Confocal microscopic analysis reveals sprouting of primary afferent fibres in rat dorsal horn after spinal cord injury

Following high thoracic spinal cord transection (SCT) in rats, abnormal changes in arterial pressure in response to sensory stimulation (autonomic dysreflexia) are correlated with changes in neural circuitry in the injured spinal cord. Anterograde transport of wheat germ agglutinin conjugated to Texas Red (WGATR) and confocal microscopy were used to characterize the increased arbourization of Adelta and Abeta fibre populations in laminae III-V of the dorsal horn. In cord-injured animals, significantly greater areas of WGATR-labeled fibres were found in the deeper laminae of the dorsal horn than in control rats. This increased area likely reflects sprouting of the Adelta, Abeta, and possibly C fibre populations. The time course of sprouting matches the onset of autonomic dysreflexia, indicating a possible functional correlation between the two phenomena.     

Autoradiographic evidence of serotonin1 binding sites on primary afferent fibres in the dorsal horn of the rat spinal cord

Spinal serotonin1 (5-HT1)(labelled by [3H]5-HT), 5-HT1A (labelled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT)), mu- (labelled by [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAGO) and [3H]naloxone) and delta-opiate (labelled by [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr [( 3H]DSTLE] receptor binding sites were studied in adult rats using quantitative autoradiography after either neonatal treatment with capsaicin or unilateral cervical dorsal rhizotomy. Both treatments produced a significant loss of 5-HT (-20 to -30%) and opiate (-30 to -45%) binding sites within the superficial layers of the dorsal horn, suggesting they are partly located presynaptically on primary afferent fibres. Thus, 5-HT, as well as opiates, might generate analgesia by acting--at least partly--on primary afferent nociceptive fibres at the spinal level.     

Myelinated and unmyelinated primary afferent axons form contacts with cholinergic interneurons in the spinal dorsal horn

Cholinergic interneurons in laminae III/IV of the dorsal horn contain co-localised gamma-aminobutyric acid (GABA) and frequently form axoaxonic synapses with terminals of primary afferents. They are therefore probably last-order interneurons involved in presynaptic inhibition. The purpose of the present investigation was to determine if these cells receive direct input from primary afferents. Relationships between primary afferents and interneurons were investigated in adult rats. Myelinated primary afferents were labelled with the B-subunit of cholera toxin (CTb). Unmyelinated afferents were labelled with IB4 lectin and an antibody to identify calcitonin-gene-related peptide (CGRP). Cholinergic neurons were labelled with an antibody raised against choline acetyltransferase and examined with a confocal microscope. Cells were reconstructed with NeuroLucida for Confocal and afferent contacts plotted. Interneurons (N=30) received an average of 20.2+/-11.9 (SD) contacts from CTb-labelled primary afferents, which were preferentially distributed on proximal and intermediate dendrites. Interneurons with dendrites which extended into lamina II (N=20) received an average of 27.4+/-19.0 IB4 contacts (on intermediate and distal dendrites) and 9.2+/-6.8 CGRP contacts. It is concluded that cholinergic interneurons receive contacts from both myelinated and unmyelinated primary afferents and different classes of afferent target particular dendritic domains. Cholinergic interneurons are likely to be components of an inhibitory feedback pathway that is monosynaptically activated by primary afferents.     

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     

大鼠中缝大核向脊髓投射神经元与神经降压素能终末的突触联系

神经降压素(NT)是中枢下行抑制系统的重要神经活性物质。本研究运用免疫组织化学与逆行追踪法相结合的双标技术,在电镜下观察NT能终末与中缝大核(NRM)向脊髓投射神经元的突触联系。在光镜下可见NT阳性纤维和终末散在分布于NRM,但未见NT阳性神经元;将HRP注入腰髓背角后,在NRM内可见比较密集的HRP逆标神经元。在电镜下可见NT阳性终末与HRP逆标神经元的胞体和树突形成以非对称性为主的轴-体突触和轴-树突触。上述结果说明NT可能调控NRM向脊髓背角投射神经元的活动,借此对伤害性信息向中枢的传递发挥抑制效应。     

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.     

