Responses of spinal neurones to cutaneous and dorsal root stimuli in rats with mechanical allodynia after contusive spinal cord injury

The firing of neurones in spinal segments adjacent to a contusive T13 spinal cord injury was characterised in anaesthetised rats. Three groups of rats were examined: (1) allodynic spinally injured, (2) non-allodynic spinally injured and (3) normal, uninjured. Spinal cord field potentials evoked by electrical dorsal root stimulation and the responses of 207 dorsal horn neurones to mechanical stimuli applied to the skin were studied. Within the lesioned spinal segment few active neurones were encountered and field potentials were absent. Depolarising field potentials recorded rostral to the lesion were reduced in both allodynic and non-allodynic animals compared to uninjured controls, while those recorded in caudal segments were enhanced in allodynic animals. Neuronal recordings revealed that allodynia was associated with exaggerated responses, including afterdischarges, to innocuous and noxious mechanical stimuli in a proportion of wide dynamic range, but not low threshold, neurones. These changes were observed both rostral and caudal to the site of injury. The results suggest that an increased responsiveness of some dorsal horn neurones in segments neighbouring a contusive spinal cord injury may contribute to the expression of mechanical allodynia. It is proposed that a relative lack of inhibition underlies altered cell responses.     

Adenosine exerts multiple effects in dorsal horn neurones of the adult rat spinal cord

In the present study, we have examined the effects of adenosine and its analogues on the electrophysiological properties of dorsal horn neurones in the rat adult spinal cord. Adenosine and the A₁ receptor agonist R-phenylisopropyl adenosine (RPIA) reversibly hyperpolarised these neurones via the generation of an outward current at −60 mV that was inhibited by pre-application of barium or Rp-adenosine 3′, 5′-cyclic monophosphothioate triethylamine. In contrast, the A_2a receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680) had no effect on the resting membrane properties of these neurones. Stimulation of the dorsal root evoked non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) at −60 mV that were completely abolished by 2,3-dihydroxy-6-nitro-7-sulophamoyl-benzo(F)quinoxalone (NBQX). Bath application of adenosine or RPIA reversibly inhibited these EPSCs in a concentration-dependent manner via a presynaptic action. In contrast, CGS21680 increased the amplitude of the EPSC in 20% of neurones tested and decreased the EPSC amplitude in 30% of neurones tested. It is concluded that adenosine exerts multiple effects upon the electrophysiological properties of dorsal horn neurones in the adult spinal cord via interaction with multiple receptors. These findings have important implications in the understanding of adenosine action in preclinical models of pain.     

Reversal of ERK activation in the dorsal horn after decompression in chronic constriction injury

Injury-induced neuropathic pain is related to changes in the central terminals of dorsal root ganglia neurons, i.e., dorsal horn plasticity. We investigated the influences of decompression by removing ligatures producing chronic constriction injury (CCI) in Sprague-Dawley rats at postoperative week (POW) 4, the decompression group; for comparison, all ligatures remained through the experimental period in the CCI group. The effect was evaluated with extracellular signal-regulated kinase (ERK) activation in the dorsal horn, i.e., number of phosphorylated ERK (+) cells in the dorsal horn. At POW 1, the dorsal horn indexes had increased to a similar degree in both groups (2.40+/-0.58 vs. 2.27+/-0.36, p=0.73). At POW 8, thermal hyperalgesia and mechanical allodynia had completely disappeared with a normalization of dorsal horn index (1.17+/-0.11 vs. 1.02+/-0.12 at POW 0, p=0.07) in the decompression group; in contrast, the dorsal horn index remained elevated in the CCI group (2.48+/-0.30, p<0.001) with persistent neuropathic pain behaviors at POW 8. This report suggests that ERK activation in the dorsal horn is correlated with neuropathic pain behaviors and its normalization reflects the reversal of neuropathic pain behaviors after decompression.     

Resistance to anoxic injury in the dorsal columns of adult rat spinal cord following demyelination

In order to examine the relationship between myelination and sensitivity to anoxia in adult white matter, we studied action potential conduction in the spinal cord dorsal column of adult rats in which focal demyelinating lesions had been produced using ethidium bromide/X-irradiation. Acutely isolated spinal cords from control rats and following demyelination were maintained in vitro at 36°C and compound action potentials were studied following supramaximal stimulation. The compound action potential was totally abolished within 12 min of the onset of anoxia in normal dorsal columns, but was not abolished until 50 min following the onset of anoxia in demyelinated dorsal columns. Compound action potentials showed significantly greater recovery (to 58.1±12.2% of control amplitude) in demyelinated dorsal columns compared to controls (30.8±5.3%) following 120 min of reoxygenation. These results show that focal demyelination is associated with reduced sensitivity to anoxia within white matter of the adult spinal cord.     

