Postoperative pain facilitates rat C-fibre activity-dependent slowing and induces thermal hypersensitivity in a sex-dependent manner.

BACKGROUND: Postoperative pain is a common clinical problem that, in preclinical studies, has almost exclusively been studied in males. Altered C-fibre activity-dependent slowing (ADS) is a potential underlying mechanism, given it is altered after tissue inflammation and nerve injury, but this has not been explored post-incision. We therefore investigated the effect of hind-paw incision on C-fibre ADS in both sexes and the involvement of voltage-gated sodium channels (NaV) as they contribute to ADS. We also assessed mechanical and thermal sensitivity post-incision in both sexes. METHODS: Dorsal roots were isolated from hind-paw incision (2-4 days post-surgery) or naive (control) juvenile rats of both sexes. Compound action potential recordings were made to assess C-fibre ADS in response to ×40 stimuli at 2 and 10 Hz and repeated in the presence of 20 nM tetrodotoxin/vehicle. Data were quantified by the normalised change in latency (negative peak) and width (positive-to-positive peak) of the triphasic C-fibre response. Hind-paw mechanical withdrawal thresholds and thermal withdrawal latencies were measured pre- and post-incision. RESULTS: Incision facilitates C-fibre ADS in both sexes, with more pronounced facilitation in females. Tetrodotoxin induces sex- and injury-dependent changes in C-fibre ADS that were distinct between latency and width measures. Hind-paw incision induced comparable mechanical hypersensitivity in both sexes but less peak heat hypersensitivity in females. CONCLUSIONS: Hind-paw incision induces sex-dependent changes in C-fibre activity-dependent slowing, which likely contribute to the observed sex difference in peak thermal hypersensitivity. This may reflect sex- and incision-induced differences in functional expression of NaV channels that differs by C-fibre subtype.

The formation of paranodal spirals at the ends of CNS myelin sheaths requires the planar polarity protein Vangl2.

During axonal ensheathment, noncompact myelin channels formed at lateral edges of the myelinating process become arranged into tight paranodal spirals that resemble loops when cut in cross section. These adhere to the axon, concentrating voltage-dependent sodium channels at nodes of Ranvier and patterning the surrounding axon into distinct molecular domains. The signals responsible for forming and maintaining the complex structure of paranodal myelin are poorly understood. Here, we test the hypothesis that the planar cell polarity determinant Vangl2 organizes paranodal myelin. We show that Vangl2 is concentrated at paranodes and that, following conditional knockout of Vangl2 in oligodendrocytes, the paranodal spiral loosens, accompanied by disruption to the microtubule cytoskeleton and mislocalization of autotypic adhesion molecules between loops within the spiral. Adhesion of the spiral to the axon is unaffected. This results in disruptions to axonal patterning at nodes of Ranvier, paranodal axon diameter and conduction velocity. When taken together with our previous work showing that loss of the apico-basal polarity protein Scribble has the opposite phenotype-loss of axonal adhesion but no effect on loop-loop autotypic adhesion-our results identify a novel mechanism by which polarity proteins control the shape of nodes of Ranvier and regulate conduction in the CNS.

Inflammatory Pain Reduces C Fiber Activity-Dependent Slowing in a Sex-Dependent Manner, Amplifying Nociceptive Input to the Spinal Cord.

