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.     

Involvement of protein kinase c in responses of rat dorsal horn neurons to mechanical stimuli and periaqueductal gray descending inhibition

 The effects of a protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on the activity and periaqueductal gray (PAG)-induced inhibition of rat dorsal horn neurons of the lumbar spinal cord were tested. A microdialysis fiber was placed through the dorsal horn for the purpose of local application of pharmacological agents. Extracellular single-unit recordings from dorsal horn neurons were made near the microdialysis fiber. TPA was tested on nociceptive dorsal horn cells. There was a significant increase in the background activity and responses to ”brush”, with no changes in responses to pressure and pinch stimuli. TPA also significantly blocked the PAG-induced inhibition of responses to brush, press, and pinch. These effects were eliminated by coadministration of the PKC inhibitor NPC-15437. The solvent, which contained dimethyl sulfoxide, was also tested for its effect on the responses to peripheral mechanical stimuli and PAG-induced inhibition of the dorsal horn neurons. There were no significant changes. This experiment suggests that activation of the PKC second messenger system might increase the activity of dorsal horn neurons and their responses to peripheral stimuli; in addition, the phorbol ester attenuated the PAG-induced descending inhibition of the dorsal horn neuron activity. Received: 15 May 1996 / Accepted: 14 November 1996     

Projection of a cutaneous nerve to the spinal cord of the pigeon II. Responses of dorsal horn neurons

Summary The responses of dorsal horn neurons to both electrical stimulation of a cutaneous nerve and natural stimulation of skin receptors have been studied in an avian species, the pigeon. Neurons located in either lamina I or lamina IV were recorded. Most lamina IV neurons had short-latency responses to electrical stimulation of a cutaneous nerve and were activated by stimulation of sensitive mechanoreceptors. This points to an input from mechanoreceptors innervated by large afferent fibers. Lamina I neurons which were usually located near the entrance zone of small fibers had longer latency responses and had often an input from several groups of afferent fibers including C-fibers. Many lamina I neurons were activated specifically by noxious stimulation. Some had an input from sensitive mechanoreceptors but possibly through an additional synapse. A few lamina I neurons responded specifically to activation of cold receptors. Some dorsal horn neurons showed segmental inhibition. Altogether, the characteristics of dorsal horn neurons in the pigeon studied so far were similar to those in mammalian species.     

PKC在成年大鼠脊髓背角C-纤维诱发电位长时程增强的诱导和维持中的作用

目的：探讨蛋白激酶C (PKC) 在大鼠脊髓背角C-纤维诱发电位长时程增强（LTP）的诱导和维持中的作用。方法： 细胞外记录技术在脊髓腰膨大部记录背角浅层神经元C-纤维诱发电位。 结果：(1) PKC的选择性抑制剂chelerythrine（200 μmol/L）或G 6983（100 μmol/L）对脊髓背角C-纤维诱发电位的基础电位没有影响，但可完全阻断脊髓背角LTP的诱导。(2) Chelerythrine或G 6983呈时间依赖性翻转脊髓背角LTP。在LTP 诱导后15 min，脊髓局部给予chelerythrine（200 μmol/L）后，LTP逐渐降低，于给药后70 min降至对照水平；而G 6983（100 μmol/L）产生同chelerythrine相似的效应，在用药后110 min，LTP降至对照水平。但同样浓度的chelerythrine或G 6983在LTP 诱导后3 h，均不能翻转业已建立的LTP。结论： PKC参与脊髓背角C-纤维诱发电位LTP的诱导和早期维持，而不影响晚期LTP的维持。     

Dendritic spread of dorsal horn neurons in cats

Summary Observations of neurons in dorsal horn laminae IV–VI of the lumbosacral segments of Golgi-stained spinal cords in kittens and adult cats revealed laminar differences in dendritic architecture. Many neurons in lamina IV had dense, bushy dendritic fields. Lamina V contained, in addition to bushy cells similar in appearance to those of lamina IV, increasing numbers of neurons with radiating dendritic fields. Lamina VI was composed almost exclusively of neurons with radiating dendritic fields. These qualitative differences among laminae were accompanied by systematic variations in mean dendritic spread, which increased more than two-fold in adult cats between laminae IV and VI. A second gradient of dendritic spread was found within individual laminae: dendritic spread, particularly medial to lateral spread, increased for successively more lateral cells within a lamina. These differences in the spread of dendrites for neurons in different regions of the dorsal horn may be related to variations in the areas of peripheral receptive fields of dorsal horn neurons.     

