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Chronic, pathological pain is a highly debilitating condition that can arise and be maintained through central sensitization. Central sensitization shares mechanistic and phenotypic parallels with memory formation. In a sensory model of memory reconsolidation, plastic changes underlying pain hypersensitivity can be dynamically regulated and reversed following the reactivation of sensitized sensory pathways. However, the mechanisms by which synaptic reactivation induces destabilization of the spinal “pain engram” are unclear. We identified nonionotropic N -methyl- d -aspartate receptor (NI-NMDAR) signaling as necessary and sufficient for the reactive destabilization of dorsal horn long-term potentiation and the reversal of mechanical sensitization associated with central sensitization. NI-NMDAR signaling engaged directly or through the reactivation of sensitized sensory networks was associated with the degradation of excitatory postsynaptic proteins. Our findings identify NI-NMDAR signaling as a putative synaptic mechanism by which engrams are destabilized in reconsolidation and as a potential means of treating underlying causes of chronic pain.

Identifying the genetic determinants of pain is a scientific imperative given the magnitude of the global health burden that pain causes. Here, we report a genetic screen for nociception, performed under the auspices of the International Mouse Phenotyping Consortium. A biased set of 110 single-gene knockout mouse strains was screened for 1 or more nociception and hypersensitivity assays, including chemical nociception (formalin) and mechanical and thermal nociception (von Frey filaments and Hargreaves tests, respectively), with or without an inflammatory agent (complete Freund's adjuvant). We identified 13 single-gene knockout strains with altered nocifensive behavior in 1 or more assays. All these novel mouse models are openly available to the scientific community to study gene function. Two of the 13 genes (Gria1 and Htr3a) have been previously reported with nociception-related phenotypes in genetically engineered mouse strains and represent useful benchmarking standards. One of the 13 genes (Cnrip1) is known from human studies to play a role in pain modulation and the knockout mouse reported herein can be used to explore this function further. The remaining 10 genes (Abhd13, Alg6, BC048562, Cgnl1, Cp, Mmp16, Oxa1l, Tecpr2, Trim14, and Trim2) reveal novel pathways involved in nociception and may provide new knowledge to better understand genetic mechanisms of inflammatory pain and to serve as models for therapeutic target validation and drug development.

The establishment and maintenance of strong affiliative relationships is fundamental for group cohesion and crucial for overall individual well-being. Empathy is considered a critical process for promoting attachment and the long-term stability of social bonds. However, it is unclear how different modalities of social communication contribute to the development of empathy. Physical contact between individuals, such as gentle touching, is a highly salient form of social communication. Despite mounting evidence that touch may be crucial for promoting social bonds, the role of touch in the development of empathy is currently not well understood. Animal models have become a powerful tool for the experimental manipulation and examination of empathy related behaviors such as emotional contagion. Here, we use the Tube Co-Occupancy Test (TCOT) to promote voluntary physical contact between mice and examine whether social, physical contact promotes emotional contagion of pain between mice. We found that repeated exposure to TCOT promoted the development of emotional contagion between mice. However, preventing physical contact in the TCOT assay also prevented the development of emotional contagion of pain. These results demonstrate that voluntary physical contact is a critical component in the formation of social bonding and emotional contagion in mice.

Background and purpose T-type voltage-gated calcium channels are an emerging potential therapeutic target for neurological disorders including epilepsy and pain. Inhibition of T-type channels reduces the excitability of peripheral nociceptive sensory neurons and reverses pain hypersensitivity in male rodent pain models. However, administration of peripherally restricted T-type antagonists has failed to show efficacy in multiple clinical and preclinical pain trials, suggesting that inhibition of peripheral T-type channels alone may be insufficient for pain relief. Experimental approach We utilized the selective and CNS-penetrant T-type channel antagonist, Z944, in electrophysiological, calcium imaging, and behavioural paradigms to determine its effect on lamina I neuron excitability and inflammatory pain behaviours. Key results Voltage-clamp recordings from lamina I spinal neurons of adult rats revealed that approximately 80% of neurons possess a low threshold T-type current, which was blocked by Z944. Due to this highly prevalent T-type current, Z944 potently blocked action-potential evoked somatic and dendritic calcium transients in lamina I neurons. Moreover, application of Z944 to spinal cord slices attenuated action potential firing rates in over half of laminae I/II neurons. Finally, we found that intraperitoneal injection of Z944 (1-10 mg/kg) dose-dependently reversed mechanical allodynia in the complete Freund's adjuvant model of persistent inflammatory pain, with a similar magnitude and time course of analgesic effects between male and female rats. Conclusion and implications Our findings demonstrate that T-type calcium channels critically shape the excitability of lamina I pain processing neurons, and inhibition of these channels by the clinical stage antagonist Z944 potently reverses pain hypersensitivity across sexes.