C fibers display activity-dependent slowing (ADS), whereby repetitive stimulation (≥1 Hz) results in a progressive slowing of action potential conduction velocity, which manifests as a progressive increase in response latency. However, the impact of ADS on spinal pain processing has not been explored, nor whether ADS is altered in inflammatory pain conditions. To investigate, compound action potentials were made, from dorsal roots isolated from rats with or without complete Freund's adjuvant (CFA) hindpaw inflammation, in response to electrical stimulus trains. CFA inflammation significantly reduced C fiber ADS at 1 and 2 Hz stimulation rates. Whole-cell patch-clamp recordings in the spinal cord slice preparation with attached dorsal roots also demonstrated that CFA inflammation reduced ADS in the monosynaptic C fiber input to lamina I neurokinin 1 receptor-expressing neurons (1-10 Hz stimulus trains) without altering the incidence of synaptic response failures. When analyzed by sex, it was revealed that females display a more pronounced ADS that is reduced by CFA inflammation to a level comparable with males. Cumulative ventral root potentials evoked by long and short dorsal root stimulation lengths, to maximize and minimize the impact of ADS, respectively, demonstrated that reducing ADS facilitates spinal summation, and this was also sex dependent. This finding correlated with the behavioral observation of increased noxious thermal thresholds and enhanced inflammatory thermal hypersensitivity in females. We propose that sex/inflammation-dependent regulation of C fiber ADS can, by controlling the temporal relay of nociceptive inputs, influence the spinal summation of nociceptive signals contributing to sex/inflammation-dependent differences in pain sensitivity.SIGNIFICANCE STATEMENT The intensity of a noxious stimulus is encoded by the frequency of action potentials relayed by nociceptive C fibers to the spinal cord. C fibers conduct successive action potentials at progressively slower speeds, but the impact of this activity-dependent slowing (ADS) is unknown. Here we demonstrate that ADS is more prevalent in females than males and is reduced in an inflammatory pain model in females only. We also demonstrate a progressive delay of C fiber monosynaptic transmission to the spinal cord that is similarly sex and inflammation dependent. Experimentally manipulating ADS strongly influences spinal summation consistent with sex differences in behavioral pain thresholds. This suggests that ADS provides a peripheral mechanism that can regulate spinal nociceptive processing and pain sensation.

Melatonin limits paclitaxel-induced mitochondrial dysfunction in vitro and protects against paclitaxel-induced neuropathic pain in the rat.

Chemotherapy-induced neuropathic pain is a debilitating and common side effect of cancer treatment. Mitochondrial dysfunction associated with oxidative stress in peripheral nerves has been implicated in the underlying mechanism. We investigated the potential of melatonin, a potent antioxidant that preferentially acts within mitochondria, to reduce mitochondrial damage and neuropathic pain resulting from the chemotherapeutic drug paclitaxel. In vitro, paclitaxel caused a 50% reduction in mitochondrial membrane potential and metabolic rate, independent of concentration (20-100 µmol/L). Mitochondrial volume was increased dose-dependently by paclitaxel (200% increase at 100 µmol/L). These effects were prevented by co-treatment with 1 µmol/L melatonin. Paclitaxel cytotoxicity against cancer cells was not affected by co-exposure to 1 µmol/L melatonin of either the breast cancer cell line MCF-7 or the ovarian carcinoma cell line A2780. In a rat model of paclitaxel-induced painful peripheral neuropathy, pretreatment with oral melatonin (5/10/50 mg/kg), given as a daily bolus dose, was protective, dose-dependently limiting development of mechanical hypersensitivity (19/43/47% difference from paclitaxel control, respectively). Melatonin (10 mg/kg/day) was similarly effective when administered continuously in drinking water (39% difference). Melatonin also reduced paclitaxel-induced elevated 8-isoprostane F2 α levels in peripheral nerves (by 22% in sciatic; 41% in saphenous) and limited paclitaxel-induced reduction in C-fibre activity-dependent slowing (by 64%). Notably, melatonin limited the development of mechanical hypersensitivity in both male and female animals (by 50/41%, respectively), and an additive effect was found when melatonin was given with the current treatment, duloxetine (75/62% difference, respectively). Melatonin is therefore a potential treatment to limit the development of painful neuropathy resulting from chemotherapy treatment.

The chemerin receptor 23 agonist, chemerin, attenuates monosynaptic C-fibre input to lamina I neurokinin 1 receptor expressing rat spinal cord neurons in inflammatory pain.

