Experimental colitis-induced visceral hypersensitivity is attenuated by GABA treatment in mice.

Ulcerative colitis (UC) is linked with inflammation of the large intestine due to an overactive response of the colon-immune system. UC is associated with weight loss, rectal bleeding, diarrhea, and abdominal pain. Given that γ-amino butyric acid (GABA) suppresses immune cell activity and the excitability of colonic afferents, and that there is a decrease in colonic GABA during UC, we hypothesized that UC pain is due to a decrease in the inhibition of colonic afferents. Thus, restoring GABA in the colon will attenuate inflammatory hypersensitivity. We tested this hypothesis in a mouse model of colitis. Colon inflammation was induced with seven days of dextran sodium sulfate (DSS, 3%) in the drinking water. GABA (40 mg/kg) was administered orally for the same period as DSS, and body weight, colon length, colon permeability, clinical progression of colitis (disease activity index or DAI), and colon histological score (HS) were assessed to determine the effects of GABA on colitis. A day after the end of GABA treatment, visceral sensitivity was assessed with balloon distention (of the colon)-evoked visceromotor response and colon samples were collected for the measurement of GABA and cytokines. Treatment with GABA reduced the DSS-induced increase in the colon permeability, DAI, HS, and decrease in body weight and colon length. Furthermore, GABA inhibited the DSS-induced increase in the proinflammatory cytokines tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-12 (IL-12), and increased the expression of the anti-inflammatory cytokine IL-10 in the colon tissue. Importantly, GABA reduced DSS-induced visceral hypersensitivity. These data suggest that increasing gastrointestinal levels of GABA may be useful for the treatment of colitis.NEW & NOTEWORTHY GABA treatment reduces the severity of colitis and inflammation and produces inhibition of visceral hypersensitivity in colon-inflamed mice. These results raise the promising possibility that GABA treatment may be an effective therapeutic strategy for the management of symptoms associated with colitis. However, clinical studies are required to corroborate whether this mouse-model data translates to human colon.

Mechanisms Underlying the Selective Therapeutic Efficacy of Carbamazepine for Attenuation of Trigeminal Nerve Injury Pain.

Different peripheral nerve injuries cause neuropathic pain through distinct mechanisms. Even the site of injury may impact underlying mechanisms, as indicated by the clinical finding that the antiseizure drug carbamazepine (CBZ) relieves pain because of compression injuries of trigeminal but not somatic nerves. We leveraged this observation in the present study hypothesizing that because CBZ blocks voltage-gated sodium channels (VGSCs), its therapeutic selectivity reflects differences between trigeminal and somatic nerves with respect to injury-induced changes in VGSCs. CBZ diminished ongoing and evoked pain behavior in rats with chronic constriction injury (CCI) to the infraorbital nerve (ION) but had minimal effect in rats with sciatic nerve CCI. This difference in behavior was associated with a selective increase in the potency of CBZ-induced inhibition of compound action potentials in the ION, an effect mirrored in human trigeminal versus somatic nerves. The increase in potency was associated with a selective increase in the efficacy of the NaV1.1 channel blocker ICA-121431 and NaV1.1 protein in the ION, but no change in NaV1.1 mRNA in trigeminal ganglia. Importantly, local ICA-121431 administration reversed ION CCI-induced hypersensitivity. Our results suggest a novel therapeutic target for the treatment of trigeminal neuropathic pain.SIGNIFICANCE STATEMENT This study is based on evidence of differences in pain and its treatment depending on whether the pain is above (trigeminal) or below (somatic) the neck, as well as evidence that voltage-gated sodium channels (VGSCs) may contribute to these differences. The focus of the present study was on channels underlying action potential propagation in peripheral nerves. There were differences between somatic and trigeminal nerves in VGSC subtypes underlying action potential propagation both in the absence and presence of injury. Importantly, because the local block of NaV1.1 in the trigeminal nerve reverses nerve injury-induced mechanical hypersensitivity, the selective upregulation of NaV1.1 in trigeminal nerves suggests a novel therapeutic target for the treatment of pain associated with trigeminal nerve injury.

