Dynamic effects of TNF-α on synaptic transmission in mice over time following sciatic nerve chronic constriction injury.

Nerve injury-induced central sensitization can manifest as an increase in excitatory synaptic transmission and/or as a decrease in inhibitory synaptic transmission in spinal dorsal horn neurons. Cytokines such as tumor necrosis factor-α (TNF-α) are induced in the spinal cord under various injury conditions and contribute to neuropathic pain. In this study we examined the effect of TNF-α in modulating excitatory and inhibitory synaptic input to spinal substantia gelatinosa (SG) neurons over time in mice following chronic constriction injury (CCI) of the sciatic nerve. Whole cell patch-clamp studies from SG neurons showed that TNF-α enhanced overall excitability of the spinal cord early in time following nerve injury 3 days after CCI compared with that in sham control mice. In contrast, the effects of TNF were blunted 14 days after CCI in nerve-injured mice compared with sham surgery mice. Immunohistochemical staining showed that the expression of TNF-α receptor 1 (TNFR1) was increased at 3 days but decreased at 14 days following CCI in the ipsilateral vs. the contralateral spinal cord dorsal horn. These results suggest that TNF-α acting at TNFR1 is important in the development of neuropathic pain by facilitating excitatory synaptic signaling in the acute phases after nerve injury but has a reduced effect on spinal neuron signaling in the later phases of nerve injury-induced pain. Failure of the facilatory effects of TNF-α on excitatory synaptic signaling in the dorsal horn to resolve following nerve injury may be an important component in the transition between acute and chronic pain conditions.

The excitability of dorsal horn neurons is affected by cerebrospinal fluid from humans with osteoarthritis.

Changes in central neural processing are thought to contribute to the development of chronic osteoarthritis pain. This may be reflected as the presence of inflammatory mediators in the cerebral spinal fluid (CSF). We therefore exposed organotypically cultured slices of rat spinal cord to CSF from human subjects with osteoarthritis (OACSF) at a ratio of 1 part CSF in 9 parts culture medium for 5-6 days, and measured changes in neuronal electrophysiological properties by means of whole-cell recording. Although OACSF had no effect on the membrane properties and excitability of neurons in the substantia gelatinosa, synaptic transmission was clearly altered. The frequency of spontaneous excitatory postsynaptic currents (sEPSC) in delay-firing putative excitatory neurons was increased, as was sEPSC amplitude and frequency in tonic-firing inhibitory neurons. These changes could affect sensory processing in the dorsal horn, and may affect the transfer of nociceptive information. Although OACSF also affected inhibitory synaptic transmission (frequency of spontaneous inhibitory synaptic currents; sIPSC), this may have little bearing on sensory processing by substantia gelatinosa neurons, as sEPSC frequency is >3× greater than sIPSC frequency in this predominantly excitatory network. These results support the clinical notion that changes in nociceptive processing at the spinal level contribute to the generation of chronic osteoarthritis pain.

Membrane-delimited coupling of TRPV1 and mGluR5 on presynaptic terminals of nociceptive neurons.

Transient receptor potential vanilloid subtype 1 (TRPV1) and metabotropic glutamate receptor 5 (mGluR5) located on peripheral sensory terminals have been shown to play critical roles in the transduction and modulation of pain sensation. To date, however, very little is known regarding the significance of functional expression of mGluR5 and TRPV1 on the central terminals of sensory neurons in the dorsal horn of the spinal cord. Here we show that TRPV1 on central presynaptic terminals is coupled to mGluR5 in a membrane-delimited manner, thereby contributing to the modulation of nociceptive synaptic transmission in the substantia gelatinosa neurons of the spinal cord. Further, our results demonstrate that TRPV1 is involved in the pain behaviors induced by spinal mGluR5 activation, and diacylglycerol produced by the activation of mGluR5 mediates functional coupling of mGluR5 and TRPV1 on the presynaptic terminals. Thus, mGluR5-TRPV1 coupling on the central presynaptic terminals of nociceptive neurons may be an important mechanism underlying central sensitization under pathological pain conditions.

Inverse relation between intensity of GFAP expression in the substantia gelatinosa and degree of chronic mechanical allodynia.

