1097-4199

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.

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 Ca2+ efflux as a pathway underlying temporal summation and persistent pain, which may be amenable to therapeutic targeting., Neuron, 110 (16)

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. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

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., Scientific Reports, 11 (1), ISSN:2045-2322

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

Previous work has shown that α2/α3GABAAR-selective modulators normalize neuropathic and inflammatory hyperalgesia. Here, we show that they also inhibit the tonic aversive pain component., 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.

Anterolateral system neurons in lamina I of the spinal cord rely on DCC for the formation of topographically appropriate nociceptive inputs to the brain., 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.

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.

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 ROSA26tdTom 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.

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.

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 (mesoSPIM) 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).

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 inGRP::creandGRP::eGFPtransgenic 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 STATEMENTDorsal 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.

Benzodiazepines (BDZ), which potentiate the action of GABA at four subtypes of GABA receptors (α1, α2, α3, and α5GABARs), 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 GABAR subtypes. While there is general consensus that activity at α1GABARs should be avoided, controversy exists as to whether α2 or α3GABARs need to be targeted for anxiolysis. While previous experiments in GABAR point-mutated mice demonstrated a critical role of α2GABARs, studies solely relying on pharmacological approaches suggested a dominant contribution of α3GABARs. As most α1GABAR-sparing BDZ site agonists discriminate little between α2 and α3GABARs, these claims rest almost exclusively on a single compound, TP003, that has been reported to be a selective α3GABAR modulator. Here, we have revisited the in vitro pharmacological profile of TP003 and, in addition, tested TP003 in GABAR triple point-mutated mice, in which only either α1, α2, or α3GABARs 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 α3GABARs (α5GABAR-mediated effects were not tested). With respect to anxiolysis, our results support a critical contribution of α2GABARs, but not of α3GABARs. TP003 should therefore not be considered an α3GABAR selective agent. Previously published studies using TP003 should be interpreted with caution.

SummaryThe acute stress response mobilizes energy to meet situational demands and re-establish homeostasis. However, little is known about the nature and dynamics of the underlying molecular cascades. We used a brief forced swim exposure to trigger a strong stress response in mice, which transiently increases anxiety, but does not lead to lasting maladaptive behavioral changes. Using multiomic profiling we characterize the stress-induced molecular events in the hippocampus over time, across molecular scales, and down to the level of cell types and single cells. Our results unveil the complexity and specificity of the healthy stress response, with widespread changes in protein phosphorylation and gene transcription, but tightly regulated protein translation. All observed molecular events resolve efficiently within 4 hours after initiation of stress. The data are accessible through an interactive web portal, creating an extensive molecular resource that will help understand how stress impacts brain function in health and disease.

Supplemental Digital Content is Available in the Text. Behavioral and electrophysiological experiments show that a single point mutation in the glycine receptor α3 subunit protects mice from the hyperalgesic actions of spinal prostaglandin E2., 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.

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., Cerebral Cortex Communications, 1 (1), ISSN:2632-7376

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., Scientific Reports, 10 (1), ISSN:2045-2322

Summary 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). PNOCARC neurons represent a previously unrecognized GABAergic population of ARC neurons distinct from well-defined feeding regulatory AgRP or POMC neurons. PNOCARC neurons arborize densely in the ARC and provide inhibitory synaptic input to nearby anorexigenic POMC neurons. Optogenetic activation of PNOCARC 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 PNOCARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia., Graphical Abstract, Highlights • Acute high-fat-diet feeding activates PNOC neurons in the arcuate nucleus (ARC) • GABAergic PNOCARC neurons inhibit anorexigenic POMC neurons • Optogenetic activation of PNOCARC neurons promotes feeding • Ablation of PNOCARC neurons protects from obesity, Calorie-rich diets induce hyperphagia and promote obesity. Here, Jais et al. report that short-term high-fat-diet (HFD) feeding in mice activates prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC). They characterize PNOCARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia.