Peripheral GABA receptors regulate colonic afferent excitability and visceral nociception.

KEY POINTS: While the presence of GABA receptors on primary afferents has been well described, most functional analyses have focused on the regulation of transmitter release from central terminals and/or signalling in the sensory neuron cell body. Evidence that GABA receptors are transported to peripheral terminals and that there are several sources of GABA in the colon raise the possibility that GABA signalling in the periphery may influence colonic afferent excitability. GABAA and GABAB are present and functional in the colon, where exogenous agonists decrease the excitability of colonic afferents and suppress visceral nociception. Endogenous GABA release within the colon is sufficient to establish the resting excitability of colonic afferents as well as the behavioural response to noxious stimulation of the colon, primarily via GABAA receptors. Peripheral GABA receptors may serve as a viable target for the treatment of visceral pain. ABSTRACT: It is well established that GABA receptors at the central terminals of primary afferent fibres regulate afferent input to the superficial dorsal horn. However, the extent to which peripheral GABA signalling may also regulate afferent input remains to be determined. The colon was used to explore this issue because of the numerous endogenous sources of GABA that have been described in this tissue. The influence of GABA signalling on colonic afferent excitability was assessed in an ex vivo mouse colorectum pelvic nerve preparation where test compounds were applied to the receptive field. The visceromotor response (VMR) evoked by noxious colorectal distension was used to assess the impact of GABA signalling on visceral nociception, where test compounds were applied directly to the colon. Application of either GABAA or GABAB receptor agonists attenuated the colonic afferent response to colon stretch. Conversely, GABAA and GABAB receptor antagonists increased the stretch response. However, while the noxious distension-induced VMR was attenuated in the presence of GABAA and GABAB receptor agonists, the VMR was only consistently increased by GABAA receptor antagonists. These results suggest that GABA receptors are present and functional in the peripheral terminals of colonic afferents and activation of these receptors via endogenous GABA release contributes to the establishment of colonic afferent excitability and visceral nociception. These results suggest that increasing peripheral GABA receptor signalling could be used to treat visceral pain.

Role of anoctamin-1 and bestrophin-1 in spinal nerve ligation-induced neuropathic pain in rats.

BACKGROUND: Calcium-activated chloride channels (CaCCs) activation induces membrane depolarization by increasing chloride efflux in primary sensory neurons that can facilitate action potential generation. Previous studies suggest that CaCCs family members bestrophin-1 and anoctamin-1 are involved in inflammatory pain. However, their role in neuropathic pain is unclear. In this investigation we assessed the involvement of these CaCCs family members in rats subjected to the L5/L6 spinal nerve ligation. In addition, anoctamin-1 and bestrophin-1 mRNA and protein expression in dorsal root ganglion (DRG) and spinal cord was also determined in the presence and absence of selective inhibitors. RESULTS: L5/L6 spinal nerve ligation induced mechanical tactile allodynia. Intrathecal administration of non-selective CaCCs inhibitors (NPPB, 9-AC and NFA) dose-dependently reduced tactile allodynia. Intrathecal administration of selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) also dose-dependently diminished tactile allodynia and thermal hyperalgesia. Anoctamin-1 and bestrophin-1 mRNA and protein were expressed in the dorsal spinal cord and DRG of naïve, sham and neuropathic rats. L5/L6 spinal nerve ligation rose mRNA and protein expression of anoctamin-1, but not bestrophin-1, in the dorsal spinal cord and DRG from day 1 to day 14 after nerve ligation. In addition, repeated administration of CaCCs inhibitors (T16Ainh-A01, CaCCinh-A01 or NFA) or anti-anoctamin-1 antibody prevented spinal nerve ligation-induced rises in anoctamin-1 mRNA and protein expression. Following spinal nerve ligation, the compound action potential generation of putative C fibers increased while selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) attenuated such increase. CONCLUSIONS: There is functional anoctamin-1 and bestrophin-1 expression in rats at sites related to nociceptive processing. Blockade of these CaCCs suppresses compound action potential generation in putative C fibers and lessens established tactile allodynia. As CaCCs activity contributes to neuropathic pain maintenance, selective inhibition of their activity may function as a tool to generate analgesia in nerve injury pain states.

