Tachykinin Actions on Deep Dorsal Horn Neurons In Wm: An Electrophysiological and Morphological Study in the Immature Rat

To assess whether functional neurokinin receptors exist in the deep dorsal horn of the rat, the actions of the selective neurokinin-1 receptor (NK1R) agonist [Sar⁹,Met(O₂)¹¹]substance P [Sar⁹,Met(O₂)¹¹]]SP), the neurokinin-2 receptor (NK2R) agonists [β-Ala⁸]NKA_4-10 and GR64349 and the neurokinin-3 receptor (NK3R) agonist senktide were examined intracellularly in vitro. [Sar⁹,Met(O₂)¹¹]]SP (1–4 μM) and senktide (1-2 μM) elicited slow depolarizations (40 mV) associated with increased synaptic activity and cell firing. [β-Ala⁸]NKA_4-10 (10-20 μM) and GR64349 (0.25-10 μM) caused small depolarizations (<2.0 mV) and no firing. Neurons were categorized as either ‘tonic’ or ‘phasic’ depending on their firing response to direct current step depolarizations. Tonic neurons, which, unlike phasic neurons, display no spike firing accommodation, generated a significantly larger depolarization to the NK1R and NK3R agonists. The putative contribution of these receptors to primary afferent-mediated synaptic transmission was assessed by testing the NKIR antagonist GR82334 (1 μM), the NK2R antagonist MEN10,376 (1 μM) and the NK3R antagonist [Trp⁷,β-Ala⁸]NKA4_-10 (1 μM) against the dorsal root-evoked excitatory postsynaptic potential (DR-EPSP). GR82334 and [Trp⁷,β-Ala⁸]NKA_4-10 significantly reduced (P ≤ 0.05) the duration but not the amplitude of the DR-EPSP. MEN10,376 (1 μM) had no effect on DR-EPSP amplitude or duration. Morphological detail was obtained for seven biocytin-filled deep dorsal horn neurons tested with [Sar⁹,Met(O₂)¹¹]SP. Five neurons responded to the NKIR agonist, and two of these had dorsally directed dendrites into the substantia gelatinosa. The other three [Sar⁹,Met(O₂)¹¹]SP-sensitive neurons had dendrites within deeper laminae. These data support the existence of functional NK1Rs and NK3Rs in the deep dorsal horn which may be involved in mediating sensory afferent inputs from nociceptors.     

Pre- and Post-synaptic Actions of 5-Hydroxytryptamine in the Rat Lumbar Dorsal Horn In Vitro: Implications for Somatosensory Transmission

Since the relative contribution of pre- versus post-synaptic actions of 5-hydroxytryptamine (5-HT) to modulation of somatosensory processing in the dorsal horn is not known, recordings fro m primary afferents and dorsal horn neurons from in vitro rat spinal cord were used to address this issue. 5-HT produced a depression of spontaneous dorsal root potentials and a slow primary afferent depolarization (PAD): the PAD versus 5-HT concentration-response curve was bell shaped (maximum at 5 μM; 250±C 41.5 μV). In 28/40 dorsal horn neurons, 5-HT elicited a slow depolarization not clearly associated with a specific input resistance change. Excitatory synaptic transmission from primary afferents to dorsal horn neurons was depressed by 5-HT in 40/45 neurons. 5-HT ≥ 5 μM significantly ( P ≤ 0.05) decreased the amplitude, shortened the total duration and half-decay time of the excitatory post-synaptic potential (EPSP). A dominant effect of 5-HT on longer latency EPSP components was evident. There was no direct relationship between the magnitude of PAD and the reduction of the EPSP by 5-HT. 5-Carboxamidotryptamine, an agonist for 5-HT¹ receptors, mimicked the depression of neurotransmission in the dorsal horn without producing PAD. A sample of dorsal horn neurons ( n = 8) was injected with biocytin and their morphology described. All had somata within laminae III-VI. In five of these neurons 5-HT depressed the EPSP but in one interneuron-like and one unclassed neuron the EPSP was potentiated. These data suggest that whilst depression of synaptic transmission is the predominant effect of 5-HT in the deep dorsal horn, this is not easily related to PAD or cellular actions of 5-HT on dorsal horn neurons.     