Caudal end of the rat spinal dorsal horn

We have previously demonstrated that the transformation of the caudal spinal cord through the conus medullaris to the filum terminale takes place in three steps. In the conus medullaris the twin layers of CGRP-immunoreactive and IB4-labeled primary afferent fibers as well as the translucent portion of the superficial dorsal horn equivalent to the substantia gelatinosa discontinue before the complete removal of the dorsal horn. Parallel with these changes VGLUT1-immunoreactive myelinated primary afferent fibers arborize not only in the deep layers but also in the entire extension of the remaining dorsal horn, while scattered CGRP fibers still remains at the margin of and deep in the dorsal horn. PKCgamma-immunoreactive dorsal horn neurons discontinue parallel with the disappearance of the IB4-labeled nerve fibers. These observations suggest that in the dorsal horn certain neurons are linked to the substantia gelatinosa, while others are substantia gelatinosa-independent neurons.     

Monosynaptic connections between primary afferents and giant neurons in the turtle spinal dorsal horn

This paper reports the occurrence of monosynaptic connections between dorsal root afferents and a distinct cell type — the giant neuron — deep in the dorsal horn of the turtle spinal cord. Light microscope studies combining Nissl stain and transganglionic HRP-labeling of the primary afferents have revealed the occurrence of axosomatic and axodendritic contacts between labeled boutons and giant neurons. The synaptic nature of these contacts has been confirmed by use of electron microscope procedures involving the partial three-dimensional reconstruction of identified giant neurons. Intracellular recording in spinal cord slices provided functional evidence indicating the monosynaptic connections between dorsal root afferents and giant neurons. The recorded neurons were morphologically identified by means of biocytin injection and with avidin conjugates. Electrical stimulation of the ipsilateral dorsal roots evoked synaptic responses with short, fixed latencies (1.6–5.6 ms), which remained unchanged at high frequencies (10 Hz). Excitatory polysynaptic potentials were also observed. By means of pharmacological procedures the short-latency response was dissected in two components: one insensitive to tetrodotoxin, the other abolished by the drug. The toxin-resistant component was presumed to be sustained by small-diameter C fibers. The synaptic response was mainly mediated by the glutamate-AMPA receptor subtype; however, a small component mediated by NMDA receptor was also present.     

Synaptic architecture of glomeruli in superficial dorsal horn of rat spinal cord, as shown in serial reconstructions

Summary Using serial section EM analysis, synaptic organization of glomeruli in lamina II of the dorsal horn of the rat has been examined. Four C_I-terminals (small, dark and sinuous), four C_IIa (large, light and regular, without neurofilaments) and four C_IIb, (with neurofilaments) at the centres of synaptic glomeruli of types I, IIa and IIb, respectively, were serially sectioned and reconstructed. Asymmetrical synapses between the central terminal (C) and dendritic profiles without synaptic vesicles (D) prevailed in all types of glomeruli. Symmetrical dendroaxonic contacts with presynaptic dendrites (V₁ C) occurred practically only in type I glomeruli in which there were also more asymmetrical C V₁ contacts than in type II glomeruli. Symmetrical axoaxonic synapses V₂ C were more abundant in type IIa and IIb glomeruli. Type IIa glomeruli had a significantly larger number of C D synapses and of all synapses per unit area of C surface, than type IIb glomeruli.Triadic systems with C and D postsynaptic to V₂ were nearly as numerous as those involving V₁ in type I glomeruli. Triads with V₂ were however largely preponderant in type IIa and virtually exclusive in type IIb. It thus seems that each of the three types of glomerulus has its own pattern of synaptic interactions which might reflect specific complexes of feed-forward and feed-back mechanisms. In type I glomeruli, excitation of second-order neurons by nociceptive C_I terminals may be controlled in similar proportions by presynaptic dendrites excited within the glomerulus by the C terminal itself, or by peripheral axons excited from outside the glomerulus. This kind of control is likely to prevail in type IIa glomeruli and to be the only efficient modulatory mechanism in type IIb glomeruli.     