α(5)GABA(A) receptors mediate primary afferent fiber tonic excitability in the turtle spinal cord.

γ-Amino butyric acid (GABA) plays a key role in the regulation of central nervous system by activating synaptic and extrasynaptic GABAA receptors. It is acknowledged that extrasynaptic GABAA receptors located in the soma, dendrites, and axons may be activated tonically by low extracellular GABA concentrations. The activation of these receptors produces a persistent conductance that can hyperpolarize or depolarize nerve cells depending on the Cl(-) equilibrium potential. In an in vitro preparation of the turtle spinal cord we show that extrasynaptic α5GABAA receptors mediate the tonic state of excitability of primary afferents independently of the phasic primary afferent depolarization mediated by synaptic GABAA receptors. Blockade of α5GABAA receptors with the inverse agonist L-655,708 depressed the dorsal root reflex (DRR) without affecting the phasic increase in excitability of primary afferents. Using RT-PCR and Western blotting, we corroborated the presence of the mRNA and the α5GABAA protein in the dorsal root ganglia of the turtle spinal cord. The receptors were localized in primary afferents in dorsal root, dorsal root ganglia, and peripheral nerve terminals using immunoconfocal microscopy. Considering the implications of the DRR in neurogenic inflammation, α5GABAA receptors may serve as potential pharmacological targets for the treatment of pain.

Peripheral GABAA receptor signaling contributes to visceral hypersensitivity in a mouse model of colitis.

ABSTRACT: Pain is a common and debilitating symptom of inflammatory bowel disease (IBD). Based on evidence that peripheral GABAA receptor (GAR) inhibition plays an important role in establishing colonic afferent excitability and nociceptive threshold, we hypothesized that the increase in pain associated with IBD is due to, at least in part, a decrease in peripheral GAR-mediated inhibition. Acute colitis was induced with 5 days of dextran sodium sulfate (DSS, 3%) in the drinking water. Visceral sensitivity was assessed with the visceromotor response (VMR) evoked with balloon distention of the colon in control and DSS-treated mice before and after intracolonic administration of GAR agonist muscimol, the high-affinity GAR preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP), the GAR positive allosteric modulator diazepam, or the GAR antagonists gabazine and bicuculline. Low concentrations of muscimol or THIP increased the VMR in DSS-treated mice but not in control mice. However, high concentrations of muscimol decreased the VMR in both control and DSS-treated mice. Diazepam decreased the VMR in both DSS-treated and control mice. By contrast, at a concentration of gabazine that blocks only low-affinity GAR, there was no effect on the VMR in either DSS-treated or control mice, but at concentrations of the antagonist that block low-affinity and high-affinity GAR, the VMR was increased in control mice and decreased in DSS-treated mice. Furthermore, bicuculline increased the VMR in control mice but decreased it in DSS-treated mice. These data suggest that activating of low-affinity GAR or blocking high-affinity GAR may be effective therapeutic strategies for the management of pain in IBD.

Voltage-gated calcium currents in human dorsal root ganglion neurons.

ABSTRACT: Voltage-gated calcium channels in sensory neurons underlie processes ranging from neurotransmitter release to gene expression and remain a therapeutic target for the treatment of pain. Yet virtually all we know about voltage-gated calcium channels has been obtained through the study of rodent sensory neurons and heterologously expressed channels. To address this, high voltage-activated (HVA) Ca2+ currents in dissociated human and rat dorsal root ganglion neurons were characterized with whole-cell patch clamp techniques. The HVA currents from both species shared basic biophysical and pharmacological properties. However, HVA currents in human neurons differed from those in the rat in at least 3 potentially important ways: (1) Ca2+ current density was significantly smaller, (2) the proportion of nifedipine-sensitive currents was far greater, and (3) a subpopulation of human neurons displayed relatively large constitutive current inhibition. These results highlight the need to for the study of native proteins in their native environment before initiating costly clinical trials.

Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors.

GABA(A) receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α(5) has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α(5) subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α(5) subunit-containing GABA(A) receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α(4/6) subunit-containing GABA(A) receptors, but is sensitive to L-655708, an antagonist of the GABA(A) receptors containing α(5) subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α(5) subunit in the turtle spinal cord. Together, these results suggest that GABA(A) receptors containing the α(5) subunit mediate the tonic inhibitory currents observed in VHIs.

γ-Aminobutyric acid (GABA) from satellite glial cells tonically depresses the excitability of primary afferent fibers.

Primary afferent fibers express extrasynaptic GABAA and GABAB receptors in the axons and soma. However, whether these receptors are tonically activated by ambient GABA and the source of the neurotransmitter is presently unknown. Here, we show that GABA release from dorsal root ganglia (DRG) does not depend on extracellular calcium, but depends upon calcium released from intracellular stores, and is mediated by Best1 channels. Using a preparation consisting of the spinal nerve in continuity with the DRG and the dorsal root, we found that endogenous GABA tonically activates GABA receptors, depressing the excitability of the primary afferents. In addition, using HPLC we found that GABA is released in the DRG, and by immunofluorescence microscopy we show the presence of GABA, the Best1 channel, and some enzymes of the putrescine pathway of GABA biosynthesis, in glutamine synthase- and GFAP-positive satellite glial cells. Last, we found that the blockade of the Best1 channel activity reduced the excitability of primary afferents and prevented the activation of the GABA receptors. These results suggest that satellite glial cells may be the source of endogenous GABA released in the DRG via Best1 channels, which tonically activates extrasynaptic GABA receptors.

Optogenetic inhibition of the colon epithelium reduces hypersensitivity in a mouse model of inflammatory bowel disease.

ABSTRACT: Visceral pain is a prevalent symptom of inflammatory bowel disease that can be difficult to treat. Pain and hypersensitivity are mediated by extrinsic primary afferent neurons (ExPANs) that innervate the colon. Recent studies indicate that the colon epithelium contributes to initiating ExPAN firing and nociceptive responses. Based on these findings, we hypothesized that the epithelium contributes to inflammation-induced hypersensitivity. A key prediction of this hypothesis is that inhibition of the epithelium would attenuate nociceptive signaling and inflammatory hypersensitivity. To test this hypothesis, the inhibitory yellow light-activated protein archaerhodopsin was targeted to the intestinal epithelium (villin-Arch) or the ExPANs (TRPV1-Arch) that innervate the colon. Visceral sensitivity was assessed by measuring the visceromotor response (VMR) to colorectal distension (CRD), with and without yellow light illumination of the colon lumen. Inhibition of the colon epithelium in healthy villin-Arch mice significantly diminished the CRD-induced VMR. Direct inhibition of ExPANs during CRD using TRPV1-Arch mice showed that ExPAN and epithelial inhibition were similarly effective in reducing the VMR to CRD. We then investigated the effect of epithelial and ExPAN inhibition in the dextran sulfate sodium model of inflammatory bowel disease. Inhibition of the colon epithelium significantly decreased dextran sulfate sodium-induced hypersensitivity and was comparable with the inhibition of ExPANs. Together, these results reveal the potential of targeting the colon epithelium for the treatment of pain.

Distribution of functional opioid receptors in human dorsal root ganglion neurons.