Background: Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that begins in childhood and often persists into adulthood. ADHD increases the risk of various negative impacts, and pharmacists are well positioned to address these issues in the community. Objectives: This survey study aims to first identify pharmacists’ ADHD knowledge gaps and experience with ADHD management and to second assess their preferences for continuing education and their experience with sleep-related issues in ADHD. Methods: A survey was sent to Part A Ontario pharmacists with active licenses who opted in to receive research-related emails ( n = 6022). Descriptive statistics were used to analyze survey data, while free-form answers were pooled and evaluated for common themes and trends. Results: A total of 238 complete responses were received. The average self-reported ADHD knowledge was 5.8 ± 1.96 on a 10-point scale. There was no correlation between the number of years of practice as a pharmacist, the number of working hours per week or the location of practice on pharmacists’ self-reported knowledge scores. There was a significant difference in self-reported knowledge of ADHD between pharmacists who were not aware of the Canadian ADHD Resource Alliance (CADDRA) guidelines (5.1 ± 2.1) and those who refer to it for standard of care (7.1 ± 1.5). Almost all pharmacists (95%) indicated they could benefit from additional ADHD education, with a strong preference for “online continuing education modules” (81%). The majority of responders considered psychostimulant ADHD medication as the major possible contributor to sleep disturbances (47%) in ADHD, highlighting a need for further education on the inconclusive link between ADHD medication effects on sleep. Conclusion: The study results raise the concern that pharmacists may require additional ADHD education but also show the lack of awareness of available resources, such as the CADDRA guidelines. Can Pharm J (Ott) 2021;154:xx-xx.

Supplemental Digital Content is Available in the Text. Cage-lid hanging behavior is impaired by sustained pain in mice and can be used as an ethologically valid and translationally relevant pain outcome measure., The development of new analgesic drugs has been hampered by the inability to translate preclinical findings to humans. This failure is due in part to the weak connection between commonly used pain outcome measures in rodents and the clinical symptoms of chronic pain. Most rodent studies rely on the use of experimenter-evoked measures of pain and assess behavior under ethologically unnatural conditions, which limits the translational potential of preclinical research. Here, we addressed this problem by conducting an unbiased, prospective study of behavioral changes in mice within a natural homecage environment using conventional preclinical pain assays. Unexpectedly, we observed that cage-lid hanging, a species-specific elective behavior, was the only homecage behavior reliably impacted by pain assays. Noxious stimuli reduced hanging behavior in an intensity-dependent manner, and the reduction in hanging could be restored by analgesics. Finally, we developed an automated approach to assess hanging behavior. Collectively, our results indicate that the depression of hanging behavior is a novel, ethologically valid, and translationally relevant pain outcome measure in mice that could facilitate the study of pain and analgesic development.

Reconsolidation enables the activity-dependent modification of memory traces and has been used to reverse addiction, fear memory, and pain hypersensitivity in animal models. We demonstrate that non-ionotropic NMDA receptor signalling in the spinal dorsal horn is sufficient to reverse pain hypersensitivity and necessary for pain modulation by spinal reconsolidation. These findings reveal a key process by which reconsolidation disrupts memory traces that may be exploited in the treatment of pain and other disorders.

Wide dynamic range (WDR) neurons of the spinal dorsal horn respond to a wide range of innocuous and noxious mechanical stimulation and encode the intensity of mechanical stimuli as changes in firing rate. However, there are inconsistent findings regarding whether WDR neuron stimulus encoding activity is altered in pathological pain states. This inconsistency may arise from differences in the pain models used or in the experimental conditions themselves. In this study, we use a meta-regression approach to examine which variables modulate and determine WDR activity. We pooled data from in vivo electrophysiological studies of WDR activity evoked by von Frey filament stimulation of the hind paw in rats across a number of pathological pain models. We observed that WDR firing rate was better predicted by the calculated pressure of von Frey stimulation rather than applied filament force, as reported in all studies. The pressure-evoked firing rate of WDR neurons was not altered by any experimental pain model except for arthritis and inflammation models, where mechanical stimuli evoked a higher firing rate than controls. Conversely, there was a consistent increase in the spontaneous firing rate of WDR neurons in neuropathic pain, arthritis and inflammation, and chemoneuropathy pain models. Overall, these data indicate that changes in WDR encoding of applied pressure are unlikely to significantly contribute to pathological sensory processing but suggest a possible role for these neurons in spontaneous pain.