BACKGROUND: Recent evidence has shown that the chemerin receptor 23 (ChemR23) represents a novel inflammatory pain target, whereby the ChemR23 agonists, resolvin E1 and chemerin, can inhibit inflammatory pain hypersensitivity, by a mechanism that involves normalisation of potentiated spinal cord responses. This study has examined the ability of the ChemR23 agonist, chemerin, to modulate synaptic input to lamina I neurokinin 1 receptor expressing (NK1R+) dorsal horn neurons, which are known to be crucial for the manifestation of inflammatory pain. RESULTS: Whole-cell patch-clamp recordings from pre-identified lamina I NK1R+ neurons, in rat spinal cord slices, revealed that chemerin significantly attenuates capsaicin potentiation of miniature excitatory postsynaptic current (mEPSC) frequency, but is without effect in non-potentiated conditions. In tissue isolated from complete Freund's adjuvant (CFA) treated rats, chemerin significantly reduced the peak amplitude of monosynaptic C-fibre evoked excitatory postsynaptic currents (eEPSCs) in a subset of lamina I NK1R+ neurons, termed chemerin responders. However, chemerin did not alter the peak amplitude of monosynaptic C-fibre eEPSCs in control tissue. Furthermore, paired-pulse recordings in CFA tissue demonstrated that chemerin significantly reduced paired-pulse depression in the subset of neurons classified as chemerin responders, but was without effect in non-responders, indicating that chemerin acts presynaptically to attenuate monosynaptic C-fibre input to a subset of lamina I NK1R+ neurons. CONCLUSIONS: These results suggest that the reported ability of ChemR23 agonists to attenuate inflammatory pain hypersensitivity may in part be due to a presynaptic inhibition of monosynaptic C-fibre input to lamina I NK1R+ neurons and provides further evidence that ChemR23 represents a promising inflammatory pain target.

Childhood Reading Ability and Pain in Childhood Through to Midlife.

Dyslexia and pain have recently been shown to correlate on a genetic level, but there has been little exploration of this association on the phenotypic level despite reports of increased pain in Attention Deficit Hyperactivity Disorder, which commonly co-occurs with dyslexia. In this study we test for an association between reading ability, which is the primary feature of dyslexia, and pain both in childhood and adulthood. Logistic regression modeling was used to test associations between reading ability in childhood and pain from childhood to midlife in a large UK birth cohort; the 1958 National Child Development Study. Associations were found between poor childhood reading ability and increased headache and abdominal pain in childhood, and between poor childhood reading ability and headache, eye pain, back pain, and rheumatism in adulthood. Mediation analyses indicated that socioeconomic status (defined by employment) fully mediated the association between poor reading ability in childhood and back pain at age 42. By contrast, the association between reading ability and eye pain acted independently of socioeconomic status. Different mechanisms were thus indicated for the association of reading with different pain types, including manual labor and a potential shared biological pathway. PERSPECTIVE: This study found a relationship between poor reading ability in childhood and pain in childhood and adulthood. Those with reading difficulties should be monitored for pain symptoms. Future research may uncover shared biological mechanisms, increasing our understanding of pain and potential treatments.

Postsurgical tactile-evoked pain: a role for brain-derived neurotrophic factor-tropomyosin receptor kinase B-dependent novel tactile corpuscles.

