Mechanisms of neurokinin₁ receptor action in the dorsal horn of the spinal cord

This study addressed the role of neurokinin] (NKi) receptors in nociceptive transmission and their participation in a series of events involving glycine and NMDA receptor-mediated effects on spinal neurones. Using an in vivo electrophysiology protocol utilising ionophoresis and extracellular recording from laminae III-V dorsal horn neurones of anaesthetised rats, the mechanisms of these interactions were assessed. The functions of the inflammatory cytokine leukaemia inhibitory factor (LIF) were also considered. Injury-induced alterations in the spinal expression pattern of this factor and the consequences of these changes to neuropeptide and excitatory amino acid expression were measured using in situ hybridisation.1. The involvement of NK] receptors in spinal pain transmission may be dependent upon the duration and intensity of the nociceptive stimulus. Since activation of spinal NK] receptors leads to increases in the concentration of glycine in the dorsal horn, the role of the inhibitory glycine receptor as a regulator of NKj receptor function was investigated. Ionophoretic application of GR82334, a selective NK] receptor antagonist did not alter activity evoked by cutaneous applications of mustard oil. Flowever in the presence of the glycine antagonists strychnine or phenylbenzene-co-phosphono- a-amino acid (PMBA), GR82334 displayed inhibitory properties. Therefore inhibitory glycine receptors may mask the contribution made by NK] receptors to nociceptive processing. This is discussed with reference to the role of NK] receptors during brief nociceptive transmission.2. NK] receptors can contribute to the processing of sustained nociceptive stimuli by modulating excitatory amino acid-mediated transmission, particularly through potentiation of NMDA receptor activity. Experiments were carried out to investigate the possible role of the glycine site of the NMDA receptor (GlyNMDA) and of protein kinase C (PKC) activation to the NK]/NMDA interaction.Ionophoresis of the NMDA receptor agonist 1 -aminocyclobutane-m-1,3-dicarboxylic acid (ACBD) produced a sustained increase in the firing rate of dorsal horn neurones that was facilitated by the NK] agonist acetyl-[Arg6,Sar9,Met(02)11]-SP6-ii (Sar9-SP). GlyNMDA site antagonists 2-carboxy-4,6-dichloro-(lH)-indole-3-propanoic acid (MDL 29951), 7-chloro-3- (cyclopropylcarbonyl)-4-hydroxy-2(lH)-quinoline (L701,252), 5,7-dinitroquinaxoline-2,3- dione (MNQX) and 7-chlorothiokynurenic acid (7-CTK) or PKC inhibitors, chelerythrine and GF109203X significantly reduced this facilitation whilst having no effect on activity driven by ACBD alone. Like the NK] receptor agonist, a selective GlyNMDA site agonist 1- aminocyclopropanecarboxylic acid (ACPC) also caused facilitation of ACBD-evoked activity and this was inhibited by the GlyNMDA site antagonist L701,252 in a similar manner. These data suggest that NKi receptors facilitate NMDA receptor by potentiating the positive influence of the GlyNMDA site. This may be bought about by the NKi receptor-induced glycine release and/or by a PKC mediated increase in the sensitivity of the GlyNMDA site. This demonstrates that co-transmitters released from fine primary afferent fibres may interact postsynaptically to alter central hyperexcitability, particularly that associated with more prolonged noxious events.3. LIF is a neuroactive cytokine that is associated with peripheral nerve injury. Using in situ hybridisation, the present study has examined the distribution of LIF mRNA in the spinal cord, normally or following peripheral inflammation or nerve injury and determined the consequences of intrathecally applied LIF on spinal expression of NK, receptor and the high affinity glutamate transporter, EAAT2.In control animals, dorsal horn expression of LIF was high in superficial laminae I-II with only light expression in the deeper laminae III-V and in the ventral horn. Both peripheral inflammation and neuropathy significantly increased levels of LIF mRNA in the dorsal horn and this was most evident in the lateral parts of laminae I and II. NKi and EAAT2 expression was normally associated with cells in laminae I-II, IV-V and ventral horn motorneurones. Intrathecal LIF administration significantly increased this expression for all three mRNAs and resulted in dense hybridisation throughout the dorsal horn.These results show that LIF is normally expressed in the spinal cord in a specific laminar pattern that increases dramatically following peripheral inflammation or nerve damage. Furthermore, LIF upregulates the expression of NKi receptors and EAAT2 transporters indicating that it may be a critical regulator of the central changes that occur following peripheral injury.Interactions within the spinal cord may underlie the plasticity of the dorsal horn in sensory processing. This has been discussed with reference to the regulation of short-term co¬ operation between NIG and NMDA receptors by glycine and to long-term modifications of peptide and excitatory amino acid neurotransmission by altered LIF gene expression

Cutaneous nociceptors lack sensitisation, but reveal \u3bc-opioid receptor-mediated reduction in excitability to mechanical stimulation in neuropathy

