Search

Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with the loss of cortical and spinal motor neurons (MNs) and muscle degeneration (Kiernan et al. in Lancet 377:942–955, 2011). In the preclinical setting, functional tests that can detect early changes in motor function in rodent models of ALS are critical to understanding the etiology of the disease and treatment development. Here, we established a string-pulling paradigm that can detect forelimb and hindlimb motor deficits in the SOD1 mouse model of ALS earlier than traditional motor performance tasks. Additionally, our findings indicate that early loss of forelimb and hindlimb function is correlated with cortical and spinal MN loss, respectively. This task is not only ecological, low-cost, efficient, and non-onerous, it also requires little animal handling and reduces the stress placed on the animal. It has long been a concern in the field that the SOD1 mouse does not display forelimb motor deficits and does not give researchers a complete picture of the disease. Here, we provide evidence that the SOD1 model does in fact develop early forelimb motor deficits due to the task’s ability to assess fine-motor function, reconciling this model with the various clinical presentation of ALS. Taken together, the string-pulling paradigm may provide novel insights into the pathogenesis of ALS, offer nuanced evaluation of prospective treatments, and has high translational potential to the clinic.

Summary: Neuropathic pain is a debilitating condition resulting from damage to the nervous system. Imbalance of spinal excitation and inhibition has been proposed to contribute to neuropathic pain. However, the structural basis of this imbalance remains unknown. Using a preclinical model of neuropathic pain, we show that microglia selectively engulf spinal synapses that are formed by central neurons and spare those of peripheral sensory neurons. Furthermore, we reveal that removal of inhibitory and excitatory synapses exhibits distinct temporal patterns, in which microglia-mediated inhibitory synapse removal precedes excitatory synapse removal. We also find selective and gradual increase in complement depositions on dorsal horn synapses that corresponds to the temporal pattern of microglial synapse pruning activity and type-specific synapse loss. Together, these results define a specific role for microglia in the progression of neuropathic pain pathogenesis and implicate these immune cells in structural remodeling of dorsal horn circuitry.

The encoding of noxious stimuli into action potential firing is largely mediated by nociceptive free nerve endings. Tissue inflammation, by changing the intrinsic properties of the nociceptive endings, leads to nociceptive hyperexcitability and thus to the development of inflammatory pain. Here, we showed that tissue inflammation–induced activation of the mammalian target of rapamycin complex 2 (mTORC2) triggers changes in the architecture of nociceptive terminals and leads to inflammatory pain. Pharmacological activation of mTORC2 induced elongation and branching of nociceptor peripheral endings and caused long-lasting pain hypersensitivity. Conversely, nociceptor-specific deletion of the mTORC2 regulatory protein rapamycin-insensitive companion of mTOR (Rictor) prevented inflammation-induced elongation and branching of cutaneous nociceptive fibers and attenuated inflammatory pain hypersensitivity. Computational modeling demonstrated that mTORC2-mediated structural changes in the nociceptive terminal tree are sufficient to increase the excitability of nociceptors. Targeting mTORC2 using a single injection of antisense oligonucleotide against Rictor provided long-lasting alleviation of inflammatory pain hypersensitivity. Collectively, we showed that tissue inflammation–induced activation of mTORC2 causes structural plasticity of nociceptive free nerve endings in the epidermis and inflammatory hyperalgesia, representing a therapeutic target for inflammatory pain.

