Superficial dorsal horn neurons with double spike activity in the rat

Superficial dorsal horn neurons promote the transfer of nociceptive information from the periphery to supraspinal structures. The membrane and discharge properties of spinal cord neurons can alter the reliability of peripheral signals. In this paper, we analyze the location and response properties of a particular class of dorsal horn neurons that exhibits double spike discharge with a very short interspike interval (2.01+/-0.11 ms). These neurons receive nociceptive C-fiber input and are located in laminae I-II. Double spikes are generated spontaneously or by depolarizing current injection (interval of 2.37+/-0.22). Cells presenting double spike (interval 2.28+/-0.11) increased the firing rate by electrical noxious stimulation, as well as, in the first minutes after carrageenan injection into their receptive field. Carrageenan is a polysaccharide soluble in water and it is used for producing an experimental model of semi-chronic pain. In the present study carrageenan also produces an increase in the interval between double spikes and then, reduced their occurrence after 5-10 min. The results suggest that double spikes are due to intrinsic membrane properties and that their frequency is related to C-fiber nociceptive activity. The present work shows evidence that double spikes in superficial spinal cord neurones are related to the nociceptive stimulation, and they are possibly part of an acute pain-control mechanism.     

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors modulate release of inhibitory amino acids in rat spinal cord dorsal horn

Local inhibition within the spinal cord dorsal horn is mediated by the neurotransmitters GABA and glycine and strongly influences nociceptive and temperature signaling. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are expressed by inhibitory interneurons and have been shown to modulate GABA release in other regions of the CNS. In the spinal cord, there is morphological evidence for presynaptic AMPA receptor subunits in GABAergic dorsal horn neurons, but functional data are lacking. To determine if AMPA receptors are indeed functional at presynaptic terminals of inhibitory neurons, we recorded evoked and miniature inhibitory postsynaptic currents (mIPSPs) in the superficial dorsal horn of the rat spinal cord. We show that AMPA receptor activation enhances spontaneous release of inhibitory amino acids in the presence of tetrodotoxin onto both lamina II neurons and NK1 receptor-expressing (NK1R+) lamina I neurons. This effect is sensitive to the concentration of extracellular Ca2+, yet is not fully blocked in most neurons in the presence of Cd2+, suggesting possible Ca2+ entry through AMPA receptors. Postsynaptic Ca2+ elevation is not required for these changes. AMPA-induced increases in mIPSP frequency are also seen in more mature dorsal horn neurons, indicating that these receptors may play a role in nociceptive processing in the adult. In addition, we have observed AMPA-induced depression of evoked release of GABA and glycine onto lamina I NK1R+ neurons. Taken together these data support a role for presynaptic AMPA receptors in modulating release of GABA and glycine in the superficial dorsal horn. Because inhibition in the dorsal horn is important for controlling pain signaling, presynaptic AMPA receptors acting to modulate the inhibitory inputs onto dorsal horn neurons would be expected to impact upon pain signaling in the spinal cord dorsal horn.     

Oxytocin,but not vassopressin,modulates nociceptive responses in dorsal horn neurons

Oxytocin (OT) and vasopressin (VP) are synthesized and secreted by the paraventricular hypothalamic nucleus (PVN), and both peptides have been implicated in the pain modulatory system. In the spinal cord, activation of OT-containing axons modulates nociceptive neuronal responses in dorsal horn neurons; however, it is not known whether the direct VPergic descending projection participates. Here, we show that both PVN electrical stimulation and topical application of OT in the vicinity of identified and recorded dorsal horn WDR selectively inhibit Aδ and C-fiber responses. In contrast, the topical administration of VP on the same neurons did not affect the nociceptive responses. In addition, the reduction in nociceptive responses caused by PVN stimulation or OT administration was blocked with a selective OT antagonist. The results suggest that the VP descending projection does not modulate the antinociceptive effects mediated by the PVN on dorsal horn neurons; instead, it is the hypothalamic-spinal OT projection that regulates nociceptive information.     