Systemic lidocaine induces expansion of the receptive field of spinal dorsal horn neurons in rats

 The effect of intravenous administration of the local anaesthetic lidocaine (1, 3 or 5 mg/kg) on the responsiveness and size of the cutaneous receptive fields of 18 lumbar dorsal horn neurons was examined in intact urethane-anaesthetized rats. Lidocaine induced expansion of the receptive field in the majority of neurons examined, particularly after the two higher doses. The expansion occurred usually within 10 min after lidocaine injection and the effect was reversible. Lidocaine also altered the responsiveness of dorsal horn neurons to peripheral mechanical stimulation. The responses of wide-dynamic-range neurons to noxious pinch were usually inhibited by lidocaine. However, some low-threshold neurons started to react to noxious mechanical stimulation and some high-threshold neurons started to respond to innocuous brushing after lidocaine injection. The present results show that moderate doses of systemic lidocaine induce complex changes in the excitability of dorsal horn neurons, including an increase in the size of the receptive field and altered response characteristics to mechanical stimulation. Received: 16 July 1997 / Accepted: 21 October 1997     

Cellular interactions in the thymus regulate the protein kinase C signaling pathway

We provide evidence that thymocytes receive signals from the thymic microenvironment which regulate the protein kinase C (PKC) signaling pathway. Thus, phorbol 12-myristate 13-acetate (PMA) causes a PKC-dependent down-regulation of CD4 expression and induces apoptosis in isolated thymocytes but has little effect on thymocytes maintained within intact thymic lobes or in reaggregate lobes containing purified thymocytes with either thymic or non-thymic stromal cells. Moreover, compact pellets of thymocytes alone are protected from the effects of PMA. This protection is maintained when the compacted thymocytes are rigorously depleted of MHC class II-expressing cells. We conclude that signals arising from thymocyte-thymocyte contact control the utilization of the PKC cascade. These observations have implications for thymocyte signaling in general as well as for the interpretation of studies carried out on thymocyte suspensions.     

Intersegmental synchronization of spontaneous activity of dorsal horn neurons in the cat spinal cord

Extracellular recordings of neuronal activity made in the lumbosacral spinal segments of the anesthetized cat have disclosed the existence of a set of neurons in Rexed's laminae III–VI that discharged in a highly synchronized manner during the occurrence of spontaneous negative cord dorsum potentials (nCDPs) and responded to stimulation of low-threshold cutaneous fibers (<1.5×T) with mono- and polysynaptic latencies. The cross-correlation between the spontaneous discharges of pairs of synchronic neurons was highest when they were close to each other, and decreased with increasing longitudinal separation. Simultaneous recordings of nCDPs from several segments in preparations with the peripheral nerves intact have disclosed the existence of synchronized spontaneous nCDPs in segments S1–L4. These potentials lasted between 25 and 70 ms and were usually larger in segments L7–L5, where they attained amplitudes between 50 and 150 μV. The transection of the intact ipsilateral hindlimb cutaneous and muscle nerves, or the section of the dorsal columns between the L5 and L6, or between the L6 and L7 segments in preparations with already transected nerves, had very small effects on the intersegmental synchronization of the spontaneous nCDPs and on the power spectra of the cord dorsum potentials recorded in the lumbosacral enlargement. In contrast, sectioning the ipsilateral dorsal horn and the dorsolateral funiculus at these segmental levels strongly decoupled the spontaneous nCDPs generated rostrally from those generated caudally to the lesion and reduced the magnitude of the power spectra throughout the whole frequency range. These results indicate that the lumbosacral intersegmental synchronization between the spontaneous nCDPs does not require sensory inputs and is most likely mediated by intra- and intersegmental connections. It is suggested that the occurrence of spontaneous synchronized nCDPs is due to the activation of tightly coupled arrays of neurons, each comprising one or several spinal segments. This system of neurons could be involved in the modulation of the information transmitted by cutaneous and muscle afferents to functionally related, but rostrocaudally distributed spinal interneurons and motoneurons, as well as in the selection of sensory inputs during the execution of voluntary movements or during locomotion. Electronic Publication     