Upregulation of Group I metabotropic glutamate receptors in neurons and astrocytes in the dorsal horn following spinal cord injury

Of the glutamate receptor types, the metabotropic glutamate receptors (mGluRs) are G proteins coupled and can initiate a number of intracellular pathways leading to hyperexcitability of spinal neurons. In this study, we tested the expression of mGluRs to determine which cell types might contribute to sustained neuronal hyperexcitability in the lumbar enlargement with postoperative day (POD) 7 (early), 14 (late), and 30 (chronic phase) following spinal cord injury (SCI) by unilateral hemisection at T13 in Sprague-Dawley rats. Expression was determined by confocal analyses of immunocytochemical reaction product of neurons (NeuN positive) and astrocytes (GFAP positive) in the dorsal horn on both sides of the L4 segment. Neurons were divided into two sizes: small (<20 microm) and large (>35 microm), for physiological reasons. We report a significant increase of mGluR(1) expression in large and small neurons of the dorsal horn on both sides of the cord in late and chronic phases when compared to control sham groups. Expression of mGluR(2/3) significantly increased in large neurons on the ipsilateral (hemisected) side in the late phase. Expression of mGluR(5) significantly increased in large neurons in early, late, and chronic phases. In addition, mGluR(1) and mGluR(5) expression after hemisection was significantly increased in astrocytes in early, late, and chronic phases; whereas mGluR(2/3) did not display any significant changes. In conclusion, our data demonstrate long-term changes in expression levels of Group I mGluRs (mGluR(1) and mGluR(5)) in both neurons and astrocytes in segments below a unilateral SCI. Thus, permanent alterations in dorsal horn receptor expression may play important roles in transmission of nociceptive responses in the spinal cord following SCI.     

Temporal and spatial distribution of Fos protein in the lumbar spinal dorsal horn neurons in the rat with chronic constriction injury to the sciatic nerve

The temporal and spatial expression pattern of Fos protein in spinal dorsal horn neurons was examined by immunohistochemistry in rats with chronic constriction injury (CCI) to the sciatic nerve. In normal animals, a few Fos-immunoreactive (-IR) neurons were detected in the dorsal horn of the lumbar spinal cord. Following induction of CCI, a very large number of Fos-IR neurons appeared in the spinal dorsal horn, but a significant number of Fos-IR neurons were also observed in the contralateral dorsal horn where primary afferents of the injured sciatic nerve rarely project. Sham-operated animals also had a significant number of Fos-IR neurons in the dorsal horn bilaterally. The number of Fos-IR neurons reached its maximal level 1 day following placement of the ligatures (PO 1d). The ratio of the number of Fos-IR neurons in the ipsilateral dorsal horn to the contralateral dorsal horn, however, had its peak level 3 days following CCI (3.1-fold increase compared to the contralateral dorsal horn). The number of Fos-IR neurons in the dorsal horn gradually decreased, but increased again around PO 15d. On PO 30d, the number of Fos-IR neurons decreased and became comparable to that in normal animals. The present results indicate that the induction of Fos-IR neurons in the dorsal horn caused by CCI is biphasic and reaches its maximal level on PO 3d, near the time of hyperalgesia onset.     

脊髓和马尾神经损伤后慢性神经源性疼痛外科治疗策略

目的 探讨脊髓和马尾神经损伤后慢性神经源性疼痛的神经外科治疗策略.方法 脊髓和马尾神经损伤后慢性神经源性疼痛20例,年龄28～81岁,病程8个月～50年.共行镇痛手术23次,其中,脊髓背根入髓区显微外科切开术19次,脊髓电刺激术3次,鞘内靶控输注系统植入术1次.采用视觉模拟评分（VAS）来评估手术疗效,以术后疼痛缓解大于75%为疗效优秀,疼痛缓解50%～75%为良好,疼痛缓解小于50%为差.结果 随访6个月～4年,10例疼痛消失,停用镇痛剂,生活质量改善;7例疼痛明显减轻,VAS 2～4分,其中,5例停用镇痛剂,2例镇痛剂使用量明显下降,生活质量改善;3例疼痛无明显改善.结论 脊髓背根入髓区显微外科切开术和脊髓电刺激术对脊髓和马尾神经损伤后慢性神经源性疼痛患者疗效满意,但其适应证有差别,应根据患者的损伤节段、损伤程度和疼痛部位等具体情况选择手术方式.     

