Restoring neuronal chloride extrusion reverses cognitive decline linked to Alzheimer's disease mutations.

Disinhibition during early stages of Alzheimer's disease is postulated to cause network dysfunction and hyperexcitability leading to cognitive deficits. However, the underlying molecular mechanism remains unknown. Here we show that, in mouse lines carrying Alzheimer's disease-related mutations, a loss of neuronal membrane potassium-chloride cotransporter KCC2, responsible for maintaining the robustness of GABAA-mediated inhibition, occurs pre-symptomatically in the hippocampus and prefrontal cortex. KCC2 downregulation was inversely correlated with the age-dependent increase in amyloid-ß 42 (Aß42). Acute administration of Aß42 caused a downregulation of membrane KCC2. Loss of KCC2 resulted in impaired chloride homeostasis. Preventing the decrease in KCC2 using long term treatment with CLP290 protected against deterioration of learning and cortical hyperactivity. In addition, restoring KCC2, using short term CLP290 treatment, following the transporter reduction effectively reversed spatial memory deficits and social dysfunction, linking chloride dysregulation with Alzheimer's disease-related cognitive decline. These results reveal KCC2 hypofunction as a viable target for treatment of Alzheimer's disease-related cognitive decline; they confirm target engagement, where the therapeutic intervention takes place, and its effectiveness.

Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain.

The prevalence and severity of many chronic pain syndromes differ across sex, and recent studies have identified differences in immune signalling within spinal nociceptive circuits as a potential mediator. Although it has been proposed that sex-specific pain mechanisms converge once they reach neurons within the superficial dorsal horn, direct investigations using rodent and human preclinical pain models have been lacking. Here, we discovered that in the Freund's adjuvant in vivo model of inflammatory pain, where both male and female rats display tactile allodynia, a pathological coupling between KCC2-dependent disinhibition and N-methyl-D-aspartate receptor (NMDAR) potentiation within superficial dorsal horn neurons was observed in male but not female rats. Unlike males, the neuroimmune mediator brain-derived neurotrophic factor (BDNF) failed to downregulate inhibitory signalling elements (KCC2 and STEP61) and upregulate excitatory elements (pFyn, GluN2B and pGluN2B) in female rats, resulting in no effect of ex vivo brain-derived neurotrophic factor on synaptic NMDAR responses in female lamina I neurons. Importantly, this sex difference in spinal pain processing was conserved from rodents to humans. As in rodents, ex vivo spinal treatment with BDNF downregulated markers of disinhibition and upregulated markers of facilitated excitation in superficial dorsal horn neurons from male but not female human organ donors. Ovariectomy in female rats recapitulated the male pathological pain neuronal phenotype, with BDNF driving a coupling between disinhibition and NMDAR potentiation in adult lamina I neurons following the prepubescent elimination of sex hormones in females. This discovery of sexual dimorphism in a central neuronal mechanism of chronic pain across species provides a foundational step towards a better understanding and treatment for pain in both sexes.

Loss of STEP61 couples disinhibition to N-methyl-d-aspartate receptor potentiation in rodent and human spinal pain processing.

Dysregulated excitability within the spinal dorsal horn is a critical mediator of chronic pain. In the rodent nerve injury model of neuropathic pain, BDNF-mediated loss of inhibition (disinhibition) gates the potentiation of excitatory GluN2B N-methyl-d-aspartate receptor (NMDAR) responses at lamina I dorsal horn synapses. However, the centrality of this mechanism across pain states and species, as well as the molecular linker involved, remain unknown. Here, we show that KCC2-dependent disinhibition is coupled to increased GluN2B-mediated synaptic NMDAR responses in a rodent model of inflammatory pain, with an associated downregulation of the tyrosine phosphatase STEP61. The decreased activity of STEP61 is both necessary and sufficient to prime subsequent phosphorylation and potentiation of GluN2B NMDAR by BDNF at lamina I synapses. Blocking disinhibition reversed the downregulation of STEP61 as well as inflammation-mediated behavioural hypersensitivity. For the first time, we characterize GluN2B-mediated NMDAR responses at human lamina I synapses and show that a human ex vivo BDNF model of pathological pain processing downregulates KCC2 and STEP61 and upregulates phosphorylated GluN2B at dorsal horn synapses. Our results demonstrate that STEP61 is the molecular brake that is lost following KCC2-dependent disinhibition and that the decrease in STEP61 activity drives the potentiation of excitatory GluN2B NMDAR responses in rodent and human models of pathological pain. The ex vivo human BDNF model may thus form a translational bridge between rodents and humans for identification and validation of novel molecular pain targets.

Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins.

Fluorescence fluctuation spectroscopy can be used to measure the aggregation of fluorescently labeled molecules and is typically performed using time series data. Spatial intensity distribution analysis and fluorescence moment image analysis are established tools for measuring molecular brightnesses from single-color images collected with laser scanning microscopes. We have extended these tools for analysis of two-color images to resolve heteromeric interactions between molecules labeled with spectrally distinct chromophores. We call these new methods two-color spatial intensity distribution analysis and two-color spatial cumulant analysis (2c-SpCA). To implement these techniques on a hyperspectral imaging system, we developed a spectral shift filtering technique to remove artifacts due to intrinsic cross talk between detector bins. We determined that 2c-SpCA provides better resolution from samples containing multiple fluorescent species; hence, this technique was carried forward to study images of living cells. We used fluorescent heterodimers labeled with enhanced green fluorescent protein and mApple to quantify the effects of resonance energy transfer and incomplete maturation of mApple on brightness measurements. We show that 2c-SpCA can detect the interaction between two components of trimeric G-protein complexes. Thus, 2c-SpCA presents a robust and computationally expedient means of measuring heteromeric interactions in cellular environments.

Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs.

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·µm(-2) human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior.

Regulation of oligomeric organization of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor observed by spatial intensity distribution analysis.

The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor.

Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells.

Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique-spatial intensity distribution analysis (SpIDA)-that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.

Gephyrin clusters are absent from small diameter primary afferent terminals despite the presence of GABA(A) receptors.

Whereas both GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) play a role in control of dorsal horn neuron excitability, their relative contribution to inhibition of small diameter primary afferent terminals remains controversial. To address this, we designed an approach for quantitative analyses of the distribution of GABA(A)R-subunits, GlyR α1-subunit and their anchoring protein, gephyrin, on terminals of rat spinal sensory afferents identified by Calcitonin-Gene-Related-Peptide (CGRP) for peptidergic terminals, and by Isolectin-B4 (IB4) for nonpeptidergic terminals. The approach was designed for light microscopy, which is compatible with the mild fixation conditions necessary for immunodetection of several of these antigens. An algorithm was designed to recognize structures with dimensions similar to those of the microscope resolution. To avoid detecting false colocalization, the latter was considered significant only if the degree of pixel overlap exceeded that expected from randomly overlapping pixels given a hypergeometric distribution. We found that both CGRP(+) and IB4(+) terminals were devoid of GlyR α1-subunit and gephyrin. The α1 GABA(A)R was also absent from these terminals. In contrast, the GABA(A)R α2/α3/α5 and ß3 subunits were significantly expressed in both terminal types, as were other GABA(A)R-associated-proteins (α-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA(A)R ß3 subunits in small afferent terminals. Real-time quantitative PCR (qRT-PCR) confirmed the results of light microscopy immunochemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynaptic sites (nociceptive afferent terminals vs inhibitory synapses on dorsal horn neurons). The absence of gephyrin clusters from primary afferent terminals suggests a more diffuse mode of GABA(A)-mediated transmission at presynaptic than at postsynaptic sites.

Roundabout 1 exists predominantly as a basal dimeric complex and this is unaffected by binding of the ligand Slit2.