Rat trigeminal lamina I neurons that project to thalamic or parabrachial nuclei contain the mu-opioid receptor

Ligands of the mu-opioid receptor are known to inhibit nociceptive transmission in the dorsal horn, yet the cellular site(s) of action for this inhibition remain to be fully elucidated. Neurons located in lamina I of the dorsal horn are involved in distinct aspects of nociceptive transmission. Neurons projecting to the thalamus are thought to be involved in sensory-discriminative aspects of pain perception, while neurons projecting to the parabrachial nucleus are thought to be important for emotional and/or autonomic responses to noxious stimuli. The present study examined these two populations of lamina I projection neurons in the trigeminal dorsal horn to determine if the mu-opioid receptor protein (MOR1) is differentially located in these populations of neurons. Lamina I projection neurons were identified using the retrograde tracer FluoroGold (FGold). FGold was injected into either the contralateral thalamus (ventral posterolateral (VPM)/ventral posterolateral (VPL) thalamic region) or into the ipsilateral parabrachial nuclei. The distribution of MOR1 in these neurons was determined using immunocytochemistry. The distribution of MOR1-ir within these two populations of lamina I projection neurons was examined by both confocal and electron microscopy. We found that both populations of projection neurons contained MOR1. Immunogold analyses revealed the presence of MOR1-ir at membrane sites and within the cytoplasm of these neurons. Cytoplasmic receptor labeling may represent sites of synthesis, recycling or reserve populations of receptors. MOR1 was primarily found in the somata and proximal dendrites of projection neurons. In addition, these neurons rarely received synaptic input from MOR1-containing axon terminals. These results indicate that lamina I neurons in trigeminal dorsal horn that project to the thalamic and parabrachial nuclei contain MOR1 and are likely sites of action for MOR ligands that modulate sensory and/or autonomic aspects of pain transmission in the trigeminal dorsal horn.     

大鼠脊髓背角向丘脑和外侧臂旁核的神经降压素能投射

本研究应用荧光金(FG)逆行追踪结合神经降压素(NT)免疫荧光组织化学染色的双标记技术,观察了大鼠脊髓背角向丘脑(TH)和外侧臂旁核(LPb)的NT能投射。将FG注入一侧TH或LPb后,FG逆标神经元主要见于脊髓背角的I层;NT阳性神经元主要分布于脊髓背角的I层、II层外侧部及II层内侧部与III层交界处;脊髓背角I层内可观察到FG逆标记并呈NT阳性的双标记神经元。上述结果表明脊髓背角I层的NT阳性神经元向TH和LPb投射,提示脊髓背角I层内的NT阳性神经元可能向TH和LPb传递伤害性信息。     

Sensitization of the transient receptor potential vanilloid type 1 ion channel by isoflurane or sevoflurane does not result in extracellular signal-regulated kinase 1/2 activation in rat spinal dorsal horn neurons

Clinically relevant concentrations of isoflurane or sevoflurane sensitize transient receptor potential vanilloid type 1 to several of its activators, including capsaicin. It has, moreover, been suggested these volatile general anaesthetics may augment nociceptive signalling arising from surgical procedures and thereby contribute to post-operative pain. To investigate this suggestion, we have studied intraplantar capsaicin injection-induced phosphorylation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons (which is a recognized marker of spinal nociceptive processing) in rat during isoflurane or sevoflurane anaesthesia after 60 min under anaesthesia. Control animals were anaesthetized with pentobarbital (which of itself does not activate extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons). Unilateral intraplantar capsaicin injection in control animals evoked extracellular signal-regulated kinase 1/2 phosphorylation in a group of neurons in lamina I and lamina II of the ipsilateral spinal dorsal horn in a somatotopically appropriate area. In contrast, both anaesthetic gases (given for 60 min and without subsequent capsaicin injection) induced extracellular signal-regulated kinase 1/2 activation in a different group of mainly lamina I neurons bilaterally. The total number of spinal dorsal horn neurons labelled on the ipliateral side following capsaicin injection into the isoflurane-, or sevoflurane-, anaesthetized animals was significantly less than that produced by capsaicin alone. Further, capsaicin injection into isoflurane-, or sevoflurane-, anaesthetized animals reduced extracellular signal-regulated kinase 1/2 phosphorylation induced by the gases alone on both sides. These findings do not support the suggestion that isoflurane-, or sevoflurane-, induced sensitization of transient receptor potential vanilloid type 1 by capsaicin, or other agonist, is translated into induction of spinal nociceptive processing and consequential pain sensation.     

Absence of Reelin results in altered nociception and aberrant neuronal positioning in the dorsal spinal cord

Mutations in reeler, the gene coding for the Reelin protein, result in pronounced motor deficits associated with positioning errors (i.e. ectopic locations) in the cerebral and cerebellar cortices. In this study we provide the first evidence that the reeler mutant also has profound sensory defects. We focused on the dorsal horn of the spinal cord, which receives inputs from small diameter primary afferents and processes information about noxious, painful stimulation. We used immunocytochemistry to map the distribution of Reelin and Disabled-1 (the protein product of the reeler gene, and the intracellular adaptor protein, Dab1, involved in its signaling pathway) in adjacent regions of the developing dorsal horn, from early to late embryonic development. As high levels of Dab1 accumulate in cells that sustain positioning errors in reeler mutants, our findings of increased Dab1 immunoreactivity in reeler laminae I-III, lamina V and the lateral spinal nucleus suggest that there are incorrectly located neurons in the reeler dorsal horn. Subsequently, we identified an aberrant neuronal compaction in reeler lamina I and a reduction of neurons in the lateral spinal nucleus throughout the spinal cord. Additionally, we detected neurokinin-1 receptors expressed by Dab1-labeled neurons in reeler laminae I-III and the lateral spinal nucleus. Consistent with these anatomical abnormalities having functional consequences, we found a significant reduction in mechanical sensitivity and a pronounced thermal hyperalgesia (increased pain sensitivity) in reeler compared with control mice. As the nociceptors in control and reeler dorsal root ganglia are similar, our results indicate that Reelin signaling is an essential contributor to the normal development of central circuits that underlie nociceptive processing and pain.     

Antinociceptive effect of morphine on rat spinothalamic tract and other dorsal horn neurons

The importance of the spinothalamic tract in pain transmission makes it an attractive candidate for study with respect to the effects of antinociceptive compounds. We have been interested in the analgesic actions of opioids and noradrenergic agents at the spinal level and have investigated the effects of these agents on extracellularly recorded nociceptive dorsal horn neurons in the rat. Spinothalamic tract cells were identified by antidromic activation from the somatosensory thalamus. Morphine was administered by bathing the spinal cord in an artificial cerebrospinal fluid solution which contained a known concentration of drug. We observed a dose-related inhibition, naloxone-reversible in some cases, of activity produced by spinally administered morphine in identified rat spinothalamic tract cells and dorsal horn nociceptive neurons. Morphine had no effect on stimulus-evoked responses of low threshold dorsal horn neurons.     

Differential contributions of NMDA and non-NMDA receptors to spinal Fos expression evoked by superficial tissue and muscle inflammation in the rat.

The role of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the spinal cord in the transmission of nociceptive afferents from superficial tissue and muscle was studied by examining the effects of NMDA or non-NMDA receptor antagonists on Fos expression in the spinal dorsal horn. Muscle inflammation was induced by injection of turpentine oil into the gastrocnemius muscle, whereas superficial tissue inflammation was induced by an intraplantar injection of turpentine oil into the hindpaw. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP-5), the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) or normal saline were intrathecally administered 15 min before an intramuscular or intraplantar injection of turpentine oil. Muscle inflammation evoked expression of Fos-like immunoreactive neurons staining in neurons that were predominantly distributed in the middle portions of laminae I-II(outer) and the lateral portions of laminae V-VI of the ipsilateral dorsal horn at the spinal L(4)-L(5). DNQX, but not AP-5, significantly reduced the total number of Fos-like immunoreactive neurons evoked by muscle inflammation. In contrast, superficial tissue inflammation evoked expression of Fos-like immunoreactive neurons in the medial portions of laminae I-II(outer) and V-VI of the ipsilateral dorsal horn at the spinal L(4)-L(5) that was blocked by AP-5, but not by DNQX. Injection of normal saline did not influence the numbers of Fos-LI neurons.These results indicate that different glutamate receptors in the dorsal horn of the spinal cord may mediate nociceptive input from superficial tissue (particularly skin) and muscle. DNQX receptors may mediate transmission of nociceptive information originating in muscle, while NMDA receptors may preferentially mediate transmission of nociceptive information originating in skin.     

Plastic changes in nociceptive transmission of the rat spinal cord with advancing age

To understand characteristics of the pain system in the elderly, we investigated the electrophysiological properties of nociceptive neurons in the lumbar spinal dorsal horn of aged (29-34-mo old) and adult (7-13-mo old) rats. The responses of nociceptive neurons to noxious thermal stimulation, as well as the spontaneous firing rate, were significantly higher in the aged as compared with adult rats. Furthermore, the size of the high-threshold receptive field area of wide dynamic range neurons was larger (P < 0.01) and that of the low-threshold area was smaller (P < 0.05) in aged rats than in adult rats. The increased nociceptive neuronal activity in the aged rats correlated with the finding that the paw withdrawal latency was significantly shorter in the aged rats than those of the adult rats following heat stimulation of the hind paw (P < 0.05). Reversible local anesthetic block of descending pathways resulted in a dramatic increase in neuronal activity in adult rats but had little effect in aged rats. There was also a significant loss of serotoninergic and noradrenergic fibers in the spinal dorsal horn of the aged rats. These results demonstrate an age-related plasticity in spinal nociceptive processing that is related to impairment of descending modulatory pathways.     