Temporal plasticity of dorsal horn somatosensory neurons after acute and chronic spinal cord hemisection in rat

Unilateral T13 hemisection of the rat spinal cord produces a model of chronic spinal cord injury (SCI) that is characterized by bilateral hyperexcitability of lumbar dorsal horn neurons, and behavioral signs of central pain. While we have demonstrated that responsiveness of multireceptive (MR) dorsal horn neurons is dramatically increased at 28 days after injury, the effects of acute hemisection are unknown and predicted to be different than observed chronically. In the present study, the consequences of T13 hemisection are examined acutely at 45 min in MR neurons both ipsilateral and contralateral to the site of injury, and compared to the same class of cells at 28 days after injury (n=20 cells total per group: 2–3 cells/side of the cord from n=5 animals). Acutely, ipsilateral to the hemisection, both spontaneous and evoked activity of MR neurons were significantly increased, whereas contralaterally, only evoked activity was significantly increased. In animals 28 days after hemisection, spontaneous activity of MR neurons was comparable to intact levels ipsilaterally, and cells exhibited hyperexcitability to evoked stimuli bilaterally. Expansion of cutaneous receptive fields was observed only in hindpaws ipsilateral to the lesion, acutely. These results demonstrate dynamic plasticity in properties of dorsal horn somatosensory neurons after SCI.     

Neurotransmitters in the spinal cord dorsal horn in a model of painful neuropathy and in nerve crush

We tested the hypothesis that neurochemical changes in the spinal cord dorsal horn associated with neuropathic pain states differ from those seen in association with non-painful neuropathies. Immunohistochemistry was performed on spinal cord sections from rats with a chronic constriction injury (CCI), which develop hyperalgesia, and from animals with a nerve crush injury, which do not develop hyperalgesia or other signs of a painful syndrome. Immunohistochemistry was quantified by computer-assisted densitometry. Calcitonin gene-related peptide (CGRP) immunoreactivity and substance P (SP) immunoreactivity were decreased from 1 to 4 weeks after injury in CCI and from 2 to 6 weeks in crush. Gammaaminobutyric acid immunoreactivity was unchanged in both conditions at all time points. Met-enkephalin (Metenk) immunoreactivity was increased in CCI and unchanged in crush. Although SP and CGRP are involved in pain transmission, we conclude that their decrease in immunoreactivity is not specific for the CCI model, but rather a more general event in nerve de- and regeneration. The increase in immunoreactivity for the opioid peptide Met-enk, however, was only seen in the late phase of CCI, and may be specific for conditions associated with neuropathic pain and its resolution.     

The ability of inhibitory controls to ‘switch-off’ activity in dorsal horn convergent neurones in the rat

Unitary extracellular recordings were made from 51 convergent neurones in the dorsal horn of the lumbar spinal cords of urethane anaesthetized rats. All the cells tested responded to sustained noxious mechanical stimulation of their receptive fields on the ipsilateral hindpaw, but only 26/49 gave tonic responses lasting for more than 5 min. In all 26 cells, these tonic responses were depressed by diffuse noxious inhibitory controls (DNIC) triggered by applying noxious conditioning stimuli elsewhere on the body. In seven cells, the inhibitory effects could involve a complete abolition of activity and in five cells, when this occured, activity did not return during 2.5–6 min periods of observation following removal of the conditioning stimuli. However, in those cases, activity could be restored to pre-conditioning levels by further manipulations of the receptive field - either removal and re-application of the original stimulus or brief application of an additional stimulus. These results show that inhibitory controls can ‘switch-off’ activity in at least a small proportion of dorsal horn convergent neurones. One possible explanation would be that in these neurones, responses to sustained noxious stimuli may depend on activity in a positive feedback circuit within the central nervous system, which when interrupted, may be restored only by additional afferent inputs. The existence of such a loop could also explain the finding of convergent neurones which initially were not spontaneously active but which after stimulation of their receptive fields, developed on-going discharges which could be switched-off by DNIC.     