Presynaptic modulation by neuromedin U of sensory synaptic transmission in rat spinal dorsal horn neurones

Neuromedin U (NMU) is a brain–gut peptide first isolated from the spinal cord. Recent studies on NMU and its receptors have suggested a role of NMU in sensory transmission. Here we report on the localization of NMU in sensory neurones, and the actions of NMU in the substantia gelatinosa (SG) and the deep layer of the dorsal horn (laminae III–V) in adult rat spinal cord slices using the patch-clamp technique. An immunohistochemical study revealed that NMU peptide was present in most of the dorsal root ganglion neurones. In the spinal cord, NMU-immunoreactive neurones were located in the deep layer (laminae III–V), but not in the SG. However, NMU-positive axon terminals were observed in the SG as well as the deep layer. Bath-applied NMU (10 μ m ) increased the frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSCs) in the SG and deep layer neurones by 146 ± 14% ( P < 0.01, n = 17) and 174 ± 21% ( P < 0.01, n = 6), respectively, without inducing any postsynaptic membrane currents recorded in tetrodotoxin. On the other hand, NMU did not affect miniature inhibitory postsynaptic currents recorded in tetrodotoxin. These findings, taken together, suggest that NMU acts on the presynaptic terminals of the primary afferent fibres working as an autocrine/paracrine neuromodulator to increase mEPSC frequency of the SG and deep layer neurones. This may account for the spinal mechanisms of the NMU-induced hyperalgesia reported previously.     

GABA-immunoreactive neurons in the dorsal horn of the rat spinal cord

An antiserum to GABA was used on semithin resin-embedded sections of rat dorsal horn. Immunoreactive neurons were evenly distributed throughout laminae I-III and constituted between 24 and 33% of the total neuronal population within three laminae. Fifty Golgi-stained cells in lamina II were tested with the antiserum. Most of the islet cells examined were immunoreactive, although some small islet cells were not. None of the 14 stalked cells tested was immunoreactive. These results provide further evidence that the stalked and islet cells of lamina II form two distinct functional classes and suggest that the islet cells function as inhibitory interneurons.     

Action of neuropeptide Y on nociceptive transmission in substantia gelatinosa of the adult rat spinal dorsal horn

Effects of neuropeptide Y (NPY) on substantia gelatinosa neurons were investigated in adult rat spinal cord slices using blind whole-cell patch-clamp technique. Bath application of NPY (1 microM) induced a membrane hyperpolarization, resulting in a suppression of the dorsal root stimulation-induced action potentials in 24% of the substantia gelatinosa neurons tested. In voltage clamp mode, NPY produced an outward current dose-dependently in about one third of substantia gelatinosa neurons at the holding potential of -60 mV, which was not affected by tetrodotoxin (1 microM). The NPY-induced current was suppressed by perfusion with a Ba2+-containing external solution and a Cs2SO4 or tetraethylammonium-containing pipette solution. In addition, The NPY-induced outward currents reversed its polarity near the equilibrium potential of K+ ions (-93 mV). The response to NPY recorded with guanosine-5'-O-(2-thiodiphosphate)-beta-S (GDP-beta-S) containing pipette solution was abolished 30 min after patch formation, suggesting that the response was mediated by the G-protein-coupled receptors. Application of an NPY-Y1 selective agonist, [Leu(31), Pro(-34)]-NPY (1 microM), for 30 s also induced an outward current with a similar time course and amplitude to that induced by NPY. On the other hand, the NPY response was blocked by a simultaneous application of NPY-Y1 selective antagonist, BIBP 3226 (1 microM). No significant changes were found in amplitude and frequency of miniature excitatory postsynaptic currents and dorsal root evoked excitatory postsynaptic currents by NPY. In addition, NPY did not affect both of the miniature inhibitory postsynaptic currents and evoked inhibitory postsynaptic currents, mediated by either the GABA or glycine receptor. These findings, taken together, suggest that NPY produces an outward current in substantia gelatinosa neurons through G-protein coupled, and NPY-Y1 receptor-mediated activation of K+ channels without affecting presynaptic components. The inhibition of the synaptic transmission from the primary fibers to the substantia gelatinosa neurons is considered to contribute to the antinociceptive effects of NPY.     