Diminished synaptic inhibition in the superficial spinal dorsal horn contributes to exaggerated pain responses that accompany peripheral inflammation and neuropathy. α2GABA

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., Science Advances, 6 (23), ISSN:2375-2548

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., Life Science Alliance, 4 (11), ISSN:2575-1077

Acetaminophen (paracetamol) is a widely used analgesic and antipyretic drug with only incompletely understood mechanisms of action. Previous work, using models of acute nociceptive pain, indicated that analgesia by acetaminophen involves an indirect activation of CB1receptors by the acetaminophen metabolite and endocannabinoid reuptake inhibitor AM 404. However, the contribution of the cannabinoid system to antihyperalgesia against inflammatory pain, the main indication of acetaminophen, and the precise site of the relevant CB1receptors have remained elusive. Here, we analyzed acetaminophen analgesia in mice of either sex with inflammatory pain and found that acetaminophen exerted a dose-dependent antihyperalgesic action, which was mimicked by intrathecally injected AM 404. Both compounds lost their antihyperalgesic activity inCB1−/−mice, confirming the involvement of the cannabinoid system. Consistent with a mechanism downstream of proinflammatory prostaglandin formation, acetaminophen also reversed hyperalgesia induced by intrathecal prostaglandin E2. To distinguish between a peripheral/spinal and a supraspinal action, we administered acetaminophen and AM 404 tohoxB8-CB1−/−mice, which lack CB1receptors from the peripheral nervous system and the spinal cord. These mice exhibited unchanged antihyperalgesia indicating a supraspinal site of action. Accordingly, local injection of the CB1receptor antagonist rimonabant into the rostral ventromedial medulla blocked acetaminophen-induced antihyperalgesia, while local rostral ventromedial medulla injection of AM 404 reduced hyperalgesia in wild-type mice but not inCB1−/−mice. Our results indicate that the cannabinoid system contributes not only to acetaminophen analgesia against acute pain but also against inflammatory pain, and suggest that the relevant CB1receptors reside in the rostral ventromedial medulla.SIGNIFICANCE STATEMENTAcetaminophen is a widely used analgesic drug with multiple but only incompletely understood mechanisms of action, including a facilitation of endogenous cannabinoid signaling via one of its metabolites. Our present data indicate that enhanced cannabinoid signaling is also responsible for the analgesic effects of acetaminophen against inflammatory pain. Local injections of the acetaminophen metabolite AM 404 and of cannabinoid receptor antagonists as well as data from tissue-specific CB1receptor-deficient mice suggest the rostral ventromedial medulla as an important site of the cannabinoid-mediated analgesia by acetaminophen.

Glutamate-mediated excitotoxicity induces neuronal death by altering various intracellular signaling pathways and is implicated as a common pathogenic pathway in many neurodegenerative diseases. In the case of motor neuron disease, there is significant evidence to suggest that the overactivation of AMPA receptors due to deficiencies in the expression and function of glial glutamate transporters GLT1 and GLAST plays an important role in the mechanisms of neuronal death. However, a causal role for glial glutamate transporter dysfunction in motor neuron death remains unknown. Here, we developed a new animal model of excitotoxicity by conditionally deleting astroglial glutamate transporters GLT1 and GLAST in the spinal cords of mice (GLAST(+/−)/GLT1-cKO). GLAST(+/−)/GLT1-cKO mice (both sexes) exhibited nuclear irregularity and calpain-mediated degradation of nuclear pore complexes (NPCs), which are responsible for nucleocytoplasmic transport. These abnormalities were associated with progressive motor neuron loss, severe paralysis, and shortened lifespan. The nuclear export inhibitor KPT-350 slowed but did not prevent motor neuron death, whereas long-term treatment of the AMPA receptor antagonist perampanel and the calpain inhibitor SNJ-1945 had more persistent beneficial effects. Thus, NPC degradation contributes to AMPA receptor-mediated excitotoxic motor neuronal death, and preventing NPC degradation has robust protective effects. Normalization of NPC function could be a novel therapeutic strategy for neurodegenerative disorders in which AMPA receptor-mediated excitotoxicity is a contributory factor. SIGNIFICANCE STATEMENT Despite glial glutamate transporter dysfunction leading to excitotoxicity has been documented in many neurological diseases, it remains unclear whether its dysfunction is a primary cause or secondary outcome of neuronal death at disease state. Here we show the combined loss of glial glutamate transporters GLT1 and GLAST in spinal cord caused motor neuronal death and hindlimb paralysis. Further, our novel mutant exhibits the nuclear irregularities and calpain-mediated progressive nuclear pore complex degradation. Our study reveals that glial glutamate transporter dysfunction is sufficient to cause motor neuronal death in vivo.

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., The Journal of Clinical Investigation, 130 (1), ISSN:1558-8238, ISSN:0021-9738

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., Pain Reports, 4 (3)

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., Communications Biology, 2 (1), ISSN:2399-3642