脊髓背根入髓区毁损术治疗脊髓和马尾神经损伤后疼痛

目的 探讨脊髓和马尾神经损伤后疼痛的神经外科治疗方法、效果和安全性.方法 脊髓和马尾神经损伤后疼痛患者14例,年龄28～72岁,病程8个月-28年;疼痛位于下肢感觉减退和缺失区,为烧灼、压榨或痉挛样疼痛,视觉模拟疼痛评分(Visual analogy scale,VAS)8～10分;均伴有不同程度的下肢肌力下降.14例患者共行脊髓背根入髓区(dorsal root entry zone,DREZ)毁损术15次.结果 随访3个月-3年.6例疼痛消失,5例疼痛明显减轻,停用或少量使用镇痛剂,VAS 2～4分;3例疼痛无明显改善.所有病例无严重手术并发症.结论 DREZ毁损术对脊髓和马尾神经损伤后慢性神经病理性疼痛安全有效.     

Galectin-1 is involved in the potentiation of neuropathic pain in the dorsal horn

Galectin-1 is one of the endogenous-galactoside-binding lectins, suggested to be involved in a variety of functions, such as neurite outgrowth, synaptic connectivity, cell proliferation and apoptosis. This protein is expressed in the dorsal root ganglion (DRG) and the spinal cord in the developing and adult rats, especially intensely in small DRG neurons. In the present study, we examined whether galectin-1 is colocalized with TrkA or c-Ret mRNA in small DRG neurons and the effect of axotomy on the expression of galectin-1 in the spinal cord. About 20% of the DRG neurons showed intense galectin-1-immunoreactivity (IR). Of the intensely galectin-1-IR DRG neurons, 93.9% displayed c-Ret mRNA positive signals. On the other hand, only 6.8% displayed TrkA mRNA positive signals. Galectin-1-IR was increased in the dorsal horn at 1 to 2 weeks after axotomy. Intrathecal administration of anti-recombinant human galectin-1 antibody (anti-rhGAL-1 Ab) partially but significantly attenuated the upregulation of substance P receptor (SPR) in the spinal dorsal horn and the mechanical hypersensitivity induced by the peripheral nerve injury. These data suggest that endogenous galectin-1 may potentiate neuropathic pain after the peripheral nerve injury at least partly by increasing SPR in the dorsal horn.     

Increased receptive field size of dorsal horn neurons following chronic spinal cord hemisections in cats

The somatotopic organization of the 17 dorsal horn was studied using extracellular recordings in normal cats, and in cats with acute or chronic spinal cord hemisection at T13, sparing the dorsal columns. Based on data concerning recovery of function and collateral sprouting of afferents following hemisections, we predicted that the lesion would result in increases in receptive field size and decreases in the specificity of the somatotopic map. In normal animals, the usual mediolateral, rostrocaudal and dorsoventral somatotopic sequences were found. Following acute hemisections (6 h–5 days), there were changes in spontaneous and evoked activity, but receptive field sizes and somatotopic organization remained unchanged. Following chronic hemisections (88–174 days), proximal hindlimb receptive fields in the lateral dorsal horn ipsilateral to the lesion increased dramatically in size and were significantly larger than similar receptive fields on the contralateral side. The largest of these fields extended from the dorsal midline to the middle of the foot. Receptive field sizes elsewhere in the dorsal horn remained unchanged, as did somatotopic organization in general. These findings indicate that hemisections result in a complex series of changes consisting of an early stage of anatomically generalized changes in excitability and a later stage of highly localized changes in receptive field size. Possible mechanisms for these changes, as well as their relationship to recovery of function, are discussed.     

The properties of neurones recorded in the superficial dorsal horn of the rat spinal cord