Background Gabapentin is a structural analog of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Its anticonvulsant, analgesic and anxiolytic properties suggest that it increases GABAergic inhibition; however, the molecular basis for these effects is unknown as gabapentin does not directly modify GABA type A (GABAA) receptor function, nor does it modify synaptic inhibition. Here, we postulated that gabapentin increases expression of δ subunit-containing GABAA (δGABAA) receptors that generate a tonic inhibitory conductance in multiple brain regions including the cerebellum and hippocampus. Methods Cell-surface biotinylation, Western blotting, electrophysiologic recordings, behavioral assays, high-performance liquid chromatography and gas chromatography-mass spectrometry studies were performed using mouse models. Findings Gabapentin enhanced expression of δGABAA receptors and increased a tonic inhibitory conductance in neurons. This increased expression likely contributes to GABAergic effects as gabapentin caused ataxia and anxiolysis in wild-type mice but not δ subunit null-mutant mice. In contrast, the antinociceptive properties of gabapentin were observed in both genotypes. Levels of GABAA receptor agonists and neurosteroids in the brain were not altered by gabapentin. Interpretation These results provide compelling evidence to account for the GABAergic properties of gabapentin. Since reduced expression of δGABAA receptor occurs in several disorders, gabapentin may have much broader therapeutic applications than is currently recognized. Fund Supported by a Foundation Grant (FDN-154312) from the Canadian Institutes of Health Research (to B.A.O.); a NSERC Discovery Grant (RGPIN-2016-05538), a Canada Research Chair in Sensory Plasticity and Reconsolidation, and funding from the University of Toronto Centre for the Study of Pain (to R.P.B.).

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance becomes negatively reinforcing. Ventral tegmental area (VTA) GABA neurons form a point of divergence between two double dissociable pathways responsible for these respective motivational states. Here we show that this switch from drug-naïve to opiate-dependent and withdrawn (ODW) motivation is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve motivational state and a loss of opiate withdrawal aversions. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36fl(CFP)/fl(CFP)) were perpetually drug-naïve and never experienced opiate withdrawal aversions. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to ODW motivation. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.SignificanceThe motivation to seek drugs can vary depending on prior exposure. For instance, recreational and habitual drug use can stem from a desire to experience the pleasurable or relieving properties of the substance, respectively. Here we identify a subpopulation of midbrain neurons that dictate opiate-seeking motivation via expression of the gap junction protein, connexin-36. We show that connexin-36 expression increases upon opiate dependence and withdrawal. We then demonstrate that this is not merely a correlation, as pharmacological or genetic manipulations that interfere with connexin-36 function prevent the development of opiate dependence in rats and mice. Our results identify gap junctions as a critical node in the pathogenesis of opiate addiction, and a potential new target for substance use disorder pharmacotherapies.

Common approaches to studying chronic pain in pre-clinical animal models paradoxically involve measuring reflexive withdrawal responses that are more indicative of acute nociceptive pain. These methods typically do not capture the ongoing nature of chronic pain nor report on behavioral changes associated with pain. In addition, data collection and analysis protocols are often labour-intensive and require direct investigator interactions, potentially introducing bias. In this study, we develop and characterize a low-cost, easily assembled behavioral assay that yields self-reported temperature preference from mice which is sensitive to peripheral sensitization protocols. This system uses a partially automated and freely available analysis pipeline to streamline the data collection process and enable objective analysis. We found that after intraplantar administration of the TrpV1 agonist, capsaicin, mice preferred to stay in cooler temperatures than control injected mice. We further observed that gabapentin, a non-opioid analgesic commonly prescribed to treat chronic pain, reversed this aversion to higher temperatures. We further observed that optogenetic activation of the central terminals of TrpV1+ primary afferents via in vivo spinal light delivery did not induce a similar change in thermal preference, indicating a role for peripheral nociceptor activity in the modulation of temperature preference. We conclude that this easily produced and robust sensory assay provides an alternative approach to investigate the contribution of central and peripheral mechanisms to pathological sensory processing that does not rely on reflexive responses evoked by noxious stimuli.

What We Already Know about This Topic What This Article Tells Us That Is New Background Postoperative delirium is associated with poor long-term outcomes and increased mortality. General anesthetic drugs may contribute to delirium because they increase cell-surface expression and function of α5 subunit-containing γ-aminobutyric acid type A receptors, an effect that persists long after the drugs have been eliminated. Dexmedetomidine, an α2 adrenergic receptor agonist, prevents delirium in patients and reduces cognitive deficits in animals. Thus, it was postulated that dexmedetomidine prevents excessive function of α5 γ-aminobutyric acid type A receptors. Methods Injectable (etomidate) and inhaled (sevoflurane) anesthetic drugs were studied using cultured murine hippocampal neurons, cultured murine and human cortical astrocytes, and ex vivo murine hippocampal slices. γ-Aminobutyric acid type A receptor function and cell-signaling pathways were studied using electrophysiologic and biochemical methods. Memory and problem-solving behaviors were also studied. Results The etomidate-induced sustained increase in α5 γ-aminobutyric acid type A receptor cell-surface expression was reduced by dexmedetomidine (mean ± SD, etomidate: 146.4 ± 51.6% vs. etomidate + dexmedetomidine: 118.4 ± 39.1% of control, n = 8 each). Dexmedetomidine also reduced the persistent increase in tonic inhibitory current in hippocampal neurons (etomidate: 1.44 ± 0.33 pA/pF, n = 10; etomidate + dexmedetomidine: 1.01 ± 0.45 pA/pF, n = 9). Similarly, dexmedetomidine prevented a sevoflurane-induced increase in the tonic current. Dexmedetomidine stimulated astrocytes to release brain-derived neurotrophic factor, which acted as a paracrine factor to reduce excessive α5 γ-aminobutyric acid type A receptor function in neurons. Finally, dexmedetomidine attenuated memory and problem-solving deficits after anesthesia. Conclusions Dexmedetomidine prevented excessive α5 γ-aminobutyric acid type A receptor function after anesthesia. This novel α2 adrenergic receptor- and brain-derived neurotrophic factor-dependent pathway may be targeted to prevent delirium.