Robo (Roundabout) receptors and their Slit polypeptide ligands are known to play key roles in neuronal development and have been implicated in both angiogenesis and cancer. Like the other family members, Robo1 is a large single transmembrane domain polypeptide containing a series of well-defined extracellular elements. However, the intracellular domain lacks structural definition and little is known about the quaternary structure of Robo receptors or how binding of a Slit might affect this. To address these questions combinations of both autofluorescent protein-based FRET imaging and time-resolved FRET were employed. Both approaches identified oligomeric organization of Robo1 that did not require the presence of the intracellular domain. SpIDA (spatial intensity distribution analysis) of eGFP-tagged forms of Robo1 indicated that for a C-terminally deleted version approximately two-thirds of the receptor was present as a dimer and one-third as a monomer. By contrast, full-length Robo1 was present almost exclusively as a dimer. In each case this was unaffected by the addition of Slit2, although parallel studies demonstrated the biological activity of Slit2 and its interaction with Robo1. Deletion of both the immunoglobulin and fibronectin type III extracellular repeats prevented dimer formation, with the immunoglobulin repeats providing the bulk of the protein-protein interaction affinity.

Super-resolution microscopy approaches for live cell imaging.

By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells.

Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis.

Measuring protein interactions is key to understanding cell signaling mechanisms, but quantitative analysis of these interactions in situ has remained a major challenge. Here, we present spatial intensity distribution analysis (SpIDA), an analysis technique for image data obtained using standard fluorescence microscopy. SpIDA directly measures fluorescent macromolecule densities and oligomerization states sampled within single images. The method is based on fitting intensity histograms calculated from images to obtain density maps of fluorescent molecules and their quantal brightness. Because spatial distributions are acquired by imaging, SpIDA can be applied to the analysis of images of chemically fixed tissue as well as live cells. However, the technique does not rely on spatial correlations, freeing it from biases caused by subcellular compartmentalization and heterogeneity within tissue samples. Analysis of computer-based simulations and immunocytochemically stained GABA(B) receptors in spinal cord samples shows that the approach yields accurate measurements over a broader range of densities than established procedures. SpIDA is applicable to sampling within small areas (6 µm(2)) and reveals the presence of monomers and dimers with single-dye labeling. Finally, using GFP-tagged receptor subunits, we show that SpIDA can resolve dynamic changes in receptor oligomerization in live cells. The advantages and greater versatility of SpIDA over current techniques open the door to quantificative studies of protein interactions in native tissue using standard fluorescence microscopy.

Quantification of receptor tyrosine kinase transactivation through direct dimerization and surface density measurements in single cells.

Cell signaling involves dynamic changes in protein oligomerization leading to the formation of different signaling complexes and modulation of activity. Spatial intensity distribution analysis (SpIDA) is an image analysis method that can directly measure oligomerization and trafficking of endogenous proteins in single cells. Here, we show the use of SpIDA to quantify dimerization/activation and surface transport of receptor protein kinases--EGF receptor and TrkB--at early stages of their transactivation by several G protein-coupled receptors (GPCRs). Transactivation occurred on the same timescale and was directly limited by GPCR activation but independent of G-protein coupling types. Early receptor protein kinase transactivation and internalization were not interdependent for all receptor pairs tested, revealing heterogeneity between groups of GPCRs. SpIDA also detected transactivation of TrkB by dopamine receptors in intact neurons. By allowing for time and space resolved quantification of protein populations with heterogeneous oligomeric states, SpIDA provides a unique approach to undertake single cell multivariate quantification of signaling processes involving changes in protein interactions, trafficking, and activity.

Stoichiometry of the human glycine receptor revealed by direct subunit counting.

The subunit stoichiometry of heteromeric glycine-gated channels determines fundamental properties of these key inhibitory neurotransmitter receptors; however, the ratio of α1- to ß-subunits per receptor remains controversial. We used single-molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2ß. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations.

Enhanced harm detection following maternal separation: Transgenerational transmission and reversibility by inhaled amiloride.

Early-life adversities are associated with altered defensive responses. Here, we demonstrate that the repeated cross-fostering (RCF) paradigm of early maternal separation is associated with enhancements of distinct homeostatic reactions: hyperventilation in response to hypercapnia and nociceptive sensitivity, among the first generation of RCF-exposed animals, as well as among two successive generations of their normally reared offspring, through matrilineal transmission. Parallel enhancements of acid-sensing ion channel 1 (ASIC1), ASIC2, and ASIC3 messenger RNA transcripts were detected transgenerationally in central neurons, in the medulla oblongata, and in periaqueductal gray matter of RCF-lineage animals. A single, nebulized dose of the ASIC-antagonist amiloride renormalized respiratory and nociceptive responsiveness across the entire RCF lineage. These findings reveal how, following an early-life adversity, a biological memory reducible to a molecular sensor unfolds, shaping adaptation mechanisms over three generations. Our findings are entwined with multiple correlates of human anxiety and pain conditions and suggest nebulized amiloride as a therapeutic avenue.