Background: Peripheral nerve injuries often trigger a hypersensitivity to tactile stimulation. Behavioural studies demonstrated efficient and side effect-free analgesia mediated by opioid receptors on peripheral sensory neurons. However, mechanistic approaches addressing such opioid properties in painful neuropathies are lacking. Here we investigated whether opioids can directly inhibit primary afferent neuron transmission of mechanical stimuli in neuropathy. We analysed the mechanical thresholds, the firing rates and response latencies of sensory fibres to mechanical stimulation of their cutaneous receptive fields.Results: Two weeks following a chronic constriction injury of the saphenous nerve, mice developed a profound mechanical hypersensitivity in the paw innervated by the damaged nerve. Using an in vitro skin-nerve preparation we found no changes in the mechanical thresholds and latencies of sensory fibres from injured nerves. The firing rates to mechanical stimulation were unchanged or reduced following injury. Importantly, \u3bc-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5]-ol-enkephalin (DAMGO) significantly elevated the mechanical thresholds of nociceptive A\u3b4 and C fibres. Furthermore, DAMGO substantially diminished the mechanically evoked discharges of C nociceptors in injured nerves. These effects were blocked by DAMGO washout and pre-treatment with the selective \u3bc-opioid receptor antagonist Cys2-Tyr3-Orn5-Pen7-amide. DAMGO did not alter the responses of sensory fibres in uninjured nerves.Conclusions: Our findings suggest that behaviourally manifested neuropathy-induced mechanosensitivity does not require a sensitised state of cutaneous nociceptors in damaged nerves. Yet, nerve injury renders nociceptors sensitive to opioids. Prevention of action potential generation or propagation in nociceptors might represent a cellular mechanism underlying peripheral opioid-mediated alleviation of mechanical hypersensitivity in neuropathy. \ua9 2012 Schmidt et al.; licensee BioMed Central Ltd

The contribution of TRPM8 and TRPA1 channels to cold allodynia and neuropathic pain.

Cold allodynia is a common feature of neuropathic pain however the underlying mechanisms of this enhanced sensitivity to cold are not known. Recently the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified and proposed to be molecular sensors for cold. Here we have investigated the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG) and examined the cold sensitivity of peripheral sensory neurons in the chronic construction injury (CCI) model of neuropathic pain in mice.In behavioral experiments, chronic constriction injury (CCI) of the sciatic nerve induced a hypersensitivity to both cold and the TRPM8 agonist menthol that developed 2 days post injury and remained stable for at least 2 weeks. Using quantitative RT-PCR and in situ hybridization we examined the expression of TRPM8 and TRPA1 in DRG. Both channels displayed significantly reduced expression levels after injury with no change in their distribution pattern in identified neuronal subpopulations. Furthermore, in calcium imaging experiments, we detected no alterations in the number of cold or menthol responsive neurons in the DRG, or in the functional properties of cold transduction following injury. Intriguingly however, responses to the TRPA1 agonist mustard oil were strongly reduced.Our results indicate that injured sensory neurons do not develop abnormal cold sensitivity after chronic constriction injury and that alterations in the expression of TRPM8 and TRPA1 are unlikely to contribute directly to the pathogenesis of cold allodynia in this neuropathic pain model

Microglial refinement of A-fibre projections in the postnatal spinal cord dorsal horn is required for normal maturation of dynamic touch

Sensory systems are shaped in postnatal life by the refinement of synaptic connections. In the dorsal horn of the spinal cord, sensory circuits undergo postnatal activity dependent reorganisation, including the retraction of primary afferent A-fibres from superficial to deeper laminae which is accompanied by decreases in cutaneous sensitivity. Here we show that microglia, the resident immune cells in the CNS, phagocytose A-fibre terminals in superficial laminae in the first weeks of life. Genetic perturbation of microglial engulfment at that time prevents the normal process of A-fibre retraction, resulting in increased sensitivity of dorsal horn cells to dynamic tactile cutaneous stimulation, and behavioural hypersensitivity to dynamic touch. Thus, functional microglia are necessary for normal postnatal development of dorsal horn sensory circuits. In the absence of microglial engulfment, superfluous A-fibre projections remain in the dorsal horn and the balance of sensory connectivity is disrupted, leading to lifelong hypersensitivity to dynamic touch

Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons

A complex of stomatin-family proteins and acid-sensing (proton-gated) ion channel (ASIC) family members participate in sensory transduction in invertebrates and vertebrates. Here, we have examined the role of the stomatin-family protein stomatin-like protein-3 (STOML3) in this process. We demonstrate that STOML3 interacts with stomatin and ASIC subunits and that this occurs in a highly mobile vesicle pool in dorsal root ganglia (DRG) neurons and Chinese hamster ovary cells. We identify a hydrophobic region in the N-terminus of STOML3 that is required for vesicular localization of STOML3 and regulates physical and functional interaction with ASICs. We further characterize STOML3-containing vesicles in DRG neurons and show that they are Rab11-positive, but not part of the early-endosomal, lysosomal or Rab14-dependent biosynthetic compartment. Moreover, uncoupling of vesicles from microtubules leads to incorporation of STOML3 into the plasma membrane and increased acid-gated currents. Thus, STOML3 defines a vesicle pool in which it associates with molecules that have critical roles in sensory transduction. We suggest that the molecular features of this vesicular pool may be characteristic of a ‘transducosome’ in sensory neurons

Functional and Molecular Characterization of Mechanoinsensitive "Silent" Nociceptors.