Nociceptive neurones in the superficial dorsal horn of cat lumbar spinal cord and their primary afferent inputs

Summary The morphology, background activity and responses to stimulation of primary afferent inputs of small neurones in the superficial dorsal horn which could only be excited from the skin by noxious stimulation were investigated by intracellular recording and ionophoresis of HRP. Neurones which gave similar responses to afferent stimulation were morphologically heterogeneous with respect to dendritic tree geometry and axonal projection, but were located around the lamina I/II border. Cutaneous excitatory receptive fields responding to noxious stimulation were generally small; most neurones had more extensive inhibitory fields responding to innocuous mechanical stimulation, in many cases overlapping the excitatory fields. Generally, stimulation of the excitatory field resulted in depolarization of the neurone and increased action potential firing, and stimulation of the inhibitory field resulted in hyperpolarization. Electrical stimulation of peripheral nerves revealed the existence of converging excitatory inputs carried by different fibre groups, and all neurones received an inhibitory input activated at low threshold. Excitatory responses were short-lived and occurred consistently in response to repeated stimulation. Central delay measurements gave evidence of a number of A monosynaptic inputs but only one A monosynaptic input; inhibitory inputs along A fibres were polysynaptic. The constant latency and regularity of the C response suggested monosynaptic connections. Low intensity stimulation of inhibitory inputs elicited a short-lived i.p.s.p. which increased in amplitude with increasing stimulus strength until it disappeared into a more prolonged hyperpolarization. This was associated with inhibition of background action potentials, and increased in duration with increasing stimulus strength up to C levels, indicating an A and C component. It is suggested that the level of excitability of these neurones depends on the relative amounts of concurrent noxious and innocuous stimulation, and that the resultant output, which is conveyed mainly to other neurones within the spinal cord, could modulate reflex action at the spinal level as well as affecting components of ascending sensory pathways.Supported by grant no. 11853/1.5 from the Wellcome Trust     

鞘内注射右美托咪定干预慢性神经痛模型大鼠脊髓背角蛋白激酶C的表达

背景：右美托咪啶是一种高效、高选择性的α2肾上腺素受体激动剂,具有镇静、镇痛、抗焦虑等作用,对呼吸影响小。目的：观察鞘内注射右美托咪定对坐骨神经分支选择性损伤模型大鼠的镇痛作用。方法：雄性SD大鼠60只,随机均分为3组。除正常对照组外,另2组大鼠建立坐骨神经分支选择性损伤模型,右美托咪定组在造模后14 d内每天鞘内注射右美托咪定3 μg/kg,生理盐水组在同时注射等容量的生理盐水。于坐骨神经分支选择性损伤模型前、术后鞘内给药前和鞘内给药后2,7,14 d测定大鼠热刺激缩足反射潜伏期和机械刺激缩足反射痛阈值,并在术后第2,7,14天鞘内注射药物后,每组每个时间点分别处死4只大鼠,取其L_4-6脊髓,RT-PCR及Western blot法分别检测脊髓背角蛋白激酶C mRNA及蛋白水平的表达情况,苏木精-伊红染色检测脊髓背角神经元形态学变化,免疫组织化学法检测脊髓背角蛋白激酶C蛋白表达的水平及分布情况。结果与结论：与正常对照组比较,给药前、给药后各时点生理盐水组和右美托咪定组热刺激缩足反射潜伏期和机械刺激缩足反射痛阈值均明显降低(P < 0.05);与生理盐水组比较,给药后各时点右美托咪定组热刺激缩足反射潜伏期延长和机械刺激缩足反射痛阈值均明显升高(P < 0.05);右美托咪定组脊髓背角蛋白激酶C表达明显明显低于生理盐水组,且在给药14 d时达到最低接近于正常对照组。右美托咪定组脊髓背角神经元凋亡程度也较生理盐水组轻微,在给药14 d时神经元形态基本接近正常对照组。提示鞘内注射右美托咪定可减轻坐骨神经分支选择性损伤模型引起的痛敏,可能与其抑制脊髓背角蛋白激酶C的表达相关。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Inhibitors of G-proteins and protein kinases reduce the sensitization to mechanical stimulation and the desensitization to heat of spinothalamic tract neurons induced by intradermal injection of capsaicin in the primate