A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons.

Introduction: Human induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process. Methods: In this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter. Results: The retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types. Discussion: Our results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures.

Efficacy of synaptic inhibition depends on multiple, dynamically interacting mechanisms implicated in chloride homeostasis.

Chloride homeostasis is a critical determinant of the strength and robustness of inhibition mediated by GABA(A) receptors (GABA(A)Rs). The impact of changes in steady state Cl(-) gradient is relatively straightforward to understand, but how dynamic interplay between Cl(-) influx, diffusion, extrusion and interaction with other ion species affects synaptic signaling remains uncertain. Here we used electrodiffusion modeling to investigate the nonlinear interactions between these processes. Results demonstrate that diffusion is crucial for redistributing intracellular Cl(-) load on a fast time scale, whereas Cl(-)extrusion controls steady state levels. Interaction between diffusion and extrusion can result in a somato-dendritic Cl(-) gradient even when KCC2 is distributed uniformly across the cell. Reducing KCC2 activity led to decreased efficacy of GABA(A)R-mediated inhibition, but increasing GABA(A)R input failed to fully compensate for this form of disinhibition because of activity-dependent accumulation of Cl(-). Furthermore, if spiking persisted despite the presence of GABA(A)R input, Cl(-) accumulation became accelerated because of the large Cl(-) driving force that occurs during spikes. The resulting positive feedback loop caused catastrophic failure of inhibition. Simulations also revealed other feedback loops, such as competition between Cl(-) and pH regulation. Several model predictions were tested and confirmed by [Cl(-)](i) imaging experiments. Our study has thus uncovered how Cl(-) regulation depends on a multiplicity of dynamically interacting mechanisms. Furthermore, the model revealed that enhancing KCC2 activity beyond normal levels did not negatively impact firing frequency or cause overt extracellular K(-) accumulation, demonstrating that enhancing KCC2 activity is a valid strategy for therapeutic intervention.

Computational modeling of trans-synaptic nanocolumns, a modulator of synaptic transmission.

Understanding synaptic transmission is of crucial importance in neuroscience. The spatial organization of receptors, vesicle release properties and neurotransmitter molecule diffusion can strongly influence features of synaptic currents. Newly discovered structures coined trans-synaptic nanocolumns were shown to align presynaptic vesicles release sites and postsynaptic receptors. However, how these structures, spanning a few tens of nanometers, shape synaptic signaling remains little understood. Given the difficulty to probe submicroscopic structures experimentally, computer modeling is a useful approach to investigate the possible functional impacts and role of nanocolumns. In our in silico model, as has been experimentally observed, a nanocolumn is characterized by a tight distribution of postsynaptic receptors aligned with the presynaptic vesicle release site and by the presence of trans-synaptic molecules which can modulate neurotransmitter molecule diffusion. In this work, we found that nanocolumns can play an important role in reinforcing synaptic current mostly when the presynaptic vesicle contains a small number of neurotransmitter molecules. Our work proposes a new methodology to investigate in silico how the existence of trans-synaptic nanocolumns, the nanometric organization of the synapse and the lateral diffusion of receptors shape the features of the synaptic current such as its amplitude and kinetics.

Distribution of acid-sensing ion channel subunits in human sensory neurons contrasts with that in rodents.