Glial cells are known to have a large impact on neuropathic pain conditions. Within the spinal cord, microglia rapidly respond to peripheral nerve injury, resulting in central sensitization and ultimately in the onset of enhanced pain behaviour. Astroglia respond with a short delay and are thought to contribute to the early maintenance of neuropathic pain. Nevertheless, it is unknown whether the roles of these glial cell types can be influenced by the chronicity of the neuropathology. Here, the persistent responses of astroglia and microglia to peripheral nerve injury within central pain networks in the upper dorsal horn laminae were studied. At 12 weeks after complete sciatic nerve injury, upregulation of glial fibrillary acidic protein (GFAP), but not complement receptor-3, could be detected in laminae II and III. Moreover, it was found that neuropathic animals with a higher degree of mechanical allodynia had a lower intensity of GFAP expression in lamina II (substantia gelatinosa). From these data we conclude that the role of astroglial responses in mechanical allodynia after peripheral nerve injury may be less straightforward as previously thought. Although astroglia are known to play a pro-nociceptive role in early neuropathic pain states, this role may shift to anti-nociception in more chronic pain states.

Spinal cord transection enhances afferent-evoked inhibition in lamina II neurons and abolishes BDNF-induced facilitation of their sensory input.

We previously reported that the pronociceptive neurotrophin brain-derived neurotrophic factor (BDNF) induces facilitation of C-fiber evoked EPSCs and NMDA currents in lamina II neurons of rats up to P40. Here, patch-clamp recording was used to study BDNF-induced modification of synaptic and NMDA-evoked responses in transverse spinal slices from lumbar (L2-L5) spinal cord of rats from P3 to P21 following complete spinal cord transection at P2. After transection injury at either T13/L1 or L6/S1, BDNF failed to facilitate synaptic AMPA-kainate currents or agonist-induced NMDA currents. The evoked synaptic currents were smaller in amplitude, often consisting of a biphasic (excitatory-inhibitory) response. The EPSC decayed more rapidly in neurons from transected cords than in those from uninjured cords. In transected cords most neurons responded to the GABA(A) receptor antagonist bicuculline with a significant increase in duration of the excitatory synaptic response. This could subsequently be blocked by D-APV, suggesting an NMDA-receptor mediated component. These findings suggest that following spinal cord transection, BDNF spinal actions are no longer predominantly pronociceptive. It is possible that a diminished availability of full-length TrkB and an enhanced expression of truncated TrkB receptors in the injured cord could play an important role in reducing the effect of BDNF following injury. These data are compared to those obtained after contusion, and it is concluded that the physiological changes after spinal injury differ according to nature of the injury.

Stress-induced analgesia: neural and hormonal determinants.

Extensive evidence has indicated that distinct neural systems specifically designed to inhibit sensitivity to painful stimuli exist. Recent advances suggest that the endorphins, enkephalins and the opiate receptor interact with a descending serotonergic bulbospinal system to mediate the analgesic responses to opiates and electrical stimulation. In assessing the evolutionary and behavioral significance of this pain-inhibitory system, several laboratories discovered that acute exposure to a wide variety of stressful events results in a transient analgesia. Chronic exposure to a number of these stressors results in adaptation of the analgesic response. The purpose of this review is to identify and characterize the mechanisms by which these stressors activate pain-inhibition. The relationship of stress-induced analgesia to each of the following is reviewed: (a) the role of endorphins, enkephalins and the opiate receptor; (b) the role of the descending serotonergic bulbospinal system; (c) the role of the pituitary gland; and (d) the role of hypothalamic mechanisms. Data will be discussed in terms of "opiate" and "non-opiate" pain-inhibitory mechanisms, in which some stressors act through the former and other stressors act through the latter.

Second messengers, the substantia gelatinosa and injury-induced persistent pain.

Although there is now unequivocal evidence that the circuitry within the substantia gelatinosa is a major contributor to the transmission and control of nociceptive messages, this was not known 35 years ago, when Pat Wall first focussed attention on this region. In addition to being the target of neurochemically distinct nociceptors, this region contains a heterogeneous population of excitatory and inhibitory interneurons. This review focuses on the contribution of second messenger systems that are found in the substantia gelatinosa. In particular the review highlights their critical contribution to the development of persistent pain conditions in the setting of tissue and nerve injury. Several of the studies used animals with deletions of genes that encode major second messenger molecules, including protein kinase A, C and nitric oxide synthase. Our laboratory has shown that mice with a deletion of the gene that encodes the gamma isoform of protein kinase C (which is almost exclusively expressed in a population of interneurons of the inner part of the substantia gelatinosa) have completely normal acute pain responses. However, the allodynia that characteristically develops after injury does not occur in these mice, particularly when it is generated by partial sciatic nerve injury. By contrast, deletion of genes that encode protein kinase A subunits only show deficits in the development of tissue inflammation-induced pain. These differences highlight the selectivity that characterizes the contribution of different second messenger molecules. Because of the restricted distribution of these molecules, it is likely that they are activated by different populations of primary afferent nociceptor and under very different conditions of injury. Understanding the circuitry within the substantia gelatinosa is thus critical to elucidating the mechanisms through which these second messenger molecules contribute to the development of persistent pain in the setting of injury.