Introduction The COVID-19 pandemic and associated restrictive measures have caused important disruptions in economies and labour markets, changed the way we work and socialise, forced schools to close and healthcare and social services to reorganise. This unprecedented crisis forces individuals to make considerable efforts to adapt and will have psychological and social consequences, mainly on vulnerable individuals, that will remain once the pandemic is contained and will most likely exacerbate existing social and gender health inequalities. This crisis also puts a toll on the capacity of our healthcare and social services structures to provide timely and adequate care. The MAVIPAN (Ma vie et la pandémie/ My Life and the Pandemic) study aims to document how individuals, families, healthcare workers and health organisations are affected by the pandemic and how they adapt.Methods and analysis MAVIPAN is a 5-year longitudinal prospective cohort study launched in April 2020 across the province of Quebec (Canada). Quantitative data will be collected through online questionnaires (4–6 times/year) according to the evolution of the pandemic. Qualitative data will be collected with individual and group interviews and will seek to deepen our understanding of coping strategies. Analysis will be conducted under a mixed-method umbrella, with both sequential and simultaneous analyses of quantitative and qualitative data.Ethics and dissemination MAVIPAN aims to support the healthcare and social services system response by providing high-quality, real-time information needed to identify those who are most affected by the pandemic and by guiding public health authorities’ decision making regarding intervention and resource allocation to mitigate these impacts. MAVIPAN was approved by the Ethics Committees of the Primary Care and Population Health Research Sector of CIUSSS de la Capitale-Nationale (Committee of record) and of the additional participating institutions.Trial registration number NCT04575571.

Inhibition in spinal nociceptive pathways is weaker and more labile in lamina I —where thermal input is primarily processed— than in lamina II that encodes predominantly high threshold mechanical input. This explains why noxious thermal input makes spinal circuits prone to catastrophic sensitization.

Disinhibition in the dorsal horn of the spinal cord may contribute to chronic pain. Here the authors show that, despite a paradoxical increase in α2/α3 subunits of the GABAA receptor in a neuropathic pain model, inhibition eventually fails due to KCC2 hypofunction.

This paper presents the design and the utilization of a wireless electro-optic platform to perform simultaneous multimodal electrophysiological recordings and optogenetic stimulation in freely moving rodents. The developed system can capture neural action potentials (AP), local field potentials (LFP) and electromyography (EMG) signals with up to 32 channels in parallel while providing four optical stimulation channels. The platform is using commercial off-the-shelf components (COTS) and a low-power digital field-programmable gate array (FPGA), to perform digital signal processing to digitally separate in real time the AP, LFP and EMG while performing signal detection and compression for mitigating wireless bandwidth and power consumption limitations. The different signal modalities collected on the 32 channels are time-multiplexed into a single data stream to decrease power consumption and optimize resource utilization. The data reduction strategy is based on signal processing and real-time data compression. Digital filtering, signal detection, and wavelet data compression are used inside the platform to separate the different electrophysiological signal modalities, namely the local field potentials (1–500 Hz), EMG (30–500 Hz), and the action potentials (300–5,000 Hz) and perform data reduction before transmitting the data. The platform achieves a measured data reduction ratio of 7.77 (for a firing rate of 50 AP/second) and weights 4.7 g with a 100-mAh battery, an on/off switch and a protective plastic enclosure. To validate the performance of the platform, we measured distinct electrophysiology signals and performed optogenetics stimulation in vivo in freely moving rondents. We recorded AP and LFP signals with the platform using a 16-microelectrode array implanted in the primary motor cortex of a Long Evans rat, both in anesthetized and freely moving conditions. EMG responses to optogenetic Channelrhodopsin-2 induced activation of motor cortex via optical fiber were also recorded in freely moving rodents.

Summary: Trigeminal neuralgia (TN) is a common, debilitating neuropathic face pain syndrome often resistant to therapy. The familial clustering of TN cases suggests that genetic factors play a role in disease pathogenesis. However, no unbiased, large-scale genomic study of TN has been performed to date. Analysis of 290 whole exome-sequenced TN probands, including 20 multiplex kindreds and 70 parent-offspring trios, revealed enrichment of rare, damaging variants in GABA receptor-binding genes in cases. Mice engineered with a TN-associated de novo mutation (p.Cys188Trp) in the GABAA receptor Cl− channel γ-1 subunit (GABRG1) exhibited trigeminal mechanical allodynia and face pain behavior. Other TN probands harbored rare damaging variants in Na+ and Ca+ channels, including a significant variant burden in the α-1H subunit of the voltage-gated Ca2+ channel Cav3.2 (CACNA1H). These results provide exome-level insight into TN and implicate genetically encoded impairment of GABA signaling and neuronal ion transport in TN pathogenesis.