Inflammation reduces the contribution of N-type calcium channels to primary afferent synaptic transmission onto NK1 receptor-positive lamina I neurons in the rat dorsal horn

N-type calcium channels contribute to the release of glutamate from primary afferent terminals synapsing onto nocisponsive neurons in the dorsal horn of the spinal cord, but little is known of functional adaptations to these channels in persistent pain states. Subtype-selective conotoxins and other drugs were used to determine the role of different calcium channel types in a rat model of inflammatory pain. Electrically evoked primary afferent synapses onto lumber dorsal horn neurons were examined three days after induction of inflammation with intraplantar complete Freund's adjuvant. The maximal inhibitory effect of the N-type calcium channel blockers, ω-conotoxins CVID and MVIIA, were attenuated in NK1 receptor-positive lamina I neurons after inflammation, but the potency of CVID was unchanged. This was associated with reduced inhibition of the frequency of asynchronous-evoked synaptic events by CVID studied in the presence of extracellular strontium, suggesting reduced N-type channel contribution to primary afferent synapses after inflammation. After application of CVID, the relative contributions of P/Q and L channels to primary afferent transmission and the residual current were unchanged by inflammation, suggesting the adaptation was specific to N-type channels. Blocking T-type channels did not affect synaptic amplitude under control or inflamed conditions. Reduction of N-type channel contribution to primary afferent transmission was selective for NK1 receptor-positive neurons identified by post hoc immunohistochemistry and did not occur at synapses in laminae II_o or II_i, or inhibitory synapses. These results suggest that inflammation selectively downregulates N-type channels in the terminals of primary afferents synapsing onto (presumed) nociceptive lamina I NK1 receptor-positive neurons.     

Projection field of primary afferent fibers innervating the ventral portion of the lumbar intervertebral disc in the spinal cord dorsal horn

In the present study, we investigated the central projection of afferent fibers innervating the lumbar intervertebral disc using the fluorescent neurotracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). The tracer Dil was applied to the ventrolateral portion of the L5-L6 intervertebral disc in 11 adult rats. Fluorescent sites were observed microscopically on spinal cord transverse sections. Fluorescent spots in laminae I-III were plotted on the central projection map of cutaneous afferents. In six of 11 rats, Dil was restricted to the application site. Of these six rats, three showed no evident fluorescent sites. In the remaining three rats, small fluorescent spots were scattered in the dorsal horn. Fluorescent spots in dorsal horn lamina I were located in the central projection fields of the low back and groin skin. Fluorescent spots were observed, also sporadically, in Clarke's column in T12-L1 segments. The central projection of afferent fibers innervating the rat lumbar intervertebral disc was indistinct with Dil labeling. We presumed this was due to the scarcity of central terminal arbors of disc afferent fibers. Spotty projections in laminae I-IllIIere present near the central projection fields of the loin and groin, indicating that pain would be perceived in the groin.     

Synaptic noise in spike trains of normal and denervated electroreceptor organs

The sequence of interspike intervals of the spontaneous activity in denervated electroreceptor organs of the catfish is analysed with several statistical techniques: visual inspection of dot displays, interval histograms, serial correlograms, tests for stationary and trends, and tests for renewal properties, based on the spectrum of intervals. The interspike interval train of primary afferents can usually be treated as a renewal process. Following denervation, a number of non-renewal properties emerge. The interval histogram of the renewal spike trains can be fitted well with a gamma probability density function; non-renewal cases need a more complex approach. We propose that the stochastic fluctuations in interval duration arise from randomly occurring quantal depolarizations (Stein's model of stochastic neuronal excitations. Two important properties with regard to synaptic transduction are: afferent firing results from capture of a limited number of depolarizing quanta; and the quantal input rate and the threshold for firing appear to be correlated.     