Membrane Characteristics and Synaptic Responsiveness of Superficial Dorsal Horn Neurons in a Slice Preparation of Adult Rat Spinal Cord

Intracellular recordings have been made from neurons of the superficial dorsal horn in slices of the lumbar and thoracic spinal cord of young adult rats. Three broad categories of neurons could be distinguished on the basis of their firing patterns to intracellular current pulses and their afterhyperpolarizations (AHP); there was no detectable difference in the regional distribution of the three types. Category 1 cells were characterized by maintained firing to intracellular depolarizing current pulses, brief action potential durations and polyphasic AHPs. Category 2 cells showed spike adaptation, without spike attenuation, during intracellular current pulses, and had monophasic AHPs. Category 3 cells fired only 1 or 2 spikes to maintained depolarizing pulses and had smaller monophasic AHPs than category 2 neurons. Spontaneous excitatory and inhibitory postsynaptic potential (epsp and ipsp) activity was seen with psp durations varying widely. Low intensity electrical stimulation of afferent fibres, or of superficial white matter, resulted in polyphasic epsps and/or ipsps. The spike discharge in response to such afferent inputs correlated with the membrane properties of the cells, such that the synaptic responses of category 1 neurons were usually bursts of spikes, whereas category 2 and 3 neurons either failed to fire or fired only a single spike. These results in adult rat spinal cord suggest that the discharge pattern within synaptic sensory responses of superficial dorsal horn neurons is determined by postsynaptic membrane properties as well as by the pattern of the afferent input.     

GABA and Glycine in Synaptic Glomeruli of the Rat Spinal Dorsal Horn

The superficial dorsal horn of rat spinal cord contains two types of synaptic glomerulus, which are centred around the terminals of unmyelinated and myelinated primary afferents respectively. Both types of glomerulus contain GABAergic axons and dendrites, which are thought to originate from local inhibitory interneurons. Some of the dendrites contain synaptic vesicles, and may be presynaptic to the central axon at dendroaxonic synapses. In order to determine whether GABAergic structures in the two types of glomerulus are derived from different populations of interneurons, a quantitative post-embedding immunogold study of GABA- and glycine-immunoreactivity in rat dorsal horn was performed. In type I glomeruli, all of the peripheral axons and most vesicle-containing dendrites were GABA-immunoreactive, but only one of 32 axons and none of the vesicle-containing dendrites was glycine-immunoreactive. In contrast, most of the peripheral axons and some of the vesicle-containing dendrites in type II glomeruli possessed both GABA- and glycine-immunoreactivity. This strongly suggests that different types of inhibitory interneuron in the dorsal horn are associated with the two types of glomerulus. It is therefore likely that different populations of interneurons mediate presynaptic inhibition of unmyelinated and myelinated primary afferents.     

Activity-Dependent Changes in Rat Ventral Horn Neurons in vitro; Summation of Prolonged Afferent Evoked Postsynaptic Depolarizations Produce a d-2-Amino-5-Phosphonovaleric Acid Sensitive Windup