While spinal microglia play a role in early stages of neuropathic pain etiology, whether they are useful targets to reverse chronic pain at late stages remains unknown. Here, we show that microglia activation in the spinal cord persists for >3 months following nerve injury in rodents, beyond involvement of proinflammatory cytokine and chemokine signalling. In this chronic phase, selective depletion of spinal microglia in male rats with the targeted immunotoxin Mac1-saporin and blockade of brain-derived neurotrophic factor-TrkB signalling with intrathecal TrkB Fc chimera, but not cytokine inhibition, almost completely reversed pain hypersensitivity. By contrast, local spinal administration of Mac1-saporin did not affect nociceptive withdrawal threshold in control animals nor did it affect the strength of afferent-evoked synaptic activity in the spinal dorsal horn in normal conditions. These findings show that the long-term, chronic phase of nerve injury-induced pain hypersensitivity is maintained by microglia-neuron interactions. The findings also effectively separate the central signalling pathways underlying the maintenance phase of the pathology from the early and peripheral inflammatory reactions to injury, pointing to different targets for the treatment of acute vs chronic injury-induced pain.

Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide (10panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reply to The small molecule CLP257 does not modify activity of the K + –Cl − co-transporter KCC2 but does potentiate GABA A receptor activity

Neuronal inhibition mediated by GABAA receptors constrains nociceptive processing in the spinal cord, and loss of GABAergic inhibition can produce allodynia and hyperalgesia. Extrasynaptic α5 subunit-containing GABAA receptors (α5GABAA Rs) generate a tonic conductance that inhibits neuronal activity and constrains learning and memory; however, it is unclear whether α5GABAA Rs similarly generate a tonic conductance in the spinal cord dorsal horn to constrain nociception. We assessed the distribution of α5GABAA Rs in the spinal cord dorsal horn by immunohistochemical analysis, and the activity and function of α5GABAA Rs in neurons of the superficial dorsal horn using electrophysiological and behavioral approaches in male, null-mutant mice lacking the GABAA R α5 subunit (Gabra5-/-) and wild-type mice (WT). The expression of α5GABAA Rs in the superficial dorsal horn followed a laminar pattern of distribution, with a higher expression in lamina II than lamina I. Similarly, the tonic GABAA current in lamina II neurons had a larger contribution from α5GABAA Rs than in lamina I, with no significant contribution of these receptors to synaptic GABAA current. In behavioural tests, WT and Gabra5-/- mice exhibited similar acute thermal and mechanical nociception, and similar mechanical sensitization immediately following intraplantar capsaicin or Complete Freund's Adjuvant (CFA). However, Gabra5-/- mice showed prolonged recovery from sensitization in these models, and increased responses in the late phase of the formalin test. Overall, our data suggest that tonically-active α5GABAA Rs in the spinal cord dorsal horn accelerate the resolution of hyperalgesia and may therefore serve as a novel therapeutic target to promote recovery from pathological pain. © 2016 Wiley Periodicals, Inc.

Optogenetics has become an integral tool for studying and dissecting the neural circuitries of the brain using optical control. Recently, it has also begun to be used in the investigation of the spinal cord and peripheral nervous system. However, information on these regions' optical properties is sparse. Moreover, there is a lack of data on the dependence of light propagation with respect to neural tissue organization and orientation. This information is important for effective simulations and optogenetic planning, particularly in the spinal cord where the myelinated axons are highly organized. To this end, we report experimental measurements for the scattering coefficient, validated with three different methods in both the longitudinal and radial directions of multiple mammalian spinal cords. In our analysis, we find that there is indeed a directional dependence of photon propagation when interacting with organized myelinated axons. Specifically, light propagating perpendicular to myelinated axons in the white matter of the spinal cord produced a measured reduced scattering coefficient ( μ s ' ) of 3.52 ± 0.1 mm - 1 , and light that was propagated along the myelinated axons in the white matter produced a measured μ s ' of 1.57 ± 0.03 mm - 1 , across the various species considered. This 50% decrease in scattering power along the myelinated axons is observed with three different measurement strategies (integrating spheres, observed transmittance, and punch-through method). Furthermore, this directional dependence in scattering power and overall light attenuation did not occur in the gray matter regions where the myelin organization is nearly random. The acquired information will be integral in preparing future light-transport simulations and in overall optogenetic planning in both the spinal cord and the brain.