Introduction: Millions of people undergo surgical procedures each year with many developing postsurgical pain. Dynamic allodynia can arise when, for example, clothing brushing close to the surgical site elicits pain. The allodynia circuits that enable crosstalk between afferent tactile inputs and central pain circuits have been studied, but the peripheral tactile drive has not been explored. Objective: Investigate the innervation of the skin in the rat plantar hindpaw skin-muscle incision model. Results: Incision increased epidermal thickness and cell layers and reduced intraepidermal nerve fibre density, identified with PGP9.5 immunostaining. Strikingly, Collagen IV immunostaining revealed the development of dermal protrusions, oriented towards the incision site, that were reminiscent of the dermal papillae that exist in glabrous footpads. S100 immunostaining for lamellar Schwann cells revealed the presence of novel tactile corpuscles (S100-positive bulb) within incision-induced putative dermal papillae. The occurrence of these novel tactile corpuscles coincided with behavioural observations of dynamic allodynia. Tactile corpuscles require brain-derived neurotrophic factor- tropomyosin receptor kinase B (BDNF-TrkB) signalling to form during development, and an increase in BDNF-immunostaining intensity was observed close to the incision site. Local acute administration of TrkB-Fc, to block BDNF-TrkB signalling, reduced, by approximately 50%, both tactile corpuscle size (S100+ bulb area) and dynamic allodynia. Conclusion: Surgery induces the development of novel tactile corpuscles in the incision surround, in a BDNF-TrKB-dependent manner, that contributes to postsurgical tactile-evoked pain.

LGI3/2-ADAM23 interactions cluster Kv1 channels in myelinated axons to regulate refractory period.

Along myelinated axons, Shaker-type potassium channels (Kv1) accumulate at high density in the juxtaparanodal region, directly adjacent to the paranodal axon-glia junctions that flank the nodes of Ranvier. However, the mechanisms that control the clustering of Kv1 channels, as well as their function at this site, are still poorly understood. Here we demonstrate that axonal ADAM23 is essential for both the accumulation and stability of juxtaparanodal Kv1 complexes. The function of ADAM23 is critically dependent on its interaction with its extracellular ligands LGI2 and LGI3. Furthermore, we demonstrate that juxtaparanodal Kv1 complexes affect the refractory period, thus enabling high-frequency burst firing of action potentials. Our findings not only reveal a previously unknown molecular pathway that regulates Kv1 channel clustering, but they also demonstrate that the juxtaparanodal Kv1 channels that are concealed below the myelin sheath, play a significant role in modifying axonal physiology.

Inflammatory pain unmasks heterosynaptic facilitation in lamina I neurokinin 1 receptor-expressing neurons in rat spinal cord.

Central sensitization in inflammatory pain conditions results in behavioral mechanical hypersensitivity. Specifically, C-fiber-driven spinal hyperexcitability enables A fibers to gain access to specific spinal circuitry, via heterosynaptic facilitatory mechanisms, to mediate mechanical hypersensitivity. However, the precise circuitry engaged is not known. Lamina I neurokinin 1 (NK1) receptor expressing (NK1R(+)) dorsal horn neurons, many of which are projection neurons, are required for the development of this hypersensitivity and are therefore likely to be a component of this circuitry. To investigate, whole-cell patch-clamp recordings were made from lamina I NK1R(+) neurons in the spinal cord slice preparation with attached dorsal root, obtained from rats with or without complete Freund's adjuvant (CFA) hindpaw inflammation. EPSCs were recorded in response to electrical stimulation of the dorsal root. Control neurons predominantly received monosynaptic C-fiber input (69%) with a smaller proportion receiving monosynaptic Aδ-fiber input (28%). In contrast, CFA inflammation significantly increased the incidence (by twofold) and magnitude (by 75% in a subset) of monosynaptic Aδ-fiber but not monosynaptic C-fiber-evoked responses. Aß-fiber input to lamina I NK1R(+) neurons was minimal, polysynaptic in nature, and unaltered by CFA inflammation. Additional examination of control neurons revealed that a proportion received silent monosynaptic Aδ-fiber input, suggesting that these may provide the substrate for the novel Aδ inputs observed in CFA inflammation. This inflammation induced unmasking and strengthening of monosynaptic Aδ drive to lamina I NK1R(+) neurons may contribute to the heterosynaptic facilitatory mechanisms underlying mechanical hyperalgesia in inflammatory pain.

A rapamycin-sensitive signaling pathway is essential for the full expression of persistent pain states.