The synaptic responses of lumbar ventral horn neurons including identified flexor motoneurons, to graded stimulation of peripheral nerves have been recorded in vitro in the young rat spinal cord-hindlimb preparation. Single shock stimulation of low threshold myelinated afferents evoked short latency (< 20 ms) short duration (< 1.0 s, 391 +/- 42 ms n=43 SEM) compositive mono- and polysynaptic potentials. Recruitment of both thinly myelinated (A delta) and unmyelinated (C) afferent fibres elicited a prolonged postsynaptic depolarization (> 1 s) in all cells. In the majority of cells (67.4%), this depolarization exceeded 4.0 s in duration (8.01 +/- 0.4 s, n=26, maximum 14 s). In the remainder, shorter responses were evoked (< 3.0 s, mean=1.74 +/- 0.4 s, n=18). In those cells where the postsynaptic response to a single A delta or C fibre strength stimulus exceeded 4 s, low frequency (0.5 - 1.0 Hz) repetitive stimulation resulted in a temporal summation of the postsynaptic depolarizations, which generated a cumulatively increasing depolarization. This incrementing depolarization was sufficient in 33% of the cells to produce a progressive increase in spike discharge (windup). On cessation of the train of stimuli the depolarization decayed slowly (65 +/- 27 s). The N-methyl d-aspartic acid (NMDA) receptor antagonist d-2-amino-5-phosphonovaleric acid (d-APV) reduced the duration and amplitude of the prolonged postsynaptic depolarizations elicited by a single shock stimulation of small diameter afferents by 57% and 50% respectively. A smaller effect was produced on the low threshold afferent evoked early excitatory postsynaptic potentials (EPSP) (3% decrease in amplitude and 24% decrease in duration). In the presence of d-APV the cumulatively incrementing depolarization produced by repetitive stimulation was substantially reduced and windup failed to occur. Activity-dependent amplifications of primary afferent evoked responses in spinal neurons therefore involves a temporal summation of d-APV sensitive prolonged postsynaptic depolarizations.     

Developmental Loss of GABA- and Glycine-induced Depolarization and Ca2+ Transients in Embryonic Rat Dorsal Horn Neurons in Culture

More than 90% of dorsal horn neurons from embryonic day 15–16 rats responded to the inhibitory amino acids GABA and glycine by a transient elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) when maintained in culture for <1 week. This [Ca²⁺]_i response has previously been shown to be due to depolarization and subsequent Ca²⁺ entry through voltage-gated Ca²⁺ channels following activation of bicuculline-sensitive GABA_A receptors and strychnine-sensitive glycine receptors. Both the number of cells responding to GABA and glycine and the amplitude of the [Ca²⁺]_i response diminished over time in culture. By 30 days in culture, none of the cells responded to GABA, muscimol or glycine by elevation of [Ca²⁺]_i. The loss of the [Ca²⁺]_i response was not due to a change in the abundance or the properties of voltage-gated Ca²⁺ channels, since over the same period of time dorsal horn neurons showed a large increase in the amplitude of the [Ca²⁺]_i transient in response to 30 mM K⁺. Nor was the loss of the [Ca²⁺]_i response due to a loss of GABA and glycine receptors. Instead, the decrease in the [Ca²⁺]_i response over time paralleled a similar change in the electrophysiological responses. More than 90% of the neurons tested were depolarized in response to inhibitory amino acids during the first week in culture. After 30 days, all neurons tested responded to GABA and glycine with a hyperpolarization. These observations add support to the suggestion that GABA and glycine may excite dorsal horn neurons earlyin development and play a role in postmitotic differentiation.     

Activation by Cutaneous or Visceral Noxious Stimulation of Spinal Neurons Projecting to the Medullary Dorsal Reticular Nucleus in the Rat: A c-fos Study

The involvement of spinal neurons in the transmission of cutaneous and visceral nociceptive input to the medullary dorsal reticular nucleus was studied. Rats were injected with cholera toxin subunit B in the left dorsal reticular nucleus and subjected 4 days later to noxious mechanical, thermal or chemical stimulation of the proximal internal aspect of the left thigh, or to chemical stimulation of the urinary bladder. Sections of spinal segments T₁₃-L₃ were processed immunocytochemically for cholera toxin subunit B and Fos protein. The percentage of double-labelled cells in the population of Fos-positive cells was higher in lamina I (1–4%) than in deeper laminae (0–0.7%) following all stimuli. The percentage of double-labelled cells in the population of retrogradely labelled cells was 30–53% in lamina I and 0–5% in laminae III—X. Visceral stimulation activated more retrogradely labelled lamina I cells than any kind of cutaneous stimulation. Pyramidal cells were activated in higher numbers than multipolar and flattened cells after thermal cutaneous or visceral stimulation, and in lower numbers than multipolar cells after mechanical stimulation. These results suggest that, in the experimental conditions used, spinal cord cells conveying noxious input to the dorsal reticular nucleus are concentrated in lamina I. They further indicate that the spinaldorsal reticular nucleus pathway plays a major role in the transmission of nociceptive visceral input, and point to the preferential involvement of pyramidal cells in cutaneous thermal and visceral processing.     