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.

Abstract Morphine-induced hyperalgesia (MIH) is a severe adverse effect accompanying repeated morphine treatment, causing a paradoxical decrease in nociceptive threshold. Previous reports associated MIH with a decreased expression of the Cl− extruder KCC2 in the superficial dorsal horn (SDH) of the spinal cord, weakening spinal GABAA/glycine-mediated postsynaptic inhibition. Here, we tested whether the administration of small molecules enhancing KCC2, CLP257 and its pro-drug CLP290, may counteract MIH. MIH was typically expressed within 6–8 days of morphine treatment. Morphine-treated rats exhibited decreased withdrawal threshold to mechanical stimulation and increased vocalizing behavior to subcutaneous injections. Chloride extrusion was impaired in SDH neurons measured as a depolarizing shift in E GABA under Cl− load. Delivering CLP257 to spinal cord slices obtained from morphine-treated rats was sufficient to restore Cl− extrusion capacity in SDH neurons. In vivo co-treatment with morphine and oral CLP290 prevented membrane KCC2 downregulation in SDH neurons. Concurrently, co-treatment with CLP290 significantly mitigated MIH and acute administration of CLP257 in established MIH restored normal nociceptive behavior. Our data indicate that enhancing KCC2 activity is a viable therapeutic approach for counteracting MIH. Chloride extrusion enhancers may represent an effective co-adjuvant therapy to improve morphine analgesia by preventing and reversing MIH.

Summary: The behavioral features of neuropathic pain are not sexually dimorphic despite sex differences in the underlying neuroimmune signaling. This raises questions about whether neural processing is comparably altered. Here, we test whether the K+-Cl− co-transporter KCC2, which regulates synaptic inhibition, plays an equally important role in development of neuropathic pain in male and female rodents. Past studies on KCC2 tested only males. We find that inhibiting KCC2 in uninjured animals reproduces behavioral and electrophysiological features of neuropathic pain in both sexes and, consistent with equivalent injury-induced downregulation of KCC2, that counteracting chloride dysregulation reverses injury-induced behavioral and electrophysiological changes in both sexes. These findings demonstrate that KCC2 downregulation contributes equally to pain hypersensitivity in males and females. Whereas diverse (and sexually dimorphic) mechanisms regulate KCC2, regulation of intracellular chloride relies almost exclusively on KCC2. Directly targeting KCC2 thus remains a promising strategy for treatment of neuropathic pain in both sexes. : Neuropathic pain arises in males and females through distinct neuroimmune signaling. Yet the behavioral (or clinical) features of neuropathic pain are not sexually dimorphic. Mapplebeck et al. demonstrate that KCC2—though regulated in multiple, sex-specific ways—is uniquely responsible for the synaptic disinhibition causing neuropathic pain in both sexes. Keywords: K+-Cl− co-transporter KCC2, sex differences, degeneracy, neuropathic pain, disinhibition, GABA, glycine, BDNF

Cell labelling in a non-invasive and genetic engineering-free manner is crucial to cell biology applications. Here the authors develop cell labelling via photobleaching (CLaP), that uses laser illumination to label individual cells for genomics, cell-tracking, flow cytometry or ultra-microscopy.

We present a small and lightweight fully wireless optogenetic headstage capable of optical neural stimulation and electrophysiological recording. The headstage is suitable for conducting experiments with small transgenic rodents, and features two implantable fiber-coupled light-emitting diode (LED) and two electrophysiological recording channels. This system is powered by a small lithium-ion battery and is entirely built using low-cost commercial off-the-shelf components for better flexibility, reduced development time and lower cost. Light stimulation uses customizable stimulation patterns of varying frequency and duty cycle. The optical power that is sourced from the LED is delivered to target light-sensitive neurons using implantable optical fibers, which provide a measured optical power density of 70 mW/mm2 at the tip. The headstage is using a novel foldable rigid-flex printed circuit board design, which results into a lightweight and compact device. Recording experiments performed in the cerebral cortex of transgenic ChR2 mice under anesthetized conditions show that the proposed headstage can trigger neuronal activity using optical stimulation, while recording microvolt amplitude electrophysiological signals.