Translational control through the mammalian target of rapamycin (mTOR) is critical for synaptic plasticity, cell growth, and axon guidance. Recently, it was also shown that mTOR signaling was essential for the maintenance of the sensitivity of subsets of adult sensory neurons. Here, we show that persistent pain states, but not acute pain behavior, are substantially alleviated by centrally administered rapamycin, an inhibitor of the mTOR pathway. We demonstrate that rapamycin modulates nociception by acting on subsets of primary afferents and superficial dorsal horn neurons to reduce both primary afferent sensitivity and central plasticity. We found that the active form of mTOR is present in a subpopulation of myelinated dorsal root axons, but rarely in unmyelinated C-fibers, and heavily expressed in the dorsal horn by lamina I/III projection neurons that are known to mediate the induction and maintenance of pain states. Intrathecal injections of rapamycin inhibited the activation of downstream targets of mTOR in dorsal horn and dorsal roots and reduced the thermal sensitivity of A-fibers. Moreover, in vitro studies showed that rapamycin increased the electrical activation threshold of Adelta-fibers in dorsal roots. Together, our results imply that central rapamycin reduces neuropathic pain by acting both on an mTOR-positive subset of A-nociceptors and lamina I projection neurons and suggest a new pharmacological route for therapeutic intervention in persistent pain states.

AMPA receptors bring on the pain.

The role of Ca(2+)-permeable AMPA receptors in pain processing has not been extensively studied. In this issue of Neuron, Hartmann et al. show that altering the levels of these receptors has consequences for inflammatory pain hypersensitivity but not acute pain processing.

Disinhibition opens the gate to pathological pain signaling in superficial neurokinin 1 receptor-expressing neurons in rat spinal cord.

Blockade of local spinal cord inhibition mimics the behavioral hypersensitivity that manifests in chronic pain states. This suggests that there is a pathway capable of mediating allodynia/hyperalgesia that exists but is normally under strong inhibitory control. Lamina I and III neurokinin 1 (NK1) receptor expressing (NK1R+) dorsal horn neurons, many of which are projection neurons, are required for the development of this hypersensitivity and are therefore likely to be a component of this proposed pathway. To investigate, whole-cell patch-clamp recordings were made from lamina I and III NK1R+ neurons in the spinal cord slice preparation with attached dorsal root. Excitatory postsynaptic currents were recorded in response to electrical stimulation of the dorsal root. Lamina I NK1R+ neurons were shown to receive high-threshold (Adelta/C fiber) monosynaptic input, whereas lamina III NK1R+ neurons received low-threshold (Abeta fiber) monosynaptic input. In contrast, lamina I neurons lacking NK1 receptor (NK1R-) received polysynaptic A fiber input. Blockade of local GABAergic and glycinergic inhibition with bicuculline (10 microm) and strychnine (300 nm), respectively, revealed significant A fiber input to lamina I NK1R+ neurons that was predominantly Abeta fiber mediated. This novel A fiber input was polysynaptic in nature and required NMDA receptor activity to be functional. In lamina I NK1R- and lamina III NK1R+ neurons, disinhibition enhanced control-evoked responses, and this was also NMDA receptor dependent. These disinhibition-induced changes, in particular the novel polysynaptic low-threshold input onto lamina I NK1R+ neurons, may be an underlying component of the hypersensitivity present in chronic pain states.

Characterization of sensory neuron subpopulations selectively expressing green fluorescent protein in phosphodiesterase 1C BAC transgenic mice.

BACKGROUND: The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord. RESULTS: Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X3. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn. CONCLUSION: The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.

Presynaptic NMDA receptors modulate glutamate release from primary sensory neurons in rat spinal cord dorsal horn.