Immunocytochemical Localization of trkA Receptors in Chemically Identified Subgroups of Adult Rat Sensory Neurons

Immunocytochemistry has been used to examine the location of trkA, the high-affinity receptor for nerve growth factor, in adult rat dorsal root ganglia, trigeminal ganglia and spinal cord. TrkA immunoreactivity was observed in small and medium sized ganglion cells and in the dorsal horn of the spinal cord. In lumbar L4 and L5 ganglia trkA-immunoreactive cells constitute 40% of dorsal root ganglion cells and range in size from 15 to 45 μm in diameter. Double labelling using markers for various dorsal root ganglion subpopulations revealed that virtually all (92%) trkA-immunoreactive cells express calcitonin gene-related peptide (CGRP) immunoreactivity. In contrast only 4 and 13% of trkA-immunoreactive cells are labelled by the monoclonal antibody LA4 or the lectin Griffonia simplicifolia IB4, markers for small non-peptide-containing cells. Eighteen percent of trkA-immunoreactive cells belong to the 'large light'subpopulation, identified by their strong immunostaining by the neurofilament antibody RT97. TrkA immunoreactivity in the dorsal horn is heaviest in laminae I and II outer, has a similar distribution to CGRP, and is depleted by dorsal rhizotomy. Our results show that trkA-expressing cells in dorsal root ganglia correspond almost exactly with the CGRP, peptide-producing population. The receptor is present not only on cell bodies but also on central terminals. Non-peptide-containing small cells, which constitute 30% of dorsal root ganglion cells, are not trkA-immunoreactive and therefore most probably are functionally independent of nerve growth factor.     

Correlation of monosynaptic field potentials evoked by single action potentials in single primary afferent axons and their bouton distributions in the dorsal horn

The relationship between structure and function of the projections of single identified primary cutaneous axons was investigated by recording cord dorsum potentials at 4 sites in response to electrical stimulation of the single axon and visualizing the boutons of the axon stained by intracellular injection of horseradish peroxidase. The rostrocaudal extent of boutons differed from fiber to fiber ranging from 4.14-11.50 mm; their location in the dorsal horn also varied in agreement with the known somatotopy of the presynaptic neuropil and dorsal horn neurons. Rostrocaudal distributions of cord dorsum potentials and boutons of individual fibers revealed good agreement. Cord dorsum potential amplitude and length of the spinal projection were positively correlated with number of boutons, but no correlation with bouton density was found. The spinal projection of afferents innervating slowly adapting type 1 mechanoreceptors exhibited a greater rostrocaudal extent (mean: 8.48 mm) than those innervating rapidly adapting mechanoreceptors (i.e., hair follicle and field receptors: mean: 5.87 mm). Although the mean total number of boutons was greater for axons with slowly adapting receptors (7,250/fiber) than for axons of rapidly adapting receptors (4,677/fiber), no differences in the longitudinal density of boutons (boutons/mm) were observed. Likewise, summed amplitudes of cord dorsum potentials at the 4 recording electrodes were larger for SA1 afferents than for those of field and hair follicle afferents. A major role for the number of boutons in determining these differences is supported by the finding that the calculated average contribution per bouton to cord dorsum potentials (expressed as an amplitude coefficient a) was similar for slowly and rapidly adapting afferents. No evidence was found for regions in which boutons did not contribute to the cord dorsum potential.     