KCC2 is a neuron-specific K+-Cl– cotransporter essential for establishing the Cl- gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain. Using functional proteomics, we identified the KCC2-interactome in the mouse brain to determine KCC2-protein interactions that regulate KCC2 function. Our analysis revealed that KCC2 interacts with diverse proteins, and its most predominant interactors play important roles in postsynaptic receptor recycling. The most abundant KCC2 interactor is a neuronal endocytic regulatory protein termed PACSIN1 (SYNDAPIN1). We verified the PACSIN1-KCC2 interaction biochemically and demonstrated that shRNA knockdown of PACSIN1 in hippocampal neurons increases KCC2 expression and hyperpolarizes the reversal potential for Cl-. Overall, our global native-KCC2 interactome and subsequent characterization revealed PACSIN1 as a novel and potent negative regulator of KCC2.

Type 1 cannabinoid receptors (CB1Rs) are widely expressed in the vertebrate retina, but the role of endocannabinoids in vision is not fully understood. Here, we identified a novel mechanism underlying a CB1R-mediated increase in retinal ganglion cell (RGC) intrinsic excitability acting through AMPK-dependent inhibition of NKCC1 activity. Clomeleon imaging and patch clamp recordings revealed that inhibition of NKCC1 downstream of CB1R activation reduces intracellular Cl− levels in RGCs, hyperpolarizing the resting membrane potential. We confirmed that such hyperpolarization enhances RGC action potential firing in response to subsequent depolarization, consistent with the increased intrinsic excitability of RGCs observed with CB1R activation. Using a dot avoidance assay in freely swimming Xenopus tadpoles, we demonstrate that CB1R activation markedly improves visual contrast sensitivity under low-light conditions. These results highlight a role for endocannabinoids in vision and present a novel mechanism for cannabinoid modulation of neuronal activity through Cl− regulation.

Activation of the mechanistic/mammalian target of rapamycin (mTOR) kinase in models of acute and chronic pain is strongly implicated in mediating enhanced translation and hyperalgesia. However, the molecular mechanisms by which mTOR regulates nociception remain unclear. Here we show that deletion of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a major mTOR downstream effector, which represses eIF4E activity and cap-dependent translation, leads to mechanical, but not thermal pain hypersensitivity. Mice lacking 4E-BP1 exhibit enhanced spinal cord expression of neuroligin 1, a cell-adhesion postsynaptic protein regulating excitatory synapse function, and show increased excitatory synaptic input into spinal neurons, and a lowered threshold for induction of synaptic potentiation. Pharmacological inhibition of eIF4E or genetic reduction of neuroligin 1 levels normalizes the increased excitatory synaptic activity and reverses mechanical hypersensitivity. Thus, translational control by 4E-BP1 downstream of mTOR effects the expression of neuroligin 1 and excitatory synaptic transmission in the spinal cord, and thereby contributes to enhanced mechanical nociception.

Microglia-neuron interactions play a crucial role in several neurological disorders characterized by altered neural network excitability, such as epilepsy and neuropathic pain. While a series of potential messengers have been postulated as substrates of the communication between microglia and neurons, including cytokines, purines, prostaglandins, and nitric oxide, the specific links between messengers, microglia, neuronal networks, and diseases have remained elusive. Brain-derived neurotrophic factor (BDNF) released by microglia emerges as an exception in this riddle. Here, we review the current knowledge on the role played by microglial BDNF in controlling neuronal excitability by causing disinhibition. The efforts made by different laboratories during the last decade have collectively provided a robust mechanistic paradigm which elucidates the mechanisms involved in the synthesis and release of BDNF from microglia, the downstream TrkB-mediated signals in neurons, and the biophysical mechanism by which disinhibition occurs, via the downregulation of the K+-Cl− cotransporter KCC2, dysrupting Cl−homeostasis, and hence the strength of GABAA- and glycine receptor-mediated inhibition. The resulting altered network activity appears to explain several features of the associated pathologies. Targeting the molecular players involved in this canonical signaling pathway may lead to novel therapeutic approach for ameliorating a wide array of neural dysfunctions.