 Intradermal injection of capsaicin results in sensitization of spinothalamic tract cells to brushing and pressure applied to the cutaneous receptive field in anesthetized monkeys. A significant increase in background activity also occurs immediately after capsaicin injection that lasts for at least 2 h. A 40–50% decrease in the response to noxious heat stimuli is also observed following capsaicin injection. This study investigated the spinal role of second messengers by extracellularly recording from spinothalamic tract cells and delivering inhibitors of second messenger pathways to the spinal cord by microdialysis. Blockade of protein kinases with the general protein kinase inhibitor, H7 (5.0 mM, n = 6), reduced the sensitization of the cells to brush and pressure. Blockade of protein kinase C with NPC15437 (10.0 mM, n = 10) reduced the increased background activity and the increased responses to brush. Blockade of protein kinase A with H89 (0.01 mM, n = 9) was most effective. H89 reduced the background activity, the increased responses to brush and press, and reversed the decreased response to noxious heat stimuli. Blockade of G-proteins with the general G-protein inhibitor, GDP-β-S (1.0 mM, n = 9), reduced the background activity and the responses to brush and pressure without affecting the decreased response to heat. Thus, multiple intracellular messengers appear to be involved in the processing of central sensitization induced by activation of C-fibers following intradermal injection of capsaicin. Received: 5 June 1996 / Accepted: 29 November 1996     

The laminar organization of dorsal horn cells responding to peripheral C fibre stimulation

Summary Cat dorsal horn was searched for all detectable units that responded to peripheral C fibre input. Fifty-seven such units were examined in detail. They were located in two main areas. One group was in the superficial laminae 1, 2, and possibly dorsal 3 (n = 29), and the other group was much deeper in laminae 5 and 6 (n = 24). Only four units were situated in the region of lamina 4.Differences were found in the responses to C fibre stimulation of these two groups, both in the optimum stimulus and in the timing of responses to repeated stimulation. Superficial units often did not respond to C fibre stimulation unless a train of two or more stimuli (10 ms apart) were applied, but when responses did occur they were usually very even and regular, with precise onset latencies on repeated stimulation. Deep units tended to need only one peripheral C fibre stimulus for excitation, but the responses were irregular with latencies fluctuating with each stimulus. Some superficial and deep units showed a steady increase in latency of the late C response on repeated stimulation. Increases of up to 80 ms after 30 s of stimulation at 1 Hz were observed.The results are discussed in terms of the neuronal connections in the dorsal horn.The work was supported by the Medical Research Council and the National Institutes of HealthM. Fitzgerald is a Medical Research Council Training Fellow     

Serotoninergic-mediated inhibition of substance P sensitive deep dorsal horn neurons: a combined electrophysiological and morphological study in vitro

Dorsal horn neurons that express the neurokinin 1 receptor (NK-1R) play an important role in nociceptive processing. The targetting of NK-1R neurons by serotoninergic (5-hydroxytryptamine, 5-HT) axons would provide a straightforward means to exert an inhibitory analgesic effect at spinal level. This study used single cell electrophysiology to analyse and correlate the responses of rat deep DH neurons in vitro to both 5-HT and the NK-1R agonist [Sar⁹,Met(O₂)¹¹]-substance P (SP). Subsequently a combination of immunocytochemistry and confocal imaging was applied to biocytin-filled laminae III–VI neurons to reveal putative 5-HT innervation in this neuronal sample. A population of neurons was identified in which 5-HT (50 µM) significantly attenuated the dorsal root-evoked excitatory postsynaptic potential and [Sar⁹,Met(O₂)¹¹]-SP (2 µM) induced a direct tetrodotoxin-resistant depolarisation. Immunolabelling revealed that all of these neurons were inhibited by 5-HT, including those that were excited by [Sar⁹,Met(O₂)¹¹]-SP, were overlaid by a plexus of 5-HT immunoreactive fibres and in some instances, closely apposed putative contacts with somata and proximal dendrites identified although their incidence was low. Inhibition by 5-HT of deep DH neurons directly responsive to SP may account at least in part for monoamine-induced modulation of nociceptive processing in the spinal cord.     