NMDA receptors have the potential to produce complex activity-dependent regulation of transmitter release when localized presynaptically. In the somatosensory system, NMDA receptors have been immunocytochemically detected on presynaptic terminals of primary afferents, and these have been proposed to drive release of substance P from central terminals of a subset of nociceptors in the spinal cord dorsal horn. Here we report that functional NMDA receptors are indeed present at or near the central terminals of primary afferent fibers. Furthermore, we show that activation of these presynaptic receptors results in an inhibition of glutamate release from the terminals. Some of these NMDA receptors may be expressed in the preterminal axon and regulate the extent to which action potentials invade the extensive central arborizations of primary sensory neurons.

Aberrant dendritic branching and sensory inputs in the superficial dorsal horn of mice lacking CaMKIIalpha autophosphorylation.

Superficial dorsal horn neurones undergo marked structural and functional activity-dependent development during the early postnatal period, but little is known about the molecular mechanisms underlying these changes. Calcium signalling, through activation and autophosphorylation of CaMKII, has been shown to play a major role in the maturation of neuronal morphology and connectivity in the cortex. Here, we show that the normal structural and functional development of superficial dorsal horn neurones requires CaMKII autophosphorylation at the Thr286 residue. The dendritic branching of neurones from mice containing a point mutation at this site (T286A) was significantly increased compared with wild-type littermates. This was accompanied by significant increases in receptive field size, recorded from intact preparations. Whole-cell patch clamp recordings of superficial dorsal horn slices revealed a selective deficit in low-threshold A fibre-evoked synaptic input. These results show that CaMKII autophosphorylation is required for the normal development of spinal sensory circuits.

Age-dependent effects of peripheral inflammation on the electrophysiological properties of neonatal rat dorsal horn neurons.

The aim of this study was to investigate the postnatal development of spinal cord neurophysiological mechanisms of inflammatory pain. The effect of hindpaw inflammation on the properties of neonatal spinal dorsal horn cells was investigated in urethane-anesthetized newborn rats using in vivo single-unit extracellular recordings. Responses to cutaneous mechanical and electrical A and C fiber stimulation were recorded at postnatal day (P) 3, 10, and 21 in pups that had received a unilateral intraplantar carageenan injection (1%, 1 microl/g body wt) 2-5 h earlier and compared with age-matched controls. At all three ages, carageenan inflammation increased A fiber evoked sensitization, spontaneous activity, and the suprathreshold response magnitude of dorsal horn cells. Receptive field size, which normally decreases with postnatal age, was unaffected by inflammation in P3 and P10 pups but significantly increased at P21 so that the size distribution closely resembled that in control P3 pups. Mechanical thresholds of individual dorsal horn neurons were not altered by carageenan inflammation at any age. The results show that some dorsal horn cell properties that are likely to underlie inflammatory hypersensitivity such as increased spontaneous activity and response magnitude are observed from the earliest postnatal age examined (P3). However inflammation induced expansion of mechanical receptive field size is not observed until at least the second postnatal week. These results have implications for the postnatal processing of inflammatory pain.

Spinal dorsal horn cell receptive field size is increased in adult rats following neonatal hindpaw skin injury.

Local tissue damage in newborn rats can lead to changes in skin sensitivity that last into adulthood and this is likely to be due to plasticity of developing peripheral and central sensory connections. This study examines the functional connections of dorsal horn neurons in young and adult rats that have undergone local skin damage at birth. Newborn rat pups were halothane anaesthetised and received either a unilateral subcutaneous plantar injection of 1 % lambda-carrageenan or a unilateral plantar foot injury made by removal of 2 mm x 2 mm of skin. At 3 weeks, (postnatal day (P) 19-23) and 6 weeks (P40-44) in vivo extracellular recordings of single dorsal horn cells with plantar cutaneous receptive fields were made under urethane anaesthesia (2 g kg-1) and responses to mechanical and electrical stimulation of the skin were assessed. Following neonatal carrageenan inflammation, dorsal horn neuron properties and receptive field sizes at 3 weeks were the same as those of controls. In contrast, following neonatal skin injury, dorsal horn cell receptive field sizes were significantly greater than those of controls at 3 weeks (2.5-fold) and at 6 weeks (2.2-fold). Mechanical thresholds, mechanical response magnitudes and evoked responses to single and repeated A and C fibre stimulation remained unaffected. These results show that early skin injury can cause prolonged changes in central sensory connections that persist into adult life, long after the skin has healed. Enlarged dorsal horn neuron receptive field sizes provide a physiological mechanism for the persistent behavioural hypersensitivity that follows neonatal skin injury in rats and for the prolonged sensory changes reported in human infants after early pain and injury.