Most research laboratories abide by guidelines and mandates set by their research institution regarding the administration of analgesics to control pain during the postoperative period. Unfortunately, measuring pain originating from the head is difficult, making adequate decisions regarding pain control following stereotaxic surgery problematic. In addition, most postsurgical analgesia protocols require multiple injections over several days, which may cause stress and distress during a critical recovery period. Here we sought to (1) assess the degree of postoperative pain following craniotomy in mice, (2) compare the efficacy of three common rodent analgesics (carprofen, meloxicam and buprenorphine) for reducing this pain and (3) determine whether the route of administration (injected or self-administered through the drinking supply) influenced pain relief post-craniotomy. Using the mouse grimace scale (MGS), we found that injectable analgesics were significantly more effective at relieving post-craniotomy pain, however, both routes of administration decreased pain scores in the first 24 h postsurgery. Specifically, buprenorphine administered independently of administration route was the most effective at reducing MGS scores, however, female mice showed greater sensitivity to carprofen when administered through the water supply. Although it is necessary to provide laboratory animals with analgesics after an invasive procedure, there remains a gap in the literature regarding the degree of craniotomy-related pain in rodents and the efficacy of alternative routes of administration. Our study highlights the limitations of administering drugs through the drinking supply, even at doses that are considered to be higher than those currently recommended by most research institutions for treating pain of mild to moderate severity.

Intractable pain is the single most common cause of disability, affecting more than 20% of the population world-wide. There is accordingly a global effort to decipher how changes in nociceptive processing in the peripheral and central nervous systems contribute to the onset and maintenance of chronic pain. The past several years have brought rapid progress in the adaptation of optogenetic approaches to study and manipulate the activity of sensory afferents and spinal cord neurons in freely behaving animals, and to investigate cortical processing and modulation of pain responses. This review discusses methodological advances that underlie this recent progress, and discusses practical considerations for the optogenetic modulation of nociceptive sensory processing.

We report a novel model in which remote activation of peripheral nociceptive pathways in transgenic mice is achieved optogenetically, without any external noxious stimulus or injury. Taking advantage of a binary genetic approach, we selectively targeted Nav1.8+sensory neurons for conditional expression of channelrhodopsin-2 (ChR2) channels. Acute blue light illumination of the skin produced robust nocifensive behaviors, evoked by the remote stimulation of both peptidergic and nonpeptidergic nociceptive fibers as indicated by c-Fos labeling in laminae I and II of the dorsal horn of the spinal cord. A non-nociceptive component also contributes to the observed behaviors, as shown by c-Fos expression in lamina III of the dorsal horn and the expression of ChR2–EYFP in a subpopulation of large-diameter Nav1.8+dorsal root ganglion neurons. Selective activation of Nav1.8+afferentsin vivoinduced central sensitization and conditioned place aversion, thus providing a novel paradigm to investigate plasticity in the pain circuitry. Long-term potentiation was similarly evoked by light activation of the same afferents in isolated spinal cord preparations. These findings demonstrate, for the first time, the optical control of nociception and central sensitization in behaving mammals and enables selective activation of the same class of afferents in bothin vivoandex vivopreparations. Our results provide a proof-of-concept demonstration that optical dissection of the contribution of specific classes of afferents to central sensitization is possible. The high spatiotemporal precision offered by this non-invasive model will facilitate drug development and target validation for pain therapeutics.

The K(+)-Cl(-) cotransporter KCC2 is responsible for maintaining low Cl(-) concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABA(A) and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl(-) transport. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl(-)]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl(-)]i. CLP257 restored impaired Cl(-) transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.

Neuronal inhibition mediated by GABA

Optogenetics is currently one of the most popular technique in neuroscience. It enables cell-selective and temporally-precise control of neuronal activity. Good spatial control of the stimulated area and minimized tissue damage requires a specific knowledge about light scattering properties. Light propagation in cell cultures and brain tissue is relatively well documented and allows for a precise and reliable delivery of light to the neurons. In spinal cord, light must pass through highly organized white matter before reaching cell bodies present in grey matter, this heterogenous structure makes it difficult to predict the propagation pattern. In this work we investigate the light distribution properties through mouse and monkey spinal cord. The light propagation depends on a fibers orientation, leading to less deep penetration profile in the direction perpendicular to the fibers and lower attenuation in the direction parallel to the fibers. Additionally, the use of different illumination wavelengths results in variations of the attenuation coefficient. Next, we use Monte-Carlo simulation to study light transport. The model gives a full 3-D simulation of light distribution in spinal cord and takes into account different scattering properties related to the fibers orientation. These studies are important to estimate the minimum optical irradiance required at the fiber tip to effectively excite the optogenetic proteins in a desired region of spinal cord.