An analysis of response properties of spinal cord dorsal horn neurones to nonnoxious and noxious stimuli in the spinal rat

Summary Electrophysiological properties of neurones in the spinal cord dorsal horn were studied in decerebrated, immobilized spinal rats. Extracellular recordings were performed at the thoraco-lumbar junction level. Each track was systematically located by extracellular injection of pontamine sky blue. According to their responses to mechanical peripheral stimuli, cells were classified in four classes: Class 1 cells: Cells activated only by nonnoxious stimuli. They were divided into — 1A: hair movement and/or touch and 1B: hair movement and/or touch and pressure or pressure only. Class 2 cells: Cells driven by both nonnoxious and noxious stimuli, divided into — 2A: hair movement and/or touch, pressure, pinch and/or pin-prick, and 2B: pressure, pinch and/or pin-prick. Class 3 cells: Cells only activated by noxious stimuli (pinch and/or pin-prick). Class 4 cells: Cells responding to joint movement or pressure on deep tissues.Peripheral transcutaneous or sural nerve stimulation clearly showed that class 1 cells were activated only by A fiber input while 68% of classes 2 and 3 cells received A and C input. Histological examination indicated that cells driven only by noxious input were located either in the deepest part or in the marginal zone (lamina I) of the dorsal horn. Nevertheless, some lamina I cells were also driven by both nonnoxious and noxious stimuli. In addition, there is a great deal of overlap between class 1 and class 2 cells. This fact was confirmed by considering the wide distribution in the dorsal horn of cells receiving A and C input. However, spinal organization of the different classes of cells consists of a preferential distribution rather than a strict lamination. This study indicates that properties of dorsal horn interneurones in the rat have a high degree of similarity with those previously described in other species (cat and monkey).This work was supported by the C.N.R.S. (E.R.A. 237).     

A direct projection from the medial vestibular nucleus to the cervical spinal dorsal horn of the rat, as demonstrated by anterograde and retrograde tracing

Summary Phaseolus vulgaris leucoagglutinin and wheat germ agglutinin-horseradish peroxidase were iontophoretically injected into different parts of the vestibular nuclear complex (VNC) of the rat. Injections centered into the caudal part of the medial vestibular nucleus revealed a vestibulospinal projection predominantly to the dorsal horn of the cervical spinal cord, besides the expected projection to the intermediate zone (IZ) and ventral horn (VH). While most of the anterogradely labelled fibres could be localized in laminae III to V, some scattered fibres were also seen in laminae I and VI. Lamina II remained free of labelling. The dorsal horn (DH) area with detectable anterograde labelling showed a rostrocaudal extension from C₁-C₆. Injections into other parts of the VNC labelled fibres and terminals in the IZ and VH while the DH remained almost free of labelling. Additionally, fluorogold and wheat germ agglutininhorseradish peroxidase were pressure- or iontophoretically injected at different levels into the spinal cord to confirm the projection to the dorsal horn by means of retrograde tracing. Labelled neurons in the area of the medial vestibular nucleus (MVN), from which anterograde labelling in the DH was obtained, were only detectable after fluorogold and wheat germ agglutinin-horseradish peroxidase injections into the cervical spinal cord, in particular its DH. This projection from the caudal medial vestibular nucleus to the dorsal horn of the cervical spinal cord probably enables the VNC to influence sensory processing in the DH, in addition to its well-established influence on posture and locomotion via projections to the intermediate zone and ventral horn.Abbreviations BSA bovine serum albumin - CuN cuneate nucleus - CCN central cervical nucleus - cMVN caudal medial vestibular nucleus - cVST caudal vestibulospinal tract - DAB diaminobenzidine - DH dorsal horn - DRG dorsal root ganglia - DVN descending vestibular nucleus - ECN external cuneate nucleus - FCS fetal calf serum - FG fluorogold - HRP horseradish peroxidase - icp inferior cerebellar peduncle - In intercalated nucleus - IZ intermediate zone - LVN lateral vestibular nucleus - LVST lateral vestibulospinal tract - mlf medial longitudinal fasciculus - MVN medial vestibular nucleus - MVST medial vestibulospinal tract - PHA-L Phaseolus vulgaris leucoagglutinin - PrH praepositus hypoglossi nucleus - rMVN rostral medial vestibular nucleus - Ro Roller's nucleus - SVN superior vestibular nucleus - TMB tetramethylbenzidine - VH ventral horn - VNC vestibular nuclear complex - WGAHRP wheat germ agglutinin-horseradish peroxidase - 12 hypoglossal nucleus Dedicated to Prof. J.W. Rohen on the occasion of his 70th birthday     