Microelectrodes have been very instrumental and minimally invasive for in vivo functional studies from deep brain structures. However they are limited in the amount of information they provide. Here, we describe a, aluminum-coated, fibre optic-based glass microprobe with multiple electrical and optical detection capabilities while retaining tip dimensions that enable single cell measurements (diameter ≤10 µm). The probe enables optical separation from individual cells in transgenic mice expressing multiple fluorescent proteins in distinct populations of neurons within the same deep brain nucleus. It also enables color conversion of photoswitchable fluorescent proteins, which can be used for post-hoc identification of the recorded cells. While metal coating did not significantly improve the optical separation capabilities of the microprobe, the combination of metal on the outside of the probe and of a hollow core within the fiber yields a microelectrode enabling simultaneous single unit and population field potential recordings. The extended range of functionalities provided by the same microprobe thus opens several avenues for multidimensional structural and functional interrogation of single cells and their surrounding deep within the intact nervous system.

Besides memory deficits, Alzheimer's disease (AD) patients suffer from neuropsychiatric symptoms, including alterations in social interactions, which are subject of a growing number of investigations in transgenic models of AD. Yet the biological mechanisms underlying these behavioural alterations are poorly understood. Here, a social interaction paradigm was used to assess social dysfunction in the triple-transgenic mouse model of AD (3xTg-AD). We observed that transgenic mice displayed dimorphic behavioural abnormalities at different ages. Social disinhibition was observed in 18 months old 3xTg-AD males compared to age and sex-matched control mice. In 3xTg-AD females, social disinhibition was present at 12 months followed by reduced social interactions at 18 months. These dimorphic behavioural alterations were not associated with alterations in AD neuropathological markers such as Aβ or tau levels in the frontal cortex. However, patch-clamp recordings revealed that enhanced social interactions coincided temporally with an increase in both excitatory and inhibitory basal synaptic inputs to layer 2-3 pyramidal neurons in the prefrontal cortex. These findings uncover a novel pattern of occurrence of psychiatric-like symptoms between sexes in an AD model. Our results also reveal that functional alterations in synapse activity appear as a potentially significant substrate underlying behavioural correlates of AD.

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.

In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca(2+) imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements.

Transduction of graded synaptic input into trains of all-or-none action potentials (spikes) is a crucial step in neural coding. Hodgkin identified three classes of neurons with qualitatively different analog-to-digital transduction properties. Despite widespread use of this classification scheme, a generalizable explanation of its biophysical basis has not been described. We recorded from spinal sensory neurons representing each class and reproduced their transduction properties in a minimal model. With phase plane and bifurcation analysis, each class of excitability was shown to derive from distinct spike initiating dynamics. Excitability could be converted between all three classes by varying single parameters; moreover, several parameters, when varied one at a time, had functionally equivalent effects on excitability. From this, we conclude that the spike-initiating dynamics associated with each of Hodgkin's classes represent different outcomes in a nonlinear competition between oppositely directed, kinetically mismatched currents. Class 1 excitability occurs through a saddle node on invariant circle bifurcation when net current at perithreshold potentials is inward (depolarizing) at steady state. Class 2 excitability occurs through a Hopf bifurcation when, despite net current being outward (hyperpolarizing) at steady state, spike initiation occurs because inward current activates faster than outward current. Class 3 excitability occurs through a quasi-separatrix crossing when fast-activating inward current overpowers slow-activating outward current during a stimulus transient, although slow-activating outward current dominates during constant stimulation. Experiments confirmed that different classes of spinal lamina I neurons express the subthreshold currents predicted by our simulations and, further, that those currents are necessary for the excitability in each cell class. Thus, our results demonstrate that all three classes of excitability arise from a continuum in the direction and magnitude of subthreshold currents. Through detailed analysis of the spike-initiating process, we have explained a fundamental link between biophysical properties and qualitative differences in how neurons encode sensory input.