The physiological properties of neurones in the superficial laminae of the dorsal horn of the fourth and fifth lumbar segments of the rat spinal cord have been investigated in decerebrate spinal animals. Both extracellular recordings with platinum-plated tungsten microelectrodes (n = 72) and intracellular recordings with glass microelectrodes (N = 79) were made. Attempts were made to fill cells intracellularly with horseradish peroxidase or Lucifer Yellow. Thirty-seven percent of the intracellularly injected neurones were recovered after histological processing and their cell bodies found to be in lamina 1 or 2 and in the dorsal white matter overlying lamina 1. The dendritic spread of the stained neurones was maximal in the rostrocaudal plane with a restricted mediolateral spread. The physiological properties of the extracellularly recorded units, the intracellularly unidentified units, and the intracellularly stained units were the same. The neurones were characterized by low background activity and all had excitatory receptive fields on the lower limb. Some neurones responded only to low-threshold mechanical stimulation of the skin or only to noxious skin stimulation but the majority of units (58%) were wide-dynamic-range cells responding to both types of stimuli. Receptive field classification was made questionable, however, by the existence of cells (9%) that exhibited a spontaneous shift in the size of their receptive fields and in the type of stimulus that elicited a response. The neurones in the superficial dorsal horn commonly showed a marked inhibition to repeated cutaneous stimuli (27%) or a prolonged afterdischarge followed a single stimulus (20%). Afferent input from the sural nerve was found to be from A and C fibres in both extra- and intracellular recordings. Aδ- and C-mediated excitations were most common although convergent inputs from Ab?-fibres occurred in 40% of units. No correlation was found between cell structure or distribution of dendritic fields and physiological properties in our small sample of intracellularly stained cells. The morphology of the cells was highly diverse, as were the different receptive fields. There was, however, some correlation between the location of cell bodies and their responses. Neurones responding only to low-threshold stimuli were distributed either in the dorsal white matter or in inner lamina 2. Wide-dynamic-range cells were distributed throughout the superficial dorsal horn. These results suggest that neurones of different shapes and positions may subserve the same function and, conversely, that neurones of the same shape and position may subserve different functions.     

Evidence for two populations of rat spinal dorsal horn neurons excited by urinary bladder distension

Spinal L6-S2 dorsal horn neurons of cervical spinal cord-transected, decerebrate female rats were characterized using urinary bladder distension (UBD) as a visceral stimulus. Constant pressure, phasic, graded (20-80 mm Hg, 20 s) air UBD was delivered via a transurethral catether and extracellular single-unit recordings obtained from all neurons excited by UBD. Responses to graded UBD and noxious/non-noxious cutaneous stimuli were determined in 258 neurons which could be stratified into two groups based on their effect of a counterirritation stimulus: Type I neurons (n=112) were inhibited by noxious pinch presented in a non-segmental field; Type II neurons (n=146) were not similarly inhibited. Both Types of neurons were identified in both superficial and deep recording sites and demonstrated graded responses to graded UBD. All UBD-excited neurons had convergent cutaneous receptive fields (RFs) excited by non-noxious and/or noxious stimuli. As a group, Type I neurons had a period of decreased activity following termination of the distending stimulus whereas Type II neurons typically had a sustained afterdischarge. UBD-evoked activity in Type II neurons was inhibited more than similar activity in Type I neurons by both intravenous morphine and lidocaine. These results support the assertion that at least two different populations of spinal dorsal horn neurons exist which encode for a stimulus of urinary bladder distension. These populations are an analogue to previously characterized, similar neuronal populations excited by colorectal distension and suggest that they are representative of the overall phenomenon of visceral sensory processing, a component of which is nociception.     

Segmental hypersensitivity and spinothalamic function in spinal cord injury pain

The mechanisms underlying central pain following spinal cord injury (SCI) are unsettled. The purpose of the present study was to examine differences in spinothalamic tract function below injury level and evoked pain in incomplete SCI patients with neuropathic pain below injury level (central pain) versus those without such pain. A clinical examination, quantitative sensory testing and magnetic resonance imaging (MRI) were performed in 10 SCI patients with below-level pain and in 11 SCI patients without neuropathic pain. Patients with and without pain had similar reductions of mechanical and thermal detection thresholds below injury level. SCI patients with central pain had sensory hypersensitivity in dermatomes corresponding to the lesion level more frequently than SCI patients without pain, but this may in part be explained by the exclusion of at-level spontaneous pain in the pain-free group. The rostral-caudal extent of the lesion measured by MRI did not differ between the two patient groups, and there were no statistically significant differences in any of the predefined areas of interest on the axial plane images. This study suggests that neuronal hyperexcitability plays a key role in central SCI pain and furthermore - in contrast to previous findings - that loss of spinothalamic functions does not appear to be a predictor for central neuropathic pain in spinal cord injury.     