Preclinical evidence has highlighted the importance of the µ-opioid peptide (MOP) receptor on primary afferents for both the analgesic actions of MOP receptor agonists, as well as the development of tolerance, if not opioid-induced hyperalgesia. There is also growing interest in targeting other opioid peptide receptor subtypes (δ-opioid peptide [DOP], κ-opioid peptide [KOP], and nociceptin/orphanin-FQ opioid peptide [NOP]) on primary afferents, as alternatives to MOP receptors, which may not be associated with as many deleterious side effects. Nevertheless, results from several recent studies of human sensory neurons indicate that although there are many similarities between rodent and human sensory neurons, there may also be important differences. Thus, the purpose of this study was to assess the distribution of opioid receptor subtypes among human sensory neurons. A combination of pharmacology, patch-clamp electrophysiology, Ca imaging, and single-cell semiquantitative polymerase chain reaction was used. Our results suggest that functional MOP-like receptors are present in approximately 50% of human dorsal root ganglion neurons. δ-opioid peptide-like receptors were detected in a subpopulation largely overlapping that with MOP-like receptors. Furthermore, KOP-like and NOP-like receptors are detected in a large proportion (44% and 40%, respectively) of human dorsal root ganglion neurons with KOP receptors also overlapping with MOP receptors at a high rate (83%). Our data confirm that all 4 opioid receptor subtypes are present and functional in human sensory neurons, where the overlap of DOP, KOP, and NOP receptors with MOP receptors suggests that activation of these other opioid receptor subtypes may also have analgesic efficacy.

Evaluation of the neonatal streptozotocin model of diabetes in rats: Evidence for a model of neuropathic pain.

BACKGROUND: The purpose of this study was to evaluate the participation of satellite glial cells (SGC), microglia and astrocytes in a model of streptozotocin-induced diabetes initiated in neonatal rats (nSTZ) and to determine the pharmacological profile for pain relief. METHODS: nSTZ was used to induce experimental diabetes. Von Frey filaments were used to assess tactile allodynia. Drugs were given by systemic administration. Western blotting and immunohistochemistry were used to determine protein expression and cellular localization. RESULTS: nSTZ produced mild hyperglycemia, weight loss, glucose intolerance, and reduction of nerve conduction velocity of C fibers. Moreover, nSTZ enhanced activating transcription factor 3 (ATF3) immunoreactivity in dorsal root ganglia (DRG) and sciatic nerve of adult rats. ATF3 was found in SGC (GFAP+ cells) surrounding DRG at week 16. Late changes in ATF3 immunoreactivity in DRG correlated with up-regulation of ATF3 and GFAP protein expression. nSTZ increased GFAP and OX-42 immunoreactivity and percentage of hypertrophied and ameboid microglia in the spinal dorsal horn. These changes correlated with the presence of mechanical hypersensitivity (tactile allodynia). Administration of gabapentin (30-100mg/kg, po) and metformin (200mg/kg/day, po for 2 weeks) alleviated tactile allodynia, whereas morphine (1-3mg/kg, ip) had a modest effect. CONCLUSIONS: Results suggest that nSTZ leads to activation of SGC, microglia and astrocytes in DRG and spinal cord. Pharmacological profile in the nSTZ model resembles diabetic neuropathic pain in humans. Our findings support the conclusion that the nSTZ rat model has utility for the study of a long-lasting diabetic neuropathic pain.

Kappa Opioid Receptor Distribution and Function in Primary Afferents.

Primary afferents are known to be inhibited by kappa opioid receptor (KOR) signaling. However, the specific types of somatosensory neurons that express KOR remain unclear. Here, using a newly developed KOR-cre knockin allele, viral tracing, single-cell RT-PCR, and ex vivo recordings, we show that KOR is expressed in several populations of primary afferents: a subset of peptidergic sensory neurons, as well as low-threshold mechanoreceptors that form lanceolate or circumferential endings around hair follicles. We find that KOR acts centrally to inhibit excitatory neurotransmission from KOR-cre afferents in laminae I and III, and this effect is likely due to KOR-mediated inhibition of Ca2+ influx, which we observed in sensory neurons from both mouse and human. In the periphery, KOR signaling inhibits neurogenic inflammation and nociceptor sensitization by inflammatory mediators. Finally, peripherally restricted KOR agonists selectively reduce pain and itch behaviors, as well as mechanical hypersensitivity associated with a surgical incision. These experiments provide a rationale for the use of peripherally restricted KOR agonists for therapeutic treatment.