Membrane Potential Oscillations of Neonatal Rat Spinal Motoneurons Evoked by Electrical Stimulation of Dorsal Root Fibres

Intracellular recording from lumbar motoneurons of the neonatal rat isolated spinal cord bathed in standard saline solution was used to study membrane potential oscillations which accompanied the decay phase of excitatory postsynaptic potentials (EPSP) induced by single electrical pulses to an adjacent dorsal root. About 60% of motoneurons displayed rhythmic oscillations of 10 ± 2 mV maximal amplitude and 7 ± 0.5 Hz frequency. Ability to generate oscillations could not be correlated to the cell membrane properties or to the age of the preparation (5–13 days). The oscillation frequency was independent of membrane potential (-100 to -45 mV) or of the intensity of dorsal root stimuli. The oscillation amplitude was linearly related to the cell potential within the same voltage level. Fast Fourier transform analysis showed that the power spectrum of oscillations peaked at ˜8 Hz. Electrically evoked activities and spontaneous events displayed similar cut-off frequencies. When the cell membrane potential was steadily depolarized, a hyperpolarizing pulse applied during the decay phase of the EPSP promptly revealed the presence of oscillatory behaviour. Pharmacological block of neurokinin-1 or N -methyl-D-aspartate receptors depressed the decay phase of the EPSP and the associated oscillatory responses. It is suggested that rhythmic oscillations were probably due to summated synaptic potentials generated at the premotoneuron level and are perhaps of functional relevance to motoneuron behaviour.     

The somatotopic organization of forelimb cutaneous nerves in the brachial dorsal horn: an anatomical study in the cat

The projection of forelimb cutaneous nerves to the brachial dorsal horn was studied in the cat by the transganglionic transport method. The results demonstrate a precise somatotopic termination pattern. Afferent nerves from the paw occupy the largest area, with the palm represented most medially in the dorsal horn, followed progressively more laterally by the representations for the palmar and dorsal surfaces of the digits and the dorsum of the paw. The digits are represented in a longitudinal sequence, with the first digit in the caudal part of C6 and the fifth in the caudal part of C8. The projections of the wrist and arm are split, with the line of discontinuity located along the ventral surface of the limb, so that the radial side is represented rostral to the paw and the ulnar side caudal to the paw, with the dorsal surface of the arm represented lateral to the paw. Nerves innervating the skin of the back project to the lateralmost part of the dorsal horn. The degree of overlap or separation of the terminal fields of the nerves along the mediolateral axis of the dorsal horn seems to correspond to the degree of overlap or separation of the peripheral innervation fields. However, along the rostrocaudal axis there appears to be an overlap for which there is no counterpart peripherally.     

Neurotensin Excitation of Serotonergic Neurons in the Dorsal Raphe Nucleus of the Rat In Vitro

Neurotensin-containing terminals and radioligand binding sites are present in the dorsal raphe nucleus. The purpose of this study was to test, in brain slices containing this nucleus, the effect of neurotensin on the electrical activity of serotonergic neurons. In extracellular recordings, the cells were identified by the ability of the α₁-adrenoceptor agonist phenylephrine to induce firing, and serotonin to reduce this effect. After washout of phenylephrine, neurotensin (10 nM to 10 μM) induced a concentration-dependent increase in the firing rate of serotonergic neurons (EC₅₀= 142 nM; maximum effect ˜1 μM). The neurotensin excitation, which was mimicked by neurotensin fragments 8–13 but not neurotensin peptide fragment 1–8 and selectively blocked by SR 48692 (100 nM), was observed mainly in the ventral part of the nucleus. Most serotonergic neurons showed marked desensitization to neurotensin, even at low concentrations. The neurotensin response was occluded by supramaximal concentrations of phenylephrine. In intracellular recordings using KCl-containing electrodes, neurotensin induced an inward current associated in some cases with a decrease in apparent input conductance. In conclusion, neurotensin was found to have an excitatory action on serotonergic neurons in the ventral part of the dorsal raphe nucleus, an effect which could be subject to desensitization and was occluded by phenylephrine. This occlusion phenomenon may be important for the physiological role of neurotensin in the dorsal raphe nucleus.     