Coeruleospinal inhibition of visceral nociceptive processing in the rat spinal cord

Visceral nociceptive information is transmitted in two different areas of the spinal cord gray matter, the dorsal horn and the area near the central canal. The present study was designed to examine whether visceral nociceptive transmission in the two different areas is under the control of the centrifugal pathways from the locus coeruleus/subcoeruleus (LC/SC). Extracellular recordings were made from the L(6)-S(2) segmental level using a carbon filament glass microelectrode (4-6 MOmega). Colorectal distentions (80 mmHg) were produced by inflating a balloon inside the descending colon and rectum. In both dorsal horn and deep area neurons, responses to colorectal distention were inhibited during electrical stimulation (30, 50 and 70 microA, 100 Hz, 0.1 ms pulses) of the LC/SC. It is well known that spinothalamic tract (STT) neurons excited by visceral nociceptive stimuli are located in the dorsal horn and that postsynaptic dorsal column (PSDC) neurons which conduct visceral nociceptive signals in the dorsal column (DC) are located near the central canal of the spinal cord. The present study, therefore, suggests that the descending LC/SC system can inhibit visceral nociceptive signals ascending through the STT and the DC pathways.     

Increased nociceptive input rapidly modulates spinal GABAergic transmission through endogenously released glutamate

Stimulation of nociceptive primary afferents elicits pain by promoting glutamatergic transmission in the spinal cord. Little is known about how increased nociceptive input controls GABAergic tone in the spinal dorsal horn. In this study, we determined how increased nociceptive inflow affects GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of lamina II neurons by using whole cell recordings in rat spinal cord slices. Bath application of capsaicin for 3 min induced a long-lasting inhibition of sIPSCs in 50% of the neurons tested. In the other half of the neurons, capsaicin either increased the frequency of sIPSCs (34.6%) or had no effect on sIPSCs (15.4%). The GABA(A) current elicited by puff application of GABA was not altered by capsaicin. Capsaicin did not inhibit sIPSCs in rats treated with intrathecal pertussis toxin. Also, capsaicin failed to inhibit sIPSCs in the presence of ionotropic glutamate receptor antagonists or in the presence of both LY341495 and CPPG (group II and group III metabotropic glutamate receptor antagonists, respectively). However, when LY341495 or CPPG was used alone, capsaicin still decreased the frequency of sIPSCs in some neurons. Additionally, bradykinin significantly inhibited sIPSCs in a population of lamina II neurons and this inhibitory effect was also abolished by LY341495 and CPPG. Our study provides novel information that stimulation of nociceptive primary afferents rapidly suppresses GABAergic input to many dorsal horn neurons through endogenous glutamate and activation of presynaptic group II and group III metabotropic glutamate receptors. These findings extend our understanding of the microcircuitry of the spinal dorsal horn involved in nociception.     

Functional GABA(A)-receptor-mediated inhibition in the neonatal dorsal horn

Neonatal nociceptive circuits and dorsal horn cells are characterized by an apparent lack of inhibitory control: receptive fields are large and thresholds low in the first weeks of life. It has been suggested that this may reflect immature GABA(A)-receptor (GABA(A)R) signaling whereby an early developmental shift in transmembrane anion gradient is followed by a longer period of low Cl- extrusion capacity. To investigate whether functional GABA(A)R-mediated inhibition does indeed undergo postnatal regulation at the level of dorsal horn circuits, we applied the selective GABA(A)R antagonist gabazine to the spinal cord in anesthetized rat pups [postnatal day (P) 3 or 21] while recording spike activity in single lumbar dorsal horn cells in vivo. At both ages, blockade of GABA(A)R activity resulted in enlarged hind paw receptive field areas and increased activity evoked by low- and high-intensity cutaneous stimulation, revealing comparable inhibition of dorsal horn cell firing by spinal GABA(A)Rs at P3 and P21. This inhibition did not require descending pathways to the spinal cord because perforated patch-clamp recordings of deep dorsal horn neurons in P3 spinal cord slices also showed an increase in evoked spike activity after application of gabazine. We conclude that spinal GABAergic inhibitory transmission onto single dorsal horn cells "in vivo" is functional at P3 and that low Cl- extrusion capacity does not restrict GABAergic function over the normal range of evoked sensory activity. The excitability of neonatal spinal sensory circuits could reflect immaturity in other intrinsic or descending inhibitory networks rather than weak spinal GABAergic inhibition.     