Background Optogenetic tools enable cell selective and temporally precise control of neuronal activity; yet, difficulties in delivering sufficient light to the spinal cord of freely behaving animals have hampered the use of spinal optogenetic approaches to produce analgesia. We describe an epidural optic fiber designed for chronic spinal optogenetics that enables the precise delivery of light at multiple wavelengths to the spinal cord dorsal horn and sensory afferents. Results The epidural delivery of light enabled the optogenetic modulation of nociceptive processes at the spinal level. The acute and repeated activation of channelrhodopsin-2 expressing nociceptive afferents produced robust nocifensive behavior and mechanical sensitization in freely behaving mice, respectively. The optogenetic inhibition of GABAergic interneurons in the spinal cord dorsal horn through the activation of archaerhodopsin also produced a transient, but selective induction of mechanical hypersensitivity. Finally, we demonstrate the capacity of optogenetics to produce analgesia in freely behaving mice through the inhibition of nociceptive afferents via archaerhodopsin. Conclusion Epidural optogenetics provides a robust and powerful solution for activation of both excitatory and inhibitory opsins in sensory processing pathways. Our results demonstrate the potential of spinal optogenetics to modulate sensory behavior and produce analgesia in freely behaving animals.

BACKGROUND: Many clinical trials have demonstrated the effectiveness of gabapentin and pregabalin administration in the perioperative period as an adjunct to reduce acute postoperative pain. However, very few clinical trials have examined the use of gabapentin and pregabalin for the prevention of chronic postsurgical pain (CPSP). We (1) systematically reviewed the published literature pertaining to the prevention of CPSP (2 months after surgery) after perioperative administration of gabapentin and pregabalin and (2) performed a meta-analysis using studies that report sufficient data. A search of electronic databases (Medline, Embase, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, IPA, and CINAHL) for relevant English-language trials to June 2011 was conducted. METHODS: The following inclusion criteria for identified clinical trials were used for entry into the present systematic review: randomization; double-blind assessments of pain and analgesic use; report of pain using a reliable and valid measure; report of analgesic consumption; and an absence of design flaws, methodological problems or confounders that render interpretation of the results ambiguous. Trials that did not fit the definition of preventive analgesia and did not assess chronic pain at 2 or more months after surgery were excluded. RESULTS: The database search yielded 474 citations. Eleven studies met the inclusion criteria. Of the 11 trials, 8 studied gabapentin, 4 of which (i.e., 50%) found that perioperative administration of gabapentin decreased the incidence of chronic pain more than 2 months after surgery. The 3 trials that used pregabalin demonstrated a significant reduction in the incidence of CPSP, and 2 of the 3 trials also found an improvement in postsurgical patient function. Eight studies were included in a meta-analysis, 6 of the gabapentin trials demonstrated a moderateto-large reduction in the development of CPSP (pooled odds ratio [OR] 0.52; 95% confidence interval [CI], 0.27 to 0.98; P 0.04), and the 2 pregabalin trials found a very large reduction in the development of CPSP (pooled OR 0.09; 95% CI, 0.02 to 0.79; P 0.007). CONCLUSIONS: The present review supports the view that perioperative administration of gabapentin and pregabalin are effective in reducing the incidence of CPSP. Better-designed and appropriately powered clinical trials are needed to confirm these early findings. (Anesth Analg 2012;115:428–42)

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. DOI: http://dx.doi.org/10.7554/eLife.12002.001, eLife digest Despite the unpleasant feeling it causes, pain is necessary for survival as it helps individuals to avoid objects, environments and situations that cause damage to their body. However, millions of people experience long-lasting “chronic” pain, or are hypersensitive to pain. There are few treatments available for these conditions, but these treatments do not work well for the majority of patients, and can have serious side effects. To develop new treatments, researchers must first better understand how chronic pain develops. Pain is transmitted to the brain in the form of electrical signals “fired” along nerve fibers. Different nerves transmit information about different types of pain: for example, pain caused by a sharp object pressed against the skin activates a different set of neurons to those activated when touching something dangerously hot. Studies in mice have suggested that a protein called mTOR that is found inside neurons is important for them to fire pain signals. However, it is not clear exactly how mTOR contributes to pain signaling, although it is known to affect the activities of several other proteins in neurons. One protein that mTOR affects the activity of is called 4E-BP1. Now, Khoutorsky, Bonin, Sorge et al. show that mice that lack 4E-BP1 behave in ways that suggest they are hypersensitive to poking or pinching sensations. However, the mice did not show hypersensitivity when they touched a hot surface. Further investigation revealed that the neurons in the spinal cord of mice that lack 4E-BP1 produce abnormally high amounts of a molecule called neuroligin 1, which makes the neurons more likely to fire and thus signal pain. Khoutorsky, Bonin, Sorge et al. found that treating mice that lack 4E-BP1 with a compound that reduces neuroligin 1 production causes their neurons to fire more normally. This also reduces the animals’ apparent signs of hypersensitivity to pressure on their skin. It will be important in future studies to identify additional targets of 4E-BP1 in the spinal cord that could contribute to increased mechanical sensation, and also to study the role of 4E-BP1 in peripheral nerves. DOI: http://dx.doi.org/10.7554/eLife.12002.002