C-fos induction in rat superficial dorsal horn following cutaneous application of noxious chemical or mechanical stimuli

The method of c-fos immunodetection was used to map the distributions of neurons in the lumbar spinal dorsal horn that were activated following intracutaneous (i.c.) microinjection, or iontophoretic application, of different irritant chemicals to the lateral hindpaw of rats. Microinjections (1 microl) of histamine, serotonin (5-HT), nicotine, capsaicin, or formalin each elicited similar distributions of Fos-like immunoreactivity (FLI) in laminae I-II of the ipsilateral superficial dorsal horn, with little or no FLI in deeper laminae or contralaterally. In laminae I and II, FLI cell counts were significantly higher following i.c. histamine, 5-HT, capsaicin, formalin, and noxious pinch, compared to i.c. saline controls. Capsaicin-evoked FLI was dose-dependent. Multivariate analysis of variance revealed no significant difference in spatial distributions of FLI induced by any of the chemical or pinch stimuli. Iontophoretic application of histamine, 5-HT, or nicotine also elicited similar distributions of FLI in the superficial dorsal horn, and cell counts of FLI were significantly higher compared to controls receiving iontophoretic vehicle (methyl cellulose). These results indicate either that individual laminae I-II neurons are activated by each of the irritant chemicals, or that neurons selectively responsive to a given irritant are comingled without any apparent laminar segregation.     

Differential activation of spinal dorsal horn units by subcutaneous formalin injection in the cat: an electrophysiological study

 In our previous report we found that subcutaneous (s.c.) formalin injection into the cutaneous receptive field (RF) of dorsal horn wide-dynamic-range (WDR) units and nociceptive primary afferent units resulted in a tonic, long-lasting increase in firing. However, s.c. formalin injection only resulted in a short-lasting increase in firing of non-nociceptive primary afferent units. In the present study, by using extracellular single-unit recording techniques we further studied effects of s.c. formalin on response properties of identified superficial-layer nociceptive-specific (NS) units and deeper-layer, low-threshold mechanoreceptive (LTM) units of L₇ dorsal horn in urethane-chloralose-anesthetized cats. s.c. formalin injection into the RF of NS units resulted in a tonic, long-lasting increase in firing (7.08 ± 0.42 spikes/s, n = 5), for more than 1 h, compared with the spontaneous background (1.42 ± 0.03 spikes/s, n = 5). Formalin injection into the RF of LTM units also resulted in an increase in firing; however, the duration was short-lasting, for 25–520 s (152.92 ± 46.73 s, n = 12). The present study demonstrated that s.c. injection of dilute formalin solution resulted in activation of not only nociceptive but also non-nociceptive dorsal horn units, suggesting that tissue injury caused by s.c. formalin results in vigorous injury discharges of peripheral nerve terminals, which subsequently leads to activation of primary afferent neurons and secondary dorsal horn neurons. Received: 28 August 1996 / Accepted: 2 January 1997     

Localization of NADPH-diaphorase containing neurons in the spinal dorsal horn and spinal sensory ganglia of the turtle Chrysemys d’orbigny