Physiological properties of spinal lamina II GABAergic neurons in mice following peripheral nerve injury

Aberrant GABAergic inhibition in spinal dorsal horn may underlie some forms of neuropathic pain. Potential, but yet unexplored, mechanisms include reduced excitability, abnormal discharge patterns or altered synaptic input of spinal GABAergic neurons. To test these hypotheses, we quantitatively compared active and passive membrane properties, firing patterns in response to depolarizing current steps and synaptic input of GABAergic neurons in spinal dorsal horn lamina II of neuropathic and of control animals. Transgenic mice were used which expressed enhanced green fluorescent protein (EGFP) controlled by the GAD67 promoter, thereby labelling one-third of all spinal GABAergic neurons. In all neuropathic mice included in this study, chronic constriction injury of one sciatic nerve led to tactile allodynia and thermal hyperalgesia. Control mice were sham-operated. Membrane excitability of GABAergic neurons from neuropathic or sham-treated animals was indistinguishable. The most frequent firing patterns observed in neuropathic and sham-operated animals were the initial burst (neuropathic: 46%, sham-treated: 42%), the gap (neuropathic: 31%, sham-treated: 29%) and the tonic firing pattern (neuropathic: 16%, sham-treated: 24%). The synaptic input from dorsal root afferents was similar in neuropathic and in control animals. Thus, a reduced membrane excitability, altered firing patterns or changes in synaptic input of this group of GABAergic neurons in lamina II of the spinal cord dorsal horn are unlikely causes for neuropathic pain.     

Activation of peripheral P2X receptors is sufficient to induce central sensitization in rat medullary dorsal horn nociceptive neurons

Central sensitization and purinergic receptor mechanisms have been implicated as important processes in acute and chronic pain conditions following injury or inflammation of peripheral tissues. This study has documented that application of the P2X(1,2/3,3) receptor agonist αβ-meATP (100mM) to the rat tooth pulp induces central sensitization in medullary dorsal horn nociceptive neurons that is reflected in significant increases in mechanoreceptive field size and responses to noxious stimuli and decreased mechanical activation threshold. Furthermore, these responses can be blocked by pulp application of the P2X(1,2/3,3) antagonist TNP-ATP and also attenuated by medullary application of TNP-ATP. These results suggest that activation of P2X(1,2/3,3) receptors in orofacial tissues plays a critical role in producing central sensitization in medullary dorsal horn nociceptive neurons.     

Functional cross-excitation between afferent A- and C-neurons in dorsal root ganglia

Electrophysiological recordings were made in vitro from primary afferent neurons with unmyelinated axons (C-neurons) in excised rat dorsal root ganglia. Spike activity triggered in neurons with myelinated axons (A-neurons) by stimulation of the peripheral nerve or the dorsal root produced a transient depolarization in passive neighboring C-neurons that share the same ganglion. About 90% of neurons sampled responded with this "cross-depolarization". Cross-depolarization was associated with functional excitation as indicated by an increase in firing probability in response to previously subthreshold intracellular test pulses. Furthermore, it yielded a net increase of the input resistance of the affected C-neurons. We suggest that functional coupling among DRG neurons could serve a metabolic role, providing a functionally relevant feedback signal useful for controlling the excitability of nociceptive sensory endings. In addition, the results provide a novel mechanism whereby afferent nociceptors could be stimulated by activity in low-threshold mechanoreceptors, particularly in the event of nerve injury. Hence, the coupling between afferent A- and C-neurons in dorsal root ganglia provides a novel candidate mechanism for neuropathic pain.     

Functional cross-excitation between afferent A- and C-neurons in dorsal root ganglia

Electrophysiological recordings were made in vitro from primary afferent neurons with unmyelinated axons (C-neurons) in excised rat dorsal root ganglia. Spike activity triggered in neurons with myelinated axons (A-neurons) by stimulation of the peripheral nerve or the dorsal root produced a transient depolarization in passive neighboring C-neurons that share the same ganglion. About 90% of neurons sampled responded with this “cross-depolarization”. Cross-depolarization was associated with functional excitation as indicated by an increase in firing probability in response to previously subthreshold intracellular test pulses. Furthermore, it yielded a net increase of the input resistance of the affected C-neurons.We suggest that functional coupling among DRG neurons could serve a metabolic role, providing a functionally relevant feedback signal useful for controlling the excitability of nociceptive sensory endings. In addition, the results provide a novel mechanism whereby afferent nociceptors could be stimulated by activity in low-threshold mechanoreceptors, particularly in the event of nerve injury. Hence, the coupling between afferent A- and C-neurons in dorsal root ganglia provides a novel candidate mechanism for neuropathic pain.