Mechanical and thermal hyperalgesia (pain hypersensitivity) are cardinal signs of inflammation. Although the mechanism underlying thermal hyperalgesia is well understood, the cellular and molecular basis of mechanical hyperalgesia is poorly described. Here, we have identified a subset of peptidergic C-fiber nociceptors that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli when exposed to the inflammatory mediator nerve growth factor (NGF). Strikingly, NGF did not affect mechanosensitivity of other nociceptors. We show that these mechanoinsensitive "silent" nociceptors are characterized by the expression of the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) and that the mechanically gated ion channel PIEZO2 mediates NGF-induced mechanosensitivity in these neurons. Retrograde tracing revealed that CHRNA3+ nociceptors account for ∼50% of all peptidergic nociceptive afferents innervating visceral organs and deep somatic tissues. Hence, our data suggest that NGF-induced "un-silencing" of CHRNA3+ nociceptors significantly contributes to the development of mechanical hyperalgesia during inflammation

Generation and characterization of an Advillin-Cre driver mouse line.

Progress in the somatosensory field has been restricted by the limited number of genetic tools available to study gene function in peripheral sensory neurons. Here we generated a Cre-driver mouse line that expresses Cre-recombinase from the locus of the sensory neuron specific gene Advillin. These mice displayed almost exclusive Cre-mediated recombination in all peripheral sensory neurons. As such, the Advillin-Cre-driver line will be a powerful tool for targeting peripheral neurons in future investigations

A subpopulation of itch-sensing neurons marked by Ret and somatostatin expression

Itch, the unpleasant sensation that elicits a desire to scratch, is mediated by specific subtypes of cutaneous sensory neuron. Here, we identify a subpopulation of itch-sensing neurons based on their expression of the receptor tyrosine kinase Ret. We apply flow cytometry to isolate Ret-positive neurons from dorsal root ganglia and detected a distinct population marked by low levels of Ret and absence of isolectin B4 binding. We determine the transcriptional profile of these neurons and demonstrate that they express neuropeptides such as somatostatin (Sst), the NGF receptor TrkA, and multiple transcripts associated with itch. We validate the selective expression of Sst using an Sst-Cre driver line and ablated these neurons by generating mice in which the diphtheria toxin receptor is conditionally expressed from the sensory neuron-specific Avil locus. Sst-Cre::AviliDTR mice display normal nociceptive responses to thermal and mechanical stimuli. However, scratching behavior evoked by interleukin-31 (IL-31) or agonist at the 5HT1F receptor is significantly reduced. Our data provide a molecular signature for a subpopulation of neurons activated by multiple pruritogens. Synopsis This study shows that a subset of DRG neurons expressing the tyrosine kinase Ret and somatostatin function as itch receptor and mediate 5HT1f receptor agonist-induced scratching in mice. Flow cytometric analysis of peripheral sensory neurons is used to isolate multiple Ret-positive subsets. Transcriptional profiling of sensory neurons with low levels of Ret and an absence of IB4 binding reveals co-expression of somatostatin (Sst), interleukin-31 (IL-31) and serotonin receptor 5HT1f. Ablation of Sst-positive neurons reduces scratching responses to IL-31 and 5HT1f agonists in vivo. This study shows that a subset of DRG neurons expressing the tyrosine kinase Ret and somatostatin function as itch receptor and mediate 5HT1f receptor agonist-induced scratching in mice

microRNAs in nociceptive circuits as predictors of future clinical applications

Neuro-immune alterations in the peripheral and central nervous system play a role in the pathophysiology of chronic pain, and non-coding RNAs - and microRNAs (miRNAs) in particular - regulate both immune and neuronal processes. Specifically, miRNAs control macromolecular complexes in neurons, glia and immune cells and regulate signals used for neuro-immune communication in the pain pathway. Therefore, miRNAs may be hypothesized as critically important master switches modulating chronic pain. In particular, understanding the concerted function of miRNA in the regulation of nociception and endogenous analgesia and defining the importance of miRNAs in the circuitries and cognitive, emotional and behavioral components involved in pain is expected to shed new light on the enigmatic pathophysiology of neuropathic pain, migraine and complex regional pain syndrome. Specific miRNAs may evolve as new druggable molecular targets for pain prevention and relief. Furthermore, predisposing miRNA expression patterns and inter-individual variations and polymorphisms in miRNAs and/or their binding sites may serve as biomarkers for pain and help to predict individual risks for certain types of pain and responsiveness to analgesic drugs. miRNA-based diagnostics are expected to develop into hands-on tools that allow better patient stratification, improved mechanism-based treatment, and targeted prevention strategies for high risk individuals. \ua9 2013 Kress, H\ufcttenhofer, Landry, Kuner, Favereaux, Greenberg, Bednarik, Heppenstall, Kronenberg, Malcangio, Rittner, c\ue7eyler, Trajanoski, Mouritzen, Birklein, Sommer and Soreq

Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride.

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC 50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC 50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo