A critical brainstem relay for mediation of diffuse noxious inhibitory controls.

The CNS houses naturally occurring pathways that project from the brain to modulate spinal neuronal activity. The noradrenergic locus coeruleus (the A6 nucleus) originates such a descending control whose influence on pain modulation encompasses an interaction with a spinally projecting non-cerulean noradrenergic cell group. Hypothesizing the origin of an endogenous pain inhibitory pathway, our aim was to identify this cell group. A5 and A7 noradrenergic nuclei also spinally project. We probed their activity using an array of optogenetic manipulation techniques during in vivo electrophysiological experimentation. Interestingly, noxious stimulus evoked spinal neuronal firing was decreased upon opto-activation of A5 neurons (two-way ANOVA with Tukey post hoc, P < 0.0001). Hypothesizing that this may reflect activity in the noradrenergic diffuse noxious inhibitory control circuit, itself activated upon application of a conditioning stimulus, we opto-inhibited A5 neurons with concurrent conditioning stimulus application. Surprisingly, no spinal neuronal inhibition was observed; activity in the diffuse noxious inhibitory control circuit was abolished (two-way ANOVA, P < 0.0001). We propose that the A5 nucleus is a critical relay nucleus for mediation of diffuse noxious inhibitory controls. Given the plasticity of diffuse noxious inhibitory controls in disease, and its back and forward clinical translation, our data reveal a potential therapeutic target.

Rac1/PAK1 signaling contributes to bone cancer pain by Regulation dendritic spine remodeling in rats.

Bone cancer pain (BCP) is severe chronic pain caused by tumor metastasis to the bones, often resulting in significant skeletal remodeling and fractures. Currently, there is no curative treatment. Therefore, insight into the underlying mechanisms could guide the development of mechanism-based therapeutic strategies for BCP. We speculated that Rac1/PAK1 signaling plays a critical role in the development of BCP. Tumor cells implantation (TCI) into the tibial cavity resulted in bone cancer-associated mechanical allodynia. Golgi staining revealed changes in the excitatory synaptic structure of WDR (Wide-dynamic range) neurons in the spinal cord, including increased postsynaptic density (PSD) length and thickness, and width of the cleft. Behavioral and western blotting test revealed that the development and persistence of pain correlated with Rac1/PAK1 signaling activation in primary sensory neurons. Intrathecal injection of NSC23766, a Rac1 inhibitor, reduced the persistence of BCP as well as reversed the remodeling of dendrites. Therefore, we concluded that activation of the Rac1/PAK1 signaling pathway in the spinal cord plays an important role in the development of BCP through remodeling of dendritic spines. Modulation of the Rac1/PAK1 pathway may be a potential strategy for BCP treatment.

Distinct brainstem to spinal cord noradrenergic pathways inversely regulate spinal neuronal activity.

Brainstem to spinal cord noradrenergic pathways include a locus coeruleus origin projection and diffuse noxious inhibitory controls. While both pathways are traditionally viewed as exerting an inhibitory effect on spinal neuronal activity, the locus coeruleus was previously shown to have a facilitatory influence on thermal nocioception according to the subpopulation of coerulean neurons activated. Coupled with knowledge of its functional modular organisation and the fact that diffuse noxious inhibitory controls are not expressed in varied animal models of chronicity, we hypothesized a regulatory role for the locus coeruleus on non-coerulean, discrete noradrenergic cell group(s). We implemented locus coeruleus targeting strategies by microinjecting canine adenovirus encoding for channelrhodopsin-2 under a noradrenaline-specific promoter in the spinal cord (retrogradely labelling a coeruleospinal module) or the locus coeruleus itself (labelling the entire coerulean module). Coeruleospinal module optoactivation abolished diffuse noxious inhibitory controls (two-way ANOVA, P < 0.0001), which were still expressed following locus coeruleus neuronal ablation. We propose that the cerulean system interacts with, but does not directly govern, diffuse noxious inhibitory controls. This mechanism may underlie the role of the locus coeruleus as a 'chronic pain generator'. Pinpointing the functionality of discrete top-down pathways is crucial for understanding sensorimotor modulation in health and disease.

Neuritis and vinblastine-induced axonal transport disruption lead to signs of altered dorsal horn excitability.

BACKGROUND: Many patients with neuropathic pain present without signs of nerve injury on routine clinical examination. Some of these patients may have inflamed peripheral nerves (neuritis). In this study, we have examined whether neuritis causes changes within the dorsal horn that may contribute to a central pain mechanism. Comparisons have been made to a model of axonal transport disruption induced using vinblastine, since neuritis disrupts such processes. RESULTS: At the peak of cutaneous hypersensitivities, recordings from wide dynamic range neurons revealed increases in wind-up following neuritis but not vinblastine treatment. Ongoing activity from these neurons was unchanged. Vinblastine treatment caused a reduction in the responses of wide dynamic range neurons to noxious mechanical stimulation of the receptive field. The response of neurons to innocuous mechanical stimulation was also reduced in wide dynamic range neurons that were at a depth ≥550 µm following vinblastine treatment. An examination of the superficial dorsal horn revealed an increase in c-Fos-positive neurons in both groups following electrical stimulation of the sciatic nerve. The area of dorsal horn expressing substance P was also decreased following vinblastine treatment. CONCLUSION: These findings indicate that a minor nerve insult, such as neuritis, can lead to changes within the dorsal horn that are consistent with a central neuropathic pain mechanism.

Peripheral and central alterations affecting spinal nociceptive processing and pain at adulthood in rats exposed to neonatal maternal deprivation.

The nociceptive system of rodents is not fully developed and functional at birth. Specifically, C fibers transmitting peripheral nociceptive information establish synaptic connections in the spinal cord already during the embryonic period that only become fully functional after birth. Here, we studied the consequences of neonatal maternal deprivation (NMD, 3 h/day, P2-P12) on the functional establishment of C fiber-mediated neurotransmission in spinal cord and of pain-related behavior. In vivo recording revealed that C fiber-mediated excitation of spinal cord neurons could be observed at P14 only in control but not in NMD rats. NMD was associated with a strong alteration in the expression of growth factors controlling C nociceptor maturation as well as two-pore domain K+ channels known to set nociceptive thresholds. In good agreement, C-type sensory neurons from NMD animals appeared to be hypoexcitable but functionally connected to spinal neurons, especially those expressing TRPV1 receptors. In vivo and in vitro recordings of lamina II spinal neurons at P14 revealed that the NMD-related lack of C fiber-evoked responses resulted from an inhibitory barrage in the spinal cord dorsal horn. Eventually, C-type sensory-spinal processing could be recovered after a delay of about 10 days in NMD animals. However, animals remained hypersensitive to noxious stimulus up to P100 and this might be due to an excessive expression of Nav1.8 transcripts in DRG neurons. Together, our data provide evidence for a deleterious impact of perinatal stress exposure on the maturation of the sensory-spinal nociceptive system that may contribute to the nociceptive hypersensitivity in early adulthood.

Effects of Combined Electrical Stimulation of the Dorsal Column and Dorsal Roots on Wide-Dynamic-Range Neuronal Activity in Nerve-Injured Rats.

OBJECTIVES: Electrical stimulation at the dorsal column (DC) and dorsal root (DR) may inhibit spinal wide-dynamic-range (WDR) neuronal activity in nerve-injured rats. The objective of this study was to determine if applying electrical conditioning stimulation (CS) at both sites provides additive or synergistic benefits. MATERIALS AND METHODS: By conducting in vivo extracellular recordings of WDR neurons in rats that had undergone L5 spinal nerve ligation, we tested whether combining 50 Hz CS at the two sites in either a concurrent (2.5 min) or alternate (5 min) pattern inhibits WDR neuronal activity better than CS at DC alone (5 min). The intensities of CS were determined by recording antidromic compound action potentials to graded stimulation at the DC and DR. We measured the current thresholds that resulted in the first detectable Aα/ß waveform (Ab0) and the peak Aα/ß waveform (Ab1) to select CS intensity at each site. The same number of electrical pulses and amount of current were delivered in different patterns to allow comparison. RESULTS: At a moderate intensity of 50% (Ab0 + Ab1), different patterns of CS all attenuated the C-component of WDR neurons in response to graded intracutaneous electrical stimuli (0.1-10 mA, 2 msec) and inhibited windup in response to repetitive noxious stimuli (0.5 Hz). However, the inhibitory effects did not differ significantly between different patterns. At the lower intensity (Ab0), no CS inhibited WDR neurons. CONCLUSIONS: These findings suggest that combined stimulation of DC and DR may not be superior to DC stimulation alone for inhibition of WDR neurons.

Electroacupuncture at different intensities inhibits nociceptive discharges of wide dynamic range neurons in spinal dorsal horn of rats. / ä¸åŒå¼ºåº¦ç”µé’ˆå¯¹å¤§é¼ è„Šé«“èƒŒè§’å¹¿åŠ¨åŠ›ç¥žç»å ƒä¼¤å®³æ€§æ”¾ç”µçš„æŠ‘åˆ¶ä½œç”¨.

OBJECTIVES: To observe the effect of electroacupuncture (EA) at different intensities on nociceptive discharges of wide dynamic range (WDR) neurons in the spinal dorsal horns (DHs) of rats, so as to explore its regulatory characteristics on nociceptive signals at the spinal level. METHODS: A total of 25 male SD rats were used in the present study. A microelectrode array was used to record the discharge activity of WDR neurons in the lumbar spinal DHs of normal rats. After finding the WDR neuron, electrical stimulation (pulse width of 2 ms) was administered to the plantar receptive field (RF) for determining its response component of discharges according to the latency of action potential generation (Aß ï¼»0 to 20 msï¼½, Aδ ï¼»20 to 90 msï¼½, C ï¼»90 to 500 msï¼½ and post-discharge ï¼»500 to 800 msï¼½). High-intensity electrical stimulation was continuously applied to the RF at the paw's plantar surface to induce DHs neuronal windup response. Subsequently, EA stimulation at different intensities (1 mA and 2 mA) was applied to the left "Zusanli"(ST36) at a frequency of 2 Hz/15 Hz for 10 min. The induction of WDR neuronal windup was then repeated under the same conditions. The quantity of nociceptive discharge components and the windup response of WDR neurons before and after EA stimulations at different intensities were compared. RESULTS: Compared to pre-EA, both EA1 mA and EA2 mA significantly reduced the number of Aδ and C component discharges of WDR neurons during stimulation, as well as post-discharge (P<0.01, P<0.001). The inhibitory rate of C component by EA2 mA was significantly higher than that by EA1 mA (P<0.05). Meanwhile, both EA1 mA and EA2 mA attenuated the windup response of WDR neurons (P<0.05, P<0.01), and the effect of EA2 mA was stronger than that of EA1 mA (P<0.05). Further analysis showed that when EA1 mA and EA2 mA respectively applied to both non-receptive field (non-RF) and RF, a significant reduction in the number of Aδ component, C component and post-discharge was observed (P<0.05, P<0.01). EA2 mA at the non-RF and RF demonstrated a significant inhibitory effect on the windup response of WDR neurons (P<0.01, P<0.05), but EA1 mA only at the non-RF showed a significant inhibitory effect on the windup response (P<0.01). CONCLUSIONS: EA can suppress nociceptive discharges of spinal DHs WDR neurons in rats. The inhibitory impact of EA is strongly correlated with the location and intensity of EA stimulation, and EA2 mA has a stronger inhibitory effect than EA1 mA.

Inhibitory effect of acupoint electrostimulation with different layers and intensities on muscular inflammatory pain and spinal WDR neuron activity. / ç©´ä½ä¸åŒå±‚æ¬¡å’Œå¼ºåº¦ç”µåˆºæ¿€ç¼“è§£è‚Œè‚‰ç‚Žæ€§ç—›åŠå¯¹è„Šé«“èƒŒè§’å¹¿åŠ¨åŠ›ç¥žç»å ƒçš„æŠ‘åˆ¶ä½œç”¨.

OBJECTIVES: To observe the analgesic effects of different levels and intensities of electrical stimulation on the local acupoints in the pain source area and their impact on wide dynamic range (WDR) neurons in the spinal dorsal horn, in order to provide a basis for selecting appropriate parameters for electroacupuncture (EA) stimulation. METHODS: Wistar rats were used in 3 parts of the experiment. Complete Freund's adjuvant was used to establish a model of inflammation-induced pain in the gastrocnemius muscle. After modeling, 6 rats were randomly selected for multi-channel extracellular electrophysiological recording of the electrical activity of WDR neurons, to determine the threshold for activating the A-component (Ta) and the C-component (Tc), which were used as the intervention intensities for skin transcutaneous electrical acupoint stimulation (TEAS) or EA. Thirty-six rats were randomly divided into normal , model , TEAS-Ta , TEAS-Tc, EA-Ta , and EA-Tc groups, with 6 rats in each group. In the pain source area , Ta or Tc intensity of TEAS or EA intervention at"Chengshan"(BL57) was performed for 30 min each time, once a day, for 3 consecutive days. A small animal pressure pain measurement instrument was used to measure the mechanical pressure pain threshold of the gastrocnemius muscle in rats, and the Von Frey filament was used to measure the mechanical pain threshold of the footpad. Thirteen rats were randomly selected to observe the immediate responsiveness of WDR neurons to Ta/Tc intensity of EA or TEAS in BL57. RESULTS: The thresholds of TEAS to activate WDR neuron A-component or C-component were (2.43±0.57) mA and (7.00±1.34) mA, respectively, while the thresholds for EA to activate muscle WDR neuron A-component or C-component were (0.72±0.34) mA and (1.58±0.35) mA, respectively. After injection of CFA into the gastrocnemius muscle, compared with the normal group both the mechanical pressure pain threshold of the gastrocnemius muscle and the mechanical pain threshold of the footpad of rats in the model group were significantly decreased (P<0.001). After TEAS-Ta, TEAS-Tc or EA-Ta intervention in the BL57, both the mechanical pressure pain threshold of the gastrocnemius muscle and the mechanical pain threshold of the footpad were significantly higher than those in the model group (P<0.05, P<0.001). Compared with the normal group, the electrical threshold for evoking WDR neuron C-component discharge was significantly decreased (P<0.001) in the model group, while increased after TEAS-Ta, TEAS-Tc, or EA-Ta intervention (P<0.01) compared with the model group. The evoked discharge frequency of muscle WDR neurons decreased significantly after immediate intervention with TEAS-Ta, TEAS-Tc, or EA-Ta (P<0.01, P<0.05). EA-Tc had no significant improvement on the evoked electrical activity of WDR neurons or pain behavior. CONCLUSIONS: TEAS-Ta, TEAS-Tc, or EA-Ta can all alleviate the local and footpad mechanical pain in rats with muscle inflammation and inhibit the responsiveness of WDR neurons, indicating that different intensities are required for analgesic effects at different levels of acupoints in the pain source area.

The Effect of Pre-Electroacupuncture on Nociceptive Discharges of Spinal Wide Dynamic Range Neurons in Rat.

Purpose: Spinal wide dynamic range (WDR) neurons are well studied in pain models and they play critical roles in regulating nociception. Evidence has started to accumulate that acupuncture produces a good analgesic effect via activating different primary fibers with distinct intensities. The purpose of the present study was to compare the distinct intensities of pre-electroacupuncture (pre-EA) at local muscular receptive fields (RFs), adjacent or contralateral non-RFs regulating the nociceptive discharges of spinal WDR neurons evoked by hypertonic saline (HS). Materials and Methods: Spinal segments of electrophysiological recording were identified by neural tracers applied at the left gastrocnemius muscle. The thresholds of Aß (TAß), Aδ (TAδ) and C (TC) components of WDR neurons were measured to determine the intensity of pre-EA by extracellular recording. The discharges of WDR neurons induced by distinct intensities of pre-EA and 200 µL HS (6%) injection in left gastrocnemius muscle of rats were observed by extracellular recording. Results: The spinal segments of WDR neurons were confirmed in lumbar (L)5-6 area according to the projective segments of dorsal root ganglion. TAß, TAδ and TC of WDR neurons was determined to be 0.5, 1, and 2 mA, respectively. The pre-EA with intensities of TAß (P < 0.05), TAδ (P < 0.05), TC (P < 0.05) or 2TC (P < 0.01) at ipsilateral adjacent non-RFs significantly reduced the discharges of WDR neurons, while at local RFs only pre-EA of TAδ (P < 0.05), TC (P < 0.05) and 2TC (P < 0.01) could inhibit the nociceptive discharges. In addition, intensity of pre-EA at contralateral non-RFs should reach at least TC to effectively inhibit the firing rates of WDR neurons (P < 0.01). Conclusion: Pre-EA could suppress nociceptive discharges of WDR neurons and the inhibitory effects were dependent on the distinct intensities and locations of stimulation.

The N13 spinal component of somatosensory evoked potentials is modulated by heterotopic noxious conditioning stimulation suggesting an involvement of spinal wide dynamic range neurons.