Plastic changes in sensory inputs to rat substantia gelatinosa neurons following peripheral inflammation.

Although hyperalgesia elicited by inflammation has been shown to be partly due to central sensitization, the cellular mechanisms are not clear at the moment. The present study was designed to address this issue using the blind whole-cell patch-clamp technique; glutamatergic primary-afferent inputs to substantia gelatinosa (SG) neurons were compared between spinal cord slices of naive rats and rats inflamed by an intraplantar injection of complete Freund's adjuvant. In naive rats, a large number of SG neurons examined received monosynaptic A delta- (69% of 41 neurons innervated by A fibers) and/or polysynaptic C- (94% of 36 neurons innervated by C fibers) afferent inputs, and only a few neurons received monosynaptic A beta inputs (7%). In addition, when examined in neurons which have both of the A- and C-afferent inputs, A afferent-evoked excitatory postsynaptic currents (EPSCs) were larger in amplitude than C afferent-induced ones; a ratio (A/C ratio) of the former to latter amplitude was 1.8 +/- 0.1 (n = 36). In inflamed rats, a change in the synaptic responses was observed: (1) SG neurons receiving monosynaptic A delta-afferent inputs decreased in number (to 20% of 30 neurons tested, innervated by A fibers), whereas those having monosynaptic A beta-afferent inputs increased to 33%, and (2) the A/C ratio decreased to 0.7 +/- 0.1 (n = 33). These results suggest that after inflammation, a substantial number of A beta-afferents sprout into the SG from their original location (laminae III-V) and that sensory information that used to be conveyed directly to the SG through A delta afferents is transmitted there indirectly through interneurons. These reorganizations of sensory pathway may contribute, at least in part, to underlying mechanisms for the development of hyperalgesia due to inflammation.

The stereospecific effect of naloxone on rat dorsal horn neurones; inhibition in superficial laminae and excitation in deeper laminae.

The effect of systemic naloxone on the activity evoked by C-fibre stimulation in dorsal horn neurones of the rat spinal cord has been investigated. Recordings were made in unanaesthetized, decerebrate spinalized rats. Fifteen units were recorded from laminae 4 and 5 of the dorsal horn, 11 of these units were excited by naloxone (0.2--1.0 mg/kg). The onset of this excitation was after 20 sec to 5 min and recovery to control levels occurred within 15--40 min. Of 17 units recorded in substantia gelatinosa of the dorsal horn, 13 were inhibited by the naloxone. The latency of onset of this inhibition was short (2--10 sec) and the effect persisted for 5--10 min. The effects were largely restricted to C-fibre evoked activity although sometimes A delta responses were similarly altered. Neurones stimulated by A beta-fibre threshold, or whose sole afferent input were A beta-fibres, were unaffected by the naloxone. The stereoisomer of naloxone, (+)naloxone which is inactive in opiate receptor binding tests, failed to produce the same changes found with (-)naloxone in 17 units. These results show a differential effect of naloxone on neurones in the dorsal horn which respond to C-fibre input. Units in the substantia gelatinosa are inhibited while units in deeper laminae are excited by naloxone. These effects are likely to be mediated by the blockade of endogenous opioids in the spinal cord.

Facilitation of NMDA-induced currents and Ca2+ transients in the rat substantia gelatinosa neurons after ligation of L5-L6 spinal nerves.

This study employing a rodent model of neuropathic pain investigated the influence of partial nerve injury on the ability of NMDA receptor activation to induce membrane currents and rises in cytosolic concentration of free calcium ([Ca2+]i) in the rat substantia gelatinosa (SG) neurons using simultaneous whole-cell patch-clamp recording and fura-2 calcium imaging in spinal slices. The novel findings are that: (I) L5-L6 spinal nerve ligation produces a sustained facilitation of NMDA-mediated membrane currents and [Ca2+]i rises both in the soma and dendrites of SG neurons on the injured side on post-operative days 4-13 after injury. (2) It appears that SG neurons in slices from injured rats recover from Ca2+ load less efficiently than neurons from naive rats. (3) The membrane depolarization-induced Ca2+ transients in SG neurons are not modified following spinal nerve ligation. The temporal profile of the changes in Ca2+ transients correlated well with the development of mechanical and thermal allodynia and hyperalgesia. These results suggest an important role of NMDA-mediated calcium signalling in the pathogenesis of neuropathic pain following spinal nerve injury.