Tonically Active α5GABAA Receptors Reduce Motoneuron Excitability and Decrease the Monosynaptic Reflex.

Motoneurons, the final common path of the Central Nervous System (CNS), are under a complex control of its excitability in order to precisely translate the interneuronal pattern of activity into skeletal muscle contraction and relaxation. To fulfill this relevant function, motoneurons are provided with a vast repertoire of receptors and channels, including the extrasynaptic GABAA receptors which have been poorly investigated. Here, we confirmed that extrasynaptic α5 subunit-containing GABAA receptors localize with choline acetyltransferase (ChAT) positive cells, suggesting that these receptors are expressed in turtle motoneurons as previously reported in rodents. In these cells, α5GABAA receptors are activated by ambient GABA, producing a tonic shunt that reduces motoneurons' membrane resistance and affects their action potential firing properties. In addition, α5GABAA receptors shunted the synaptic excitatory inputs depressing the monosynaptic reflex (MSR) induced by activation of primary afferents. Therefore, our results suggest that α5GABAA receptors may play a relevant physiological role in motor control.

Functional expression of T-type Ca2+ channels in spinal motoneurons of the adult turtle.

Voltage-gated Ca2+ (CaV) channels are transmembrane proteins comprising three subfamilies named CaV1, CaV2 and CaV3. The CaV3 channel subfamily groups the low-voltage activated Ca2+ channels (LVA or T-type) a significant role in regulating neuronal excitability. CaV3 channel activity may lead to the generation of complex patterns of action potential firing such as the postinhibitory rebound (PIR). In the adult spinal cord, these channels have been found in dorsal horn interneurons where they control physiological events near the resting potential and participate in determining excitability. In motoneurons, CaV3 channels have been found during development, but their functional expression has not yet been reported in adult animals. Here, we show evidence for the presence of CaV3 channel-mediated PIR in motoneurons of the adult turtle spinal cord. Our results indicate that Ni2+ and NNC55-0396, two antagonists of CaV3 channel activity, inhibited PIR in the adult turtle spinal cord. Molecular biology and biochemical assays revealed the expression of the CaV3.1 channel isotype and its localization in motoneurons. Together, these results provide evidence for the expression of CaV3.1 channels in the spinal cord of adult animals and show also that these channels may contribute to determine the excitability of motoneurons.

Extrasynaptic GABA(A) receptors in the brainstem and spinal cord: structure and function.

γ-aminobutyric acid (GABA) plays many of its key roles in embryonic development and functioning of the central nervous system (CNS) by acting on ligand gated chloride-permeable channels known as GABAA receptors (GABAAR). Classically, GABAARmediated synaptic communication is tailored to allow rapid and precise transmission of information to synchronize the activity of large populations of cells to generate and maintain neuronal networks oscillations. An alternative type of inhibition mediated by GABAA receptors, initially described about 25 years ago, is characterized by a tonic activation of receptors that react to ambient extracellular GABA. The receptors that mediate this action are wide-spread throughout the nerve cells but are located distant from the sites of GABA release, and therefore they have been called extrasynaptic GABAA receptors. The molecular nature of the extrasynaptic GABAA receptors and the tonic inhibitory current they generate have been characterized in many brain structures, and due to its relevance in controlling neuron excitability they have become attractive pharmacological targets for a variety of neurological disorders such as schizophrenia, epilepsy and Parkinson disease. In the spinal cord, early studies have implicated these receptors in anesthesia, chronic pain, motor control, and locomotion. This review highlights past and present developments in the field of extrasynaptic GABAA receptors and emphasizes their subunit containing distribution and physiological role in the spinal cord.