OBJECTIVES: Although somatosensory evoked potentials (SEPs) after median nerve stimulation are widely used in clinical practice, the dorsal horn generator of the N13 SEP spinal component is not clearly understood. To verify whether wide dynamic range neurons in the dorsal horn of the spinal cord are involved in the generation of the N13 SEP, we tested the effect of heterotopic noxious conditioning stimulation, which modulates wide dynamic range neurons, on N13 SEP in healthy humans. METHODS: In 12 healthy subjects, we performed the cold pressor test on the left foot as a heterotopic noxious conditioning stimulus to modulate wide dynamic range neurons. To verify the effectiveness of heterotopic noxious conditioning stimulation, we tested the pressure pain threshold at the thenar muscles of the right hand and recorded SEPs after right median nerve stimulation before, during and after the cold pressor test. RESULTS: The cold pressor test increased pressure pain threshold by 15% (p = 0.04). During the cold pressor test, the amplitude of the N13 component was significantly lower than that recorded at baseline (by 25%, p = 0.04). DISCUSSION: In this neurophysiological study in healthy humans, we showed that a heterotopic noxious conditioning stimulus significantly reduced N13 SEP amplitude. This finding suggests that the N13 SEP might be generated by the segmental postsynaptic response of dorsal horn wide dynamic range neurons.

Electro-acupuncture inhibits C-fiber-evoked WDR neuronal activity of the trigeminocervical complex: Neurophysiological hypothesis of a complementary therapy for acute migraine modeled rats.

INTRODUCTION: Acupuncture has become a relevant complementary and alternative treatment for acute migraine; however, the neurophysiological mechanism (C-fibers) underlying this effect remains unclear. C-fibers play a crucial role for diffuse noxious inhibitory controls (DNIC) at wide dynamic range (WDR) neurons in the trigeminocervical complex (TCC) in migraine attacks, and we supposed that this may be the mechanism of acupuncture analgesia. This study aimed to examine the neurophysiology of acupuncture intervention in an acute migraine rat model. METHODS: Inflammatory soup (IS) or saline was injected into the dura mater to establish a migraine and control model in rats. To explore the neurobiological mechanism of acupuncture for migraine, we implemented electro-acupuncture (EA), non-electric-stimulation acupuncture, and no-acupuncture in IS and saline injected rats, and recorded the single-cell extraneural neurophysiology of the atlas (C1) spinal dorsal horn neurons in the TCC. RESULTS: Our research shows that electro-acupuncture at GB8 (Shuaigu), located in the periorbital region receptive field of the trigeminal nerve, may rapidly reduce the C-fiber evoked WDR neuronal discharges of the TCC within 60 s. DISCUSSION: This study provides pioneering evidence of a potential neurobiological mechanism for the analgesic effect on migraine attacks achieved by electro-acupuncture intervention via DNIC. The data indicates that EA may become a crucial supplementary and alternative therapy for migraineurs that failed to respond to acute medications, e.g., fremanezumab, which achieves its analgesic effect via modulating Aσ-fibers, not C-fibers.

The Stage-Specific Plasticity of Descending Modulatory Controls in a Rodent Model of Cancer-Induced Bone Pain.

Pain resulting from metastatic bone disease is a major unmet clinical need. Studying spinal processing in rodent models of cancer pain is desirable since the percept of pain is influenced in part by modulation at the level of the transmission system in the dorsal horn of the spinal cord. Here, a rodent model of cancer-induced bone pain (CIBP) was generated following syngeneic rat mammary gland adenocarcinoma cell injection in the tibia of male Sprague Dawley rats. Disease progression was classified as "early" or "late" stage according to bone destruction. Even though wakeful CIBP rats showed progressive mechanical hypersensitivity, subsequent in vivo electrophysiological measurement of mechanically evoked deep dorsal horn spinal neuronal responses revealed no change. Rather, a dynamic reorganization of spinal neuronal modulation by descending controls was observed, and this was maladaptive only in the early stage of CIBP. Interestingly, this latter observation corresponded with the degree of damage to the primary afferents innervating the cancerous tissue. Plasticity in the modulation of spinal neuronal activity by descending control pathways reveals a novel opportunity for targeting CIBP in a stage-specific manner. Finally, the data herein have translational potential since the descending control pathways measured are present also in humans.

Noradrenergic inhibition of spinal hyperexcitation elicited by cutaneous cold stimuli in rats with oxaliplatin-induced allodynia: electrophysiological and behavioral assessments.

We investigated the spinal action of noradrenaline on cold-elicited hyperexcitation detected in dorsal horn neurons of rats with allodynia induced by an oxaliplatin (6 mg/kg, i.p.) injection. In vivo extracellular recordings from the spinal dorsal horn showed that wide dynamic range neurons responded to cutaneous acetone (10 µl) stimulation in normal rats, and cold-elicited firings in oxaliplatin-administered rats were increased with a longer duration, correlated with behavioral responses. These responses were significantly attenuated by spinal administration (50 µM) of noradrenaline or its agonists, clonidine (α2), phenylephrine (α1) and isoprenaline (ß), in descending order of efficacy. Thus, the inhibitory effect of noradrenaline on spinal oxaliplatin-induced cold hyperexcitation is mediated mainly by activation of α2- and/or α1-adrenoceptors.