Acid-sensing ion channels (ASICs) play a critical role in nociception in human sensory neurons. Four genes (ASIC1, ASIC2, ASIC3, and ASIC4) encoding multiple subunits through alternative splicing have been identified in humans. Real time-PCR experiments showed strong expression of three subunits ASIC1, ASIC2, and ASIC3 in human dorsal root ganglia; however, their detailed expression pattern in different neuronal populations has not been investigated yet. In the current study, using an in situ hybridization approach (RNAscope), we examined the presence of ASIC1, ASIC2, and ASIC3 mRNA in three subpopulations of human dorsal root ganglia neurons. Our results revealed that ASIC1 and ASIC3 were present in the vast majority of dorsal root ganglia neurons, while ASIC2 was only expressed in less than half of dorsal root ganglia neurons. The distribution pattern of the three ASIC subunits was the same across the three populations of dorsal root ganglia neurons examined, including neurons expressing the REarranged during Transfection (RET) receptor tyrosine kinase, calcitonin gene-related peptide, and a subpopulation of nociceptors expressing Transient Receptor Potential Cation Channel Subfamily V Member 1. These results strongly contrast the expression pattern of Asics in mice since our previous study demonstrated differential distribution of Asics among the various subpopulation of dorsal root ganglia neurons. Given the distinct acid-sensitivity and activity dynamics among different ASIC channels, the expression differences between human and rodents should be taken under consideration when evaluating the translational potential and efficiency of drugs targeting ASICs in rodent studies.

Switch of serotonergic descending inhibition into facilitation by a spinal chloride imbalance in neuropathic pain.

Descending control from the brain to the spinal cord shapes our pain experience, ranging from powerful analgesia to extreme sensitivity. Increasing evidence from both preclinical and clinical studies points to an imbalance toward descending facilitation as a substrate of pathological pain, but the underlying mechanisms remain unknown. We used an optogenetic approach to manipulate serotonin (5-HT) neurons of the nucleus raphe magnus that project to the dorsal horn of the spinal cord. We found that 5-HT neurons exert an analgesic action in naïve mice that becomes proalgesic in an experimental model of neuropathic pain. We show that spinal KCC2 hypofunction turns this descending inhibitory control into paradoxical facilitation; KCC2 enhancers restored 5-HT-mediated descending inhibition and analgesia. Last, combining selective serotonin reuptake inhibitors (SSRIs) with a KCC2 enhancer yields effective analgesia against nerve injury-induced pain hypersensitivity. This uncovers a previously unidentified therapeutic path for SSRIs against neuropathic pain.

Differential Expression of Acid - Sensing Ion Channels in Mouse Primary Afferents in Naïve and Injured Conditions.

Injury and inflammation cause tissue acidosis, which is a common feature of various painful conditions. Acid-Sensing Ion channels (ASICs) are amongst the main excitatory channels activated by extracellular protons and expressed in the nervous system. Six transcripts of ASIC subunits including ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4 are encoded by four genes (Asic1-4) and have been identified in rodents. Most ASIC subunits are present at substantial levels in primary sensory neurons of dorsal root ganglia (DRG) except for ASIC4. However, their expression pattern in DRG neurons remains largely unclear, mainly due to the lack of antibodies with appropriate specificity. In this study, we examined in detail the expression pattern of ASIC1-3 subunits, including splice variants, in different populations of DRG neurons in adult mice using an in situ hybridization technique (RNAscope) with high sensitivity and specificity. We found that in naïve condition, all five subunits examined were expressed in the majority of myelinated, NF200-immunoreactive, DRG neurons (NF200+). However, ASIC subunits showed a very different expression pattern among non-myelinated DRG neuronal subpopulations: ASIC1 and ASIC3 were only expressed in CGRP-immunoreactive neurons (CGRP+), ASIC2a was mostly expressed in the majority of IB4-binding neurons (IB4+), while ASIC2b was expressed in almost all non-myelinated DRG neurons. We also found that at least half of sensory neurons expressed multiple types of ASIC subunits, indicating prevalence of heteromeric channels. In mice with peripheral nerve injury, the expression level of ASIC1a and ASIC1b in L4 DRG and ASIC3 in L5 DRG were altered in CGRP+ neurons, but not in IB4+ neurons. Furthermore, the pattern of change varied among DRGs depending on their segmental level, which pointed to differential regulatory mechanisms between afferent types and anatomical location. The distinct expression pattern of ASIC transcripts in naïve condition, and the differential regulation of ASIC subunits after peripheral nerve injury, suggest that ASIC subunits are involved in separate sensory modalities.