Adult rat forelimb dysfunction after dorsal cervical spinal cord injury

Repairing upper extremity function would significantly enhance the quality of life for persons with cervical spinal cord injury (SCI). Repair strategy development requires investigations of the deficits and the spontaneous recovery that occurs when cervical spinal cord axonal pathways are damaged. The present study revealed that both anatomically and electrophysiologically complete myelotomies of the C4 spinal cord dorsal columns significantly increased the adult rat's averaged times to first attend to adhesive stickers placed on the palms of their forepaws at 1 week. Complete bilateral myelotomies of the dorsal funiculi and dorsal hemisection, but not bilateral dorsolateral funiculi injuries, also similarly increased these times at 1 week. These data extend a previous finding by showing that a forepaw tactile sensory deficit that occurred in the adult rat after bilateral C4 spinal cord dorsal funiculi injury is due to damage of the dorsal columns. Averaged times to first attend to the stickers also decreased to those of sham-operated rats at 3 and 4 weeks post-dorsal hemisection with weekly testing. In contrast, a separate group of rats with dorsal hemisections had significantly increased times when tested only at 4 weeks. These data indicate that frequent assessment of this particular behavior in rats with dorsal hemisections extinguishes it and/or engenders a learned response in the absence of sensory axons in the dorsal columns and dorsolateral funiculi. This finding contrasted with weekly testing of grid walking where increased forelimb footfall numbers persisted for 4 weeks post-dorsal hemisection.     

Direct evidence of primary afferent sprouting in distant segments following spinal cord injury in the rat: colocalization of GAP-43 and CGRP

Mechanical and thermal allodynia develops after spinal cord injury in three areas relative to the lesion: below level, at level, and above level. The present study tests colocalization of CGRP, associated with nociceptive neurons, with growth-associated protein (GAP-43), expressed in growing neurites, to test for neurite sprouting as a mechanism for reorganization of pain pathways at the level of the lesion and distant segments. Male Sprague-Dawley rats were divided into three groups: sham control (N = 10), hemisected at T13 and sacrificed at 3 days (N = 5) and at 30 days (N = 5) following surgery, the spinal cord tissue was prepared for standard fluorescent immunocytochemistry using mouse monoclonal anti-GAP-43 (1:200) and/or rabbit polyclonal anti-CGRP (1:200), density of immunoreaction product (IR) was quantified using the Bioquant software and values from the hemisected group were compared to similar regions from the sham control. We report significant increases at C8 and L5, in CGRP-IR in lamina III compared to control tissue (P < 0.05). We report significant bilateral increases in GAP-43-IR at C8, T13, and L5 segments in lamina I through IV, at 3 days post hemisection, compared to control tissue (P < 0.05), some of which is colocalized with alpha-CGRP. The increased area and density of GAP-43-IR is consistent with neurite sprouting, and the colocalization with alpha-CGRP indicates that some of the sprouting neurites are nociceptive primary afferents. These data are consistent with endogenous regenerative neurite growth mechanisms that occur near and several segments from a spinal lesion, that provide one of many substrates for the development and maintenance of the dysfunctional state of allodynia after spinal cord injury.     

Cholinergic effects on spinal dorsal horn neurons in vitro: an intracellular study

The cholinoceptive properties of dorsal horn neurons (lamina III–V) were investigated by means of intracellular recordings from the rat isolated spinal cord slice preparation. In half of the neurons investigated, acetycholine (ACh) evoked a dose-dependent slow depolarization and increase in excitability; hyperpolarization was observed in 10% of neurons. Acetyl-β-methylcholine (MCh) similarly depolarized 39% and hyperpolarized 25% of neurons tested; depolarization was also observed following bethanechol. Responses to the muscarinic agonists were abolished by atropine (10⁻⁵ M). Nicotine depolarized 84% of tested neurons; dihydro-β-erythroidine (5 × 10⁻⁵M) and (+)-tubocurarine (10⁻⁶ M) antagonized this depolarization. ACh-, MCh- and nicotine-induced depolarizations, associated with changes in input resistance, were maintained in the presence of tetrodotoxin (10⁻⁶ M). Substance P, as well as repetitive electrical stimulation of the dorsal root, also evoked depolarization in ACh-sensitive neurons. Atropine, but not (+)-tubocurarine, diminished responses to both substance P and dorsal root stimulation. These results indicate that dorsal horn neurons are ACh-sensitive and possess both muscarinic and nicotinic receptors. In addition, the parallel sensitivity of neurons to muscarinic agonists, substance P and dorsal root stimulation, as well as the parallel antagonistic effect of atropine, are supportive of a common ionic mechanism underlying the activation of muscarinic and substance P receptors.