The precise mechanisms underlying the memory-blocking properties of ethanol are unknown, in part because ethanol targets a wide array of neurotransmitter receptors and transporters. The aim of this study was to determine whether the memory loss caused by ethanol is mediated, in part, by α5 subunit-containing γ-aminobutyric acid subtype A receptors. These receptors have been implicated in learning and memory processes and are targets for a variety of neurodepressive drugs. Also, since these receptors generate a tonic inhibitory current in hippocampal pyramidal neurons, we examined whether concentrations of ethanol that block memory in vivo increased the tonic current using whole-cell patch-clamp recordings in hippocampal neurons. Null mutant mice lacking the α5 subunit (Gabra5-/-) and wild-type mice were equally impaired in contextual fear conditioning by moderate (1mg/kg) and high (1.5mg/kg) doses of ethanol. The higher dose of ethanol also reduced auditory delay fear conditioning to the same extent in the two genotypes. Interestingly, wild-type mice were more sensitive than Gabra5-/- mice to the sedative effects of low (0.5mg/kg) and moderate (1mg/kg) doses of ethanol in the open-field task. Concentrations of ethanol that impaired memory performance in vivo did not increase the amplitude of the tonic current. Together, the results suggest that the α5-subunit containing γ-aminobutyric acid subtype A receptors are not direct targets for positive modulation by ethanol nor do they contribute to ethanol-induced memory loss. In contrast, these receptors may contribute to the sedative properties of ethanol.

General anesthetics produce a constellation of behavioral responses and widespread neurodepression. Recent studies have implicated the gamma-aminobutyric acid (GABA) subtype A receptor as a primary anesthetic target. During the past decade, considerable progress has been made in dissecting the behavioral effects of anesthetics according to the subunit composition of GABA(A) receptors. In this review, we describe how particular GABA(A) receptor subtypes expressed in different brain regions are critical for the expression of behavioral endpoints, such as amnesia, sedation, and hypnosis.

GABAA receptors generate both phasic and tonic forms of inhibition. In hippocampal pyramidal neurons, GABAA receptors that contain the α5 subunit generate a tonic inhibitory conductance. The physiological role of this tonic inhibition is uncertain, although α5GABAA receptors are known to influence hippocampal-dependent learning and memory processes. Here we provide evidence that α5GABAA receptors regulate the strength of the depolarizing stimulus that is required to generate an action potential in pyramidal neurons. Neurons from α5 knock-out (α5−/−) and wild-type (WT) mice were studied in brain slices and cell cultures using whole cell and perforated-patch-clamp techniques. Membrane resistance was 1.6-fold greater in α5−/− than in WT neurons, but the resting membrane potential and chloride equilibrium potential were similar. Membrane hyperpolarization evoked by an application of exogenous GABA was greater in WT neurons. Inhibiting the function of α5GABAA receptor with nonselective (picrotoxin) or α5 subunit-selective (L-655,708) compounds depolarized WT neurons by ∼3 mV, whereas no change was detected in α5−/− neurons. The depolarizing current required to generate an action potential was twofold greater in WT than in α5−/− neurons, whereas the slope of the input-output relationship for action potential firing was similar. We conclude that shunting inhibition mediated by α5GABAA receptors regulates the firing of action potentials and may synchronize network activity that underlies hippocampal-dependent behavior.

Memory reconsolidation is a protein synthesis-dependent process that preserves, in some form, memories that have been destabilized through recall. Reconsolidation is a nearly universal phenomenon, occurring in a diverse array of species and learning tasks. The function of reconsolidation remains unclear but it has been proposed as a mechanism for updating or strengthening memories. Observations of an analog of reconsolidation in vitro and in sensory systems indicate that reconsolidation is unlikely to be a learning-specific phenomenon and may serve a broader function. We propose that reconsolidation arises from the activity-dependent induction of two coincident but opposing processes: the depotentiation and repotentiation of strengthened synapses. These processes suggest that reconsolidation reflects a fundamental mechanism that regulates and preserves synaptic strength.

Hyperalgesia arising from sensitization of pain relays in the spinal dorsal horn shares many mechanistic and phenotypic parallels with memory formation. We discovered that mechanical hyperalgesia could be rendered labile and reversible in mice after reactivation of spinal pain pathways in a process analogous to memory reconsolidation. These findings reveal a previously unknown regulatory mechanism underlying hyperalgesia and demonstrate the existence of reconsolidation-like processes in a sensory system.

Background The measurement of mechanosensitivity is a key method for the study of pain in animal models. This is often accomplished with the use of von Frey filaments in an up-down testing paradigm. The up-down method described by Chaplan et al. (J Neurosci Methods 53:55–63, 1994) for mechanosensitivity testing in rodents remains one of the most widely used methods for measuring pain in animals. However, this method results in animals receiving a varying number of stimuli, which may lead to animals in different groups receiving different testing experiences that influences their later responses. To standardize the measurement of mechanosensitivity we developed a simplified up-down method (SUDO) for estimating paw withdrawal threshold (PWT) with von Frey filaments that uses a constant number of five stimuli per test. We further refined the PWT calculation to allow the estimation of PWT directly from the behavioral response to the fifth stimulus, omitting the need for look-up tables. Results The PWT estimates derived using SUDO strongly correlated (r > 0.96) with the PWT estimates determined with the conventional up-down method of Chaplan et al., and this correlation remained very strong across different levels of tester experience, different experimental conditions, and in tests from both mice and rats. The two testing methods also produced similar PWT estimates in prospective behavioral tests of mice at baseline and after induction of hyperalgesia by intraplantar capsaicin or complete Freund’s adjuvant. Conclusion SUDO thus offers an accurate, fast and user-friendly replacement for the widely used up-down method of Chaplan et al.

Neuronal inhibition in nociceptive relays of the spinal cord is essential for the proper processing of nociceptive information. In the spinal cord dorsal horn, the activity of synaptic and extrasynaptic GABAA and glycine receptors generates rapid, Cl(-)-dependent neuronal inhibition. A loss of this ionotropic inhibition, particularly through the collapse of the inhibitory Cl(-)-gradient, is a key mechanism by which pathological pain conditions develop. This review summarizes the roles of ionotropic inhibition in the regulation of nociception, and explores recent evidence that the potentiation of GABAA or glycine receptor activity or the enhancement of inhibitory drive can reverse pathological pain.

The notion that drug treatments can improve memory performance has moved from the realm of science fiction to that of serious investigation. A popular working hypothesis is that cognition can be improved by altering the balance between excitatory and inhibitory neurotransmission. This review focuses on the unique physiological and pharmacological properties of GABAARs [GABA (γ-aminobutyric acid) subtype A receptors] that contain the α5 subunit (α5-GABAAR), as these receptors serve as candidate targets for memory-enhancing drugs. Abbreviations: GABA, γ-aminobutyric acid; GABAAR, GABA subtype A receptor; LTP, long-term potentiation

The mechanisms by which insulin modulates neuronal plasticity and pain processes remain poorly understood. Here we report that insulin rapidly increases the function of glycine receptors in murine spinal neurons and recombinant human glycine receptors expressed in human embryonic kidney cells. Whole-cell patch-clamp recordings showed that insulin reversibly enhanced current evoked by exogenous glycine and increased the amplitude of spontaneous glycinergic miniature inhibitory postsynaptic currents recorded in cultured spinal neurons. Insulin (1 microM) also shifted the glycine concentration-response plot to the left and reduced the glycine EC(50) value from 52 to 31 microM. Currents evoked by a submaximal concentration of glycine were increased to approximately 140% of control. The glycine receptor alpha subunit was sufficient for the enhancement by insulin because currents from recombinant homomeric alpha(1) receptors and heteromeric alpha(1)beta receptors were both increased. Insulin acted at the insulin receptor via pathways dependent on tyrosine kinase and phosphatidylinositol 3 kinase because the insulin effect was eliminated by the insulin receptor antagonist, hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester, the tyrosine kinase inhibitor lavendustin A, and the phosphatidylinositol 3 kinase antagonist wortmannin. Together, these results show that insulin has a novel regulatory action on the potency of glycine for ionotropic glycine receptors.

Background The mechanisms underlying the therapeutic actions of gabapentin remain poorly understood. The chemical structure and behavioral properties of gabapentin strongly suggest actions on inhibitory neurotransmission mediated by gamma-aminobutyric acid (GABA); however, gabapentin does not directly modulate GABAA or GABAB receptors. Two distinct forms of GABAergic inhibition occur in the brain: postsynaptic conductance and a persistent tonic inhibitory conductance primarily generated by extrasynaptic GABAA receptors. The aim of this study was to determine whether gabapentin increased the tonic conductance in hippocampal neurons in vitro. As a positive control, the effects of vigabatrin, which irreversibly inhibits GABA transaminase, were also examined. Methods GABAA receptors in hippocampal neurons from embryonic mice were studied using whole cell patch clamp recordings. Miniature inhibitory postsynaptic currents and the tonic current were recorded from cultured neurons that were treated for 36-48 h with gabapentin, vigabatrin, or gabapentin and vigabatrin. To determine whether gabapentin increased the expression of GABAA receptors, Western blots were stained with antibodies selective for alpha1, alpha2, and alpha5 subunits. Results GABAA receptors were insensitive to the acute application of gabapentin, whereas chronic treatment increased the amplitude of the tonic current threefold (EC50 = 209 microm) but did not influence miniature inhibitory postsynaptic currents. Vigabatrin increased the tonic conductance, and the maximally effective concentration did not occlude the actions of gabapentin, which suggests that these compounds act by different mechanisms. Neither gabapentin nor vigabatrin increased the expression of GABAA receptors in the neurons. Conclusions Gabapentin increases a tonic inhibitory conductance in mammalian neurons. High-affinity GABAA receptors that generate the tonic conductance may detect small increases in the ambient concentration of neurotransmitter caused by gabapentin.