 NADPH-diaphorase positive (NDP) neurons and nerve fibers were found in the spinal dorsal horn (DH) and sensory ganglia of the turtle Chrysemys d’orbigny. Three well-defined types of NDP neurons were found in the DH: (a) elongated nerve cells with two radially arranged dendritic branches, (b) neurons with rostro-caudal dendritic branches, (c) bitufted neurons with two, practically symmetric branches that project to the ipsilateral and contralateral dorsal horns. A combination of the techniques that reveal NADPH-diaphorase activity with the horseradish peroxidase transganglionic labeling of the dorsal root collaterals, suggested that NDP neurons of the DH are second-order cells of the spinal sensory pathway. NDP neurons were also found in the spinal sensory ganglia at all metameric levels. Our findings indicate that the DH of turtles, like that of mammals, contains both the enzymatic machinery and the neural connections required to postulate the participation of nitric oxide in ”plastic phenomena” such as hyperalgesia and central sensitization. Two other alternatives or complementary hypotheses are discussed: (a) NDP neurons in the DH and sensory ganglia may represent specific cell populations involved in the processing of sensory visceral information; (b) NADPH-diaphorase reactivity may indicate sustained levels of neuronal activity. Received: 12 February 1996 / Accepted: 2 August 1996     

Modulation of rat skeletal muscle chloride channels by activators and inhibitors of protein kinase C

The membrane electrical parameters and component conductances of rat extensor digitorum longus muscle fibres were studied in vitro at 30 °C with standard two microelectrode square pulse cable analysis in the presence of protein kinase C (PKC) activators and inhibitors. The PKC activator, 4--phorbol-12,13 dibutyrate (4--PDB), (2–90nM) blocked up to 67% chloride conductance (G _Cl) in rat skeletal muscle fibres and induced myotonic hyperexcitability. The concentration necessary to produce a 50% block of the membrane G _Cl was 23 nM. The inactive 4--phorbol-12,13 dibutyrate had no effect at 2 M. The blocking effect of 4--PDB on G _Cl was prevented by preincubation of the preparations with the PKC inhibitors, staurosporine (1–5 M) and tetrahydropapaverolone (50–100 M). The blocking effects on membrane G _Cl of 4--PDB and its antagonism by the inhibitors used support the concept of the involvement of PKC in regulating Cl channels of mammalian skeletal muscle fibres.     

Characterisation of axon terminals in the rat dorsal horn that are immunoreactive for serotonin 5-HT3A receptor subunits

Serotonin 5-HT₃ receptors are abundant in the superficial dorsal horn and are likely to have an involvement in processing of nociceptive information. It has been shown previously that 5-HT₃ receptors are present on primary afferent terminals and some dorsal horn cells. The primary aim of the present study was to determine what classes of primary afferent possess 5-HT₃A receptor subunits. We performed a series of double- and triple-labelling immunofluorescence experiments. Subunits were labelled with an anti-peptide antibody and primary afferent axons were identified by the presence of calcitonin gene-related peptide (CGRP) and binding of the lectin IB4. Quantitative confocal microscopic analysis revealed that approximately 10% of axons displaying 5-HT₃A immunoreactivity were also labelled for CGRP but that only 3% of these fibres bind IB4. We also investigated the relationship between immunoreactivity for the subunit and descending serotoninergic systems, axons originating from inhibitory neurons that contain glutamic acid decarboxylase, and axons of a subpopulation of excitatory neurons that contain neurotensin. None of these types of axon was associated with immunoreactivity for receptor subunits. Ultrastructural studies confirmed that punctate immunoreactive structures observed with the light microscope were axon terminals. These terminals invariably formed asymmetric synaptic junctions with dendritic profiles and often contained a mixture of granular and agranular vesicles. Some terminals formed glomerular-like arrangements. Immunoreactive cells were also examined and were found to contain intense patches of reaction product within the cytoplasm. We conclude that the majority (about 87%) of dorsal horn axons that are immunoreactive for 5-HT₃A receptor subunits do not originate from the subtypes of primary afferent fibres that bind IB4 or contain CGRP. It is likely that most of these axons have an excitatory action and they may originate from dorsal horn interneurons and/or fine myelinated primary afferent fibres. Electronic Publication