Colocalization of Oestrogen Receptor lmmunoreactivity and Preproenkephalin mRNA Expression to Neurons in the Superficial Laminae of the Spinal and Medullary Dorsal Horn of Rats

A double-labelling procedure combining immunohistochemical staining with in situ hybridization using a radiolabelled cRNA probe was employed to demonstrate oestrogen receptor-like immunoreactivity and preproenkephalin-A mRNA in the medullary and spinal dorsal horn of female rats. Both markers labelled large numbers of neurons in the substantia gelatinosa and its trigeminal homologue. Many of these neurons were double-labelled, displaying both oestrogen receptor-like- immunoreactivity and preproenkephalin-A mRNA; cell counts showed that 40–60% of the of the oestrogen receptor-like-immunoreactive cells in the superficial laminae also were labelled for preproenkephalin-A mRNA, and that 60–70% of the preproenkephalin-A mRNA-labelled neurons in the same laminae displayed oestrogen receptor-like immunoreactivity. Previous studies have shown that oestrogen receptors can bind to the promoter region of the preproenkephalin-A gene, and studies on the hypothalamus have demonstrated that oestrogen regulates enkephalin expression in select neuronal populations. The present results demonstrate that enkephalinergic neurons in the superficial dorsal horn contain oestrogen receptors and suggest that oestrogen may play an important role in the modulation of sensory and nociceptive processing in the lower medulla and spinal cord.     

Evidence for an Anuran Homologue of the Mammalian Spinocervicothalamic System: An In Vitro Tract-tracing Study in Xenopus laevis

Evidence is presented for an anuran homologue of the mammalian spinocervicothalamic system. In vitro tract-tracing experiments with biotinylated dextran amine in Xenopus laevis show that ascending spinal fibres from all levels of the spinal cord, passing via the dorsolateral funiculus, terminate in a cell area ventrolateral to the dorsal column nucleus. This cell area can be considered a possible homologue of the mammalian lateral cervical nucleus. After tracer applications to the ventral thalamus or to the torus semicircularis (both targets for somatosensory projections), the anuran lateral cervical nucleus was retrogradely labelled contralateral to the application sites. Tracer applications to the dorsolateral funiculus at the obex level and rostral spinal cord resulted in labelling of the cells of origin of the spinocervical tract. These were found, mainly ipsilaterally, in the ventral part of the dorsal horn, and were rather evenly distributed throughout the spinal cord. These data suggest the presence of an anuran homologue of the mammalian spinocervicothalamic system. A brief survey of the literature shows that such a system is much more common in vertebrates than previously thought.     

Control of Impulse Conduction in Long Range Branches of Afferents by Increases and Decreases of Primary Afferent Depolarization in the Rat

It has been shown previously that impulses in axons of the descending branches of myelinated afferents in rat dorsal columns may suffer a blockade of transmission along their course in the dorsal columns. This paper tests the effect of the mechanism of primary afferent depolarization on the orthodromic movement of impulses in descending dorsal column primary afferent axons originating in the L1 dorsal root. Orthodromic impulses were recorded in the L5 and 6 dorsal columns after stimulation of the L1 dorsal root. Twenty-seven out of 82 axons (33%) suffered a temporary transmission block if primary afferent depolarization had been induced by L5 stimulation before the L1 stimulus. The tendency to block peaked at 10–15 ms and persisted for up to 30–40 ms. The number of single unit orthodromic impulses originating from the L1 root and recorded during a search of the dorsal columns 15 mm caudal to L1 increased by a factor of 3.1 after the systemic administration of bicuculline (1 mg/kg). The number of single unit orthodromic impulses originating from the L1 root and recorded in axons descending in the dorsal columns 20 mm caudal to the root increased by a factor of 8.7 after the systemic administration of picrotoxin (5 mg/kg). It is concluded that the transmission of impulses in the long range caudally running axons from dorsal roots to dorsal columns may be blocked during primary afferent depolarization and that conduction may be restored by the administration of GABA antagonists.     