The postnatal reorganization of primary afferent input and dorsal horn cell receptive fields in the rat spinal cord is an activity-dependent process.

The dorsal horn of the spinal cord in the newborn rat is characterized by large cutaneous mechanoreceptive fields, a predominance of A-fibre synaptic inputs and diffuse primary afferent A-fibre projections, all of which are gradually reduced and refined over the first postnatal weeks. This may be partly responsible for the reduction in cutaneous flexion reflex sensitivity of rats over the postnatal period. Here we show that chronic, local exposure of the dorsal horn of the lumbar spinal cord to the NMDA antagonist MK801 from birth prevents the normal functional and structural reorganization of A-fibre connections. Dorsal horn cells in spinal MK801-treated animals, investigated at eight weeks of age by in vivo electrophysiological recording, had significantly larger cutaneous mechanoreceptive fields and greater A-fibre evoked responses than vehicle controls. C-fibre evoked responses were unaffected. Chronic MK801 also prevented the normal structural reorganization of A-fibre terminals in the spinal cord. The postnatal withdrawal of superficially projecting A-fibre primary afferents to deeper laminae did not occur in treated animals although C-fibre afferent terminals and cell density in the dorsal horn were apparently unaffected. Spinal MK801-treated animals also had significantly reduced behavioural reflex thresholds to mechanical stimulation of the hindpaw compared to naïve and vehicle-treated animals, whereas noxious heat thresholds remained unaffected. The results indicate that the normal postnatal structural and functional development of A-fibre sensory connectivity within the spinal cord is an activity-dependent process requiring NMDA receptor activation.

Localization and function of ATP and GABAA receptors expressed by nociceptors and other postnatal sensory neurons in rat.

The role of endogenous GABA and ATP in regulating transmitter release from primary afferent terminals in the superficial dorsal horn of the spinal cord is still controversial. ATP is co-released with GABA from some inhibitory dorsal horn neurons raising the possibility that ATP could act in concert with GABA to regulate transmitter release from primary afferent terminals if receptors to both transmitters are expressed there. Using electrophysiology together with immunocytochemistry, we have investigated the expression of ATP-gated P2X and GABAA receptors by identified subpopulations of dorsal root ganglion (DRG) neurons known to project primarily to the superficial dorsal horn. Expression of the heat-sensitive vanilloid receptor 1 (VR1) and sensitivity to capsaicin were used to characterize DRG neurons sensitive to noxious heat. Both P2X and GABAA receptors were expressed on the majority of DRG neurons examined. Recording compound action potentials (CAPs) from dorsal roots in the presence of muscimol, alpha,beta-methylene-ATP (alpha,beta-meATP) or capsaicin resulted in depression of CAP in the slow and medium conducting fibres, indicating cognate receptor expression on the small diameter axons. Dorsal root-evoked dorsal root potentials (DR-DRPs), reflecting depolarization of primary afferent terminals by endogenously released substances, were depressed by the GABAA receptor antagonist SR95531 and alpha,beta-meATP. These results suggest that GABAA and P2X receptors are expressed on DRG cell bodies and slow fibre axons, many of which are heat-nociceptive. These fibres project to the superficial lamina of the dorsal horn where the receptors may function to modulate transmitter release near their central terminals.