Modulation of temporal summation threshold of the nociceptive withdrawal reflex by transcutaneous spinal direct current stimulation in humans.

OBJECTIVE: Transcutaneous spinal direct current stimulation (tsDCS) modulates spinal cord pain pathways. The study is aimed to clarify the neurophysiology of the tsDCS-induced modulation of the spinal cord pain processing by evaluating the effect of the tsDCS on temporal summation threshold (TST) of the nociceptive withdrawal reflex (NWR). METHODS: In a randomized, double-blind, crossover study the effects of anodal, cathodal and sham tsDCS (2 mA, 15 min) applied on the skin overlying the thoracic spinal cord were investigated in 10 healthy subjects. RESULTS: Anodal tsDCS induced a long-lasting (up to 60 min) increase in TST of the NWR as well as a parallel decrease in related psychophysical temporal summation of pain, while cathodal and sham tsDCS resulted ineffective. CONCLUSIONS: Anodal tsDCS represents a non-invasive tool able to induce an early and long-lasting depression of the transitory facilitation of the wide dynamic range neurons activity at the basis of both the temporal summation of the NWR and the related temporal summation of pain sensation. SIGNIFICANCE: The modulation of the temporal processing of nociceptive stimuli could be effective in treating clinical pain conditions in which pain is generated by spinal cord structures.

Altered discharges of spinal neurons parallel the behavioral phenotype shown by rats with bortezomib related chemotherapy induced peripheral neuropathy.

Bortezomib is a first generation proteasome inhibitor that is the frontline chemotherapy for multiple myeloma with the chief dose-limiting side effect of painful peripheral neuropathy. The goal of this study was to define the behavioral phenotype in a preclinical model of bortezomib chemotherapy-induced peripheral neuropathy (CIPN) and to test whether this is matched by changes in the physiological responses of spinal wide dynamic range neurons. Sprague-Dawley rats were treated with four injections of bortezomib at four doses, 0.05mg/kg, 0.10mg/kg, 0.15mg/kg, 0.20mg/kg, or equal volume of saline. All doses of bortezomib above 0.05mg/kg produced showed significant dose-dependent mechanical hyperalgesia that was fully established at 30 days after treatment and that recovered to baseline levels by day 69 after treatment. Thermal, cold, and motor testing were all unaffected by treatment with bortezomib. Spinal wide dynamic range (WDR) neurons in rats with confirmed bortzomib-related CIPN showed an increase in number of evoked discharges to mechanical stimuli and exaggerated after-discharges in rats with bortezomib CIPN.

Mu-opioidergic modulation differs in deep and superficial wide-dynamic range dorsal horn neurons in mice.

The spinal cord dorsal horn is an important action site for morphine analgesia. Wide-dynamic range (WDR) neurons in the dorsal horn are essential to spinal pain transmission and show increased excitability after repetitive noxious drive (windup). In light of differences in mu-opioid receptor distribution and neurophysiological properties of WDR neurons between deep and superficial dorsal horn, we recorded extracellular single-unit activity of WDR neurons from deep (350-700 µm) and superficial (<350 µm) dorsal horn in C57BL/6 mice and compared their responses to spinal superfusion of morphine (0.5mM, 30 µl) and naloxone (1mM, 30 µl). The windup level to repetitive electrical stimulation of 1.0 Hz (16 pulses, suprathreshold for C-fiber activation, 2.0 ms) was significantly decreased by morphine in deep (n=8), but not superficial (n=11), WDR neurons. However, the steady C-component response to graded intra-cutaneous electrical stimuli (0.01-5.0 mA, 2 ms) was significantly depressed by morphine only in superficial neurons. In separate experiments, spinal administration of naloxone facilitated the development of windup to 0.2 Hz stimulation in deep (n=10), but not superficial (n=8), WDR neurons. Accordingly, morphine and naloxone modulation of neuronal activity may be related to a specific effect on neuronal sensitization/plasticity in deep WDR neurons, whereas morphine inhibition may depress acute noxious inputs to superficial WDR neurons. Our study suggests that mu-opioidergic modulation may be different in deep and superficial WDR neurons.