[Familial indifference to pain with somatosensory asymmetry: possible central anomaly]. / Forme familiale d'insensibilité à la douleur avec asymétrie de la somesthésie: anomalie centrale?

A family of seven siblings is described. The mother and six siblings have been examined, the eldest and youngest of whom suffer from congenital indifference to pain , although both were ticklish, and itched. The functions examined included somatosensory perception thresholds and autonomic functions; perception thresholds were greatly raised in the painfree subjects and to a lesser extent in some other family members, asymmetrically in all cases, being higher in the dominant hand. Painfree Subject 1 also underwent cerebrospinal fluid analysis at age 16, which showed normal B-endorphin levels but undetectable enkephalins. Electrophysiological tests when a child demonstrated notably that most (raised) measured values were lowered by naloxone. Light microscopic sural nerve biopsy performed on painfree Subject 1 in childhood did not suggest any abnormalities other than a thickened nerve sheath. Threshold asymmetry has not been observed in large numbers of subjects without neurological deficits. There were no significant autonomic changes in any tested family member, though there was some asymmetry. It is suggested that the findings may imply a congenital anomaly of the central nervous system which accounts for the somatosensory, biochemical, and electrophysiological abnormalities.

Synaptic plasticity in the substantia gelatinosa in a model of chronic neuropathic pain.

Chronic neuropathic pain (CNP) is common after peripheral nerve injuries (PNI), but is rather refractory to available anti-pain medication. Advances in neuropathic pain research have identified cellular and molecular cues triggering the onset of neuropathic pain, but the mechanisms responsible for maintenance of chronic pain states are largely unknown. Structural changes such as sprouting of injured A-fibres into the substantia gelatinosa of the dorsal horn in the spinal cord have been proposed to relate to neuropathic pain in partial PNI models. Structural changes in central pain networks may also underlie the more persistent CNP following complete sectioning of a peripheral nerve, because this type of injury results in continuous and spontaneous afferent input to the spinal cord, which can trigger central sensitization. In the present study, the left sciatic nerve was completely sectioned and a 1-cm segment was removed to maintain a chronic pathology, whereas the right sciatic nerve was left intact. Mechanical allodynia was measured up to 84 days after injury, after which synaptic changes were studied in the lumbar substantia gelatinosa. The numbers of larger sized synaptophysin-immunoreactive presynaptic boutons were found to be increased in the substantia gelatinosa ipsilateral to the nerve injury. From these data we conclude that structural synaptic changes within the substantia gelatinosa are present months after complete nerve injury and that this plasticity may be involved in maintaining neuropathic pain states.

Bright light produces Fos-positive neurons in caudal trigeminal brainstem.

Excessive discomfort after exposure to bright light often occurs after ocular injury and during headache. Although the trigeminal nerve is necessary for light-evoked discomfort, the mechanisms underlying this phenomenon, often referred to generally as photophobia, are not well defined. Quantitative Fos-like immunoreactivity (Fos-LI) was used to determine the pattern of neuronal activation in the caudal brainstem after bright light stimulation and, secondly, whether a neurovascular mechanism within the eye contributes to this response. Under barbiturate anesthesia, male rats were exposed to low (1 x 10(4) lx) or high intensity (2 x 10(4) lx) light delivered from a thermal neutral source for 30 min (30 s ON, 30 s OFF) and allowed to survive for 90 min. Intensity-dependent increases in Fos-LI were seen in laminae I-II at the trigeminal caudalis/cervical cord junction region (Vc/C1) and nucleus tractus solitarius (NTS). Fos-LI also increased at the trigeminal interpolaris/caudalis transition (Vi/Vc(vl)) and dorsal paratrigeminal (dPa5) regions independent of intensity. Intravitreal injection of norepinephrine greatly reduced light-evoked Fos-LI at the Vc/C1, dPa5 and NTS, but not at the Vi/Vc transition. Lidocaine applied to the ocular surface had no effect on Fos-LI produced in trigeminal brainstem regions. These results suggested that multiple regions of the caudal trigeminal brainstem complex integrate light-related sensory information. Fos-LI produced at the dPa5 and NTS, coupled with norepinephrine-induced inhibition, was consistent with the hypothesis that light-evoked activation of trigeminal brainstem neurons involves an intraocular neurovascular mechanism with little contribution from neurons that supply the ocular surface.