Vagal afferent modulation of spinal nociceptive transmission in the rat

1. The effects of vagal afferent stimulation (VAS) on spinal nociceptive transmission and the spinal pathway(s) mediating VAS-produced effects were examined in pentobarbital sodium- and urethane-anesthetized, paralyzed rats. The 60 units studied responded to mechanical stimuli and noxious heating (50 degrees C) of cutaneous receptive fields confined to the glabrous skin of the toes and footpads. Recording sites were located in laminae I-VI of the L3-L5 spinal segments. 2. VAS facilitated and inhibited neuronal responses to heat. In pentobarbital-anesthetized rats, responses of most (24/44) units were facilitated by low and inhibited by higher intensities of VAS. Responses of some units (15/44) were only inhibited and others (4/44) only facilitated by VAS. Inhibition produced by VAS was intensity-, pulse width-, frequency-, and stimulation duration-dependent. In urethane-anesthetized rats, responses of 6/16 units were initially facilitated, then inhibited as the intensity of VAS was increased; responses of nine units were inhibited by VAS. Quantitative comparisons of recruitment indices, mean thresholds for inhibition and mean intensities to inhibit unit responses to heat to 50% of control revealed no significant differences between the two anesthetic conditions. 3. The effects of VAS on neuronal responses to heat were dissociable from its effect on blood pressure. Regardless of the effect of VAS on unit responses to noxious heat, VAS consistently produced intensity-dependent depressor responses. The latencies to onset of inhibition and facilitation by VAS were determined by a cumulative sum technique and bin-by-bin analysis of peristimulus time histograms. The apparent latencies were 91 +/- 11 (SE) ms for inhibition and 278 +/- 59 ms for facilitation, both of which occurred before changes in blood pressure. Finally, microinjections of lidocaine into the ventrolateral funiculus (VLF) or transections of the dorsolateral funiculus (DLF) of the thoracic spinal cord attenuated VAS-produced effects on neuronal responses, but did not affect VAS-induced depressor responses. 4. The responses of 11 dorsal horn units to graded noxious heating of the skin were studied; the stimulus-response functions (SRF) were linear and monotonic throughout the temperature range examined (42-52 degrees C). VAS at intensities which inhibited unit responses to heat significantly decreased the slope of the SRF. VAS at intensities which facilitated unit responses to heat produced a leftward, parallel shift of the SRF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Statistics of midbrain dopamine neuron spike trains in the awake primate

Work in behaving primates indicates that midbrain dopamine neurons encode a prediction error, the difference between an obtained reward and the reward expected. Studies of dopamine action potential timing in the alert and anesthetized rat indicate that dopamine neurons respond in tonic and phasic modes, a distinction that has been less well characterized in the primates. We used spike train models to examine the relationship between the tonic and burst modes of activity in dopamine neurons while monkeys were performing a reinforced visuo-saccadic movement task. We studied spiking activity during four task-related intervals; two of these were intervals during which no task-related events occurred, whereas two were periods marked by task-related phasic activity. We found that dopamine neuron spike trains during the intervals when no events occurred were well described as tonic. Action potentials appeared to be independent, to occur at low frequency, and to be almost equally well described by Gaussian and Poisson-like (gamma) processes. Unlike in the rat, interspike intervals as low as 20 ms were often observed during these presumptively tonic epochs. Having identified these periods of presumptively tonic activity, we were able to quantitatively define phasic modulations (both increases and decreases in activity) during the intervals in which task-related events occurred. This analysis revealed that the phasic modulations of these neurons include both bursting, as has been described previously, and pausing. Together bursts and pauses seemed to provide a continuous, although nonlinear, representation of the theoretically defined reward prediction error of reinforcement learning.