Neonatal Sciatic Nerve Section Results in a Rearrangement of the Central Terminals of Saphenous and Axotomized Sciatic Nerve Afferents in the Dorsal Horn of the Spinal Cord of the Adult Rat

Previous studies have shown that following neonatal peripheral nerve injury, adjacent intact myelinated and unmyelinated primary afferents sprout into the central denervated terminal area. The present study investigates this in more detail and goes further, to study the fate of the central terminals of the surviving axotomized primary afferent neurons. Bulk labelling of the sciatic and saphenous nerves with horseradish peroxidase conjugated to choleragenoid (B-HRP), to label the A fibres, or wheatgerm agglutinin (WGA-HRP), to label C fibres were employed to investigate the central consequences of sciatic nerve section and ligation on the day of birth, in adult rats. Bulk labelling of the axotomized sciatic or intact saphenous nerve with either tracer and comparison with contralateral controls revealed alterations to the terminal field. The intact saphenous nerve terminal field expanded caudally from mid L4 to the L4-L5 boundary when labelled with WGA-HRP and to the sacral cord when labelled with B-HRP. Labelling the axotomized sciatic nerve with either tracer revealed little change in the overall somatotopic organization of central terminals, although labelling was less intense compared to control nerves and more variable with WGA-HRP. Invasion of the substantia gelatinosa (SG) by axotomized A fibres was observed in segments L3-5, into the area occupied by axotomized C fibres. This area was also invaded by intact saphenous A fibres in the L4–5 segments. These results demonstrate that following neonatal nerve section: (i) axotomized primary afferents are able to retain a ‘normal’ somatotopic map in the rostrocaudal plane; (ii) both A and C fibres from adjacent intact nerves sprout into the denervated territory, but A fibres sprout further caudally; (iii) axotomized A fibres and invading intact A fibres both sprout dorsally into denervated SG. As a result, there is considerable overlap between nerve territories in denervated spinal cord, suggesting that competition for laminar termination sites exists between A and C fibres and also between axotomized and intact primary afferents.     

Induction of Fictive Locomotion by Sulphur-containing Amino Acids in an In Vitro Newborn Rat Preparation

The role of the sulphur-containing amino acids (SAAs) in the initiation of fictive locomotion was tested in an isolated spinal cord preparation from newborn rats. These substances were bath-applied and the fictive locomotion was recorded in the lumbar ventral roots. It emerged from this study that all the compounds tested could trigger an organized pattern (alternating left and right bursts of activity) with a dose-dependent response. However, specific frequency and concentration ranges were observed with each of these SAAs. Moreover, a clear-cut difference between d and l isomers in the ability of the SAAs to induce this activity was observed: the SAAs of the d -forms were found to be generally more potent than those of the l -forms. The effects of the SAAs were found to be mediated by both NMDA and non-NMDA receptors, since they were blocked in a dose-dependent manner by the specific antagonists of these receptors. Moreover, it was observed that β- p -chlorophenylglutamic acid, an uptake inhibitor of homocysteic acid (HCA), potentiated the effect of exogenously applied HCA, which supports the idea that HCA may act as a transmitter. The sulphuric and non-sulphuric amino acids were also classified in their order of potency. The most potent compound turned out to be d -homocysteine sulphinic acid, while d -cysteine sulphinic acid was the least potent. It also emerged that the maximal frequencies obtained with SAAs and excitatory amino acids were in the same range, which might correspond to the maximal limits of this system.     

Active and Passive Membrane Properties of Spinal Cord Neurons that Are Rhythmically Active during Swimming in Xenopus Embryos

Cellular properties have been examined in ventrally located Xenopus spinal cord neurons that are rhythmically active during fictive swimming and presumed to be motoneurons. Resting potentials and input resistances of such neurons are - 75 +/- 2 mV (mean +/- standard error) and 118 +/- 17 M ohm respectively. Most cells fire a single impulse, 0.5 to 2.0 ms in duration and 48.5 +/- 1.8 mV in amplitude, in response to a depolarizing current step. A minority fire several spikes of diminishing amplitude to more strongly depolarizing current. Cells held above spike, threshold fire on rebound from brief hyperpolarizing pulses. Spikes are blocked by 0.1 to 1.0 microM tetrodotoxin (TTX) and are therefore Na+-dependent. Current/voltage (I/V) plots to injected current are approximately linear near the resting potential but become non-linear at more depolarized levels. Cells recorded in TTX with CsCI-filled microelectrodes show a linearized I/V plot at depolarized membrane potentials suggesting the normal presence of a voltage-dependent K+ conductance activated at relatively depolarized levels. Most cells recorded in this way but without TTX fire long trains of spikes of near constant amplitude, pointing to a role of the K+ conductance in limiting firing in normal cells. Spike blockage with TTX reveals, in some cells, a transient depolarizing Cd2+-sensitive and therefore presumably Ca2+-dependent potential that increases in amplitude with depolarization. Cells in TTX, Cd2+, and strychnine, and recorded with CsCI-filled microelectrodes to block active conductances respond to hyperpolarizing current steps with a two component exponential response. The cell time constant (tau0) obtained from the longer of these by exponential peeling is relatively long (mean 15.7 ms). These findings contribute to an increased understanding of the cellular properties involved in spinal rhythm generation in this simple vertebrate.     

On the Role of Galanin,Substance P and Other Neuropeptides in Primary Sensory Neurons of the Rat: Studies on Spinal Reflex Excitability and Peripheral Axotomy

The interaction of intrathecally (i.t.) applied galanin (GAL) with substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), somatostatin (SOM) and C-fibre conditioning stimulation (CS) with regard to their effects on the spinal nociceptive flexor reflex was studied in decerebrate, spinalized, unanaesthetized rats with intact or sectioned sciatic nerves. SP, CGRP, VIP and SOM applied onto the surface of lumbar spinal cord or a brief CS train (1 Hz, 20 s) to the sural nerve facilitated the flexor reflex for several minutes in animals with intact or sectioned nerves. Pretreatment with GAL, which by itself had a biphasic effect on the flexor reflex in a dose-dependent manner, antagonized the reflex facilitation induced by sural CS before and after sciatic nerve section. SP-induced facilitation of the flexor reflex was antagonized by GAL in rats with intact sciatic nerves, but not after nerve section. In contrast, VIP-induced reflex facilitation was antagonized by GAL only after sectioning of the sciatic nerve. GAL was effective in antagonizing the facilitatory effect of CGRP under both situations, but had no effect on SOM-induced facilitation. A parallel immunohistochemical study revealed that after sciatic nerve section GAL-like immunoreactivity (LI) and VIP-LI are increased in the dorsal root ganglia and that these two peptides coexist in many cells. The present results indicate that GAL antagonizes the excitatory effect of some neuropeptides which exist in the spinal cord. This antagonism could explain the inhibitory effect of GAL on C-fibre CS-induced facilitation of the flexor reflex, which is presumably due to the release of some of these neuropeptides from the terminals of primary afferents. Furthermore, the interaction between GAL and other neuropeptides is altered by sciatic nerve section, paralleling changes in the levels of these neuropeptides in primary afferents and their pattern of coexistence after nerve section. It is proposed that SP and CGRP are important mediators of the spinal flexor reflex in intact rats. However, after axotomy VIP may replace SP in this capacity, paralleling the decrease in SP and marked increase in VIP levels. In general the study provides further support for involvement of peptides in sensory function.