Temporal facilitation of spastic stretch reflexes following human spinal cord injury

Recent evidence suggests that alterations in ionic conductances in spinal motoneurones, specifically the manifestation of persistent inward currents, may be partly responsible for the appearance of hyperexcitable reflexes following spinal cord injury (SCI). We hypothesized that such alterations would manifest as temporal facilitation of stretch reflexes in human SCI. Controlled, triangular wave, ankle joint rotations applied at variable velocities (30–120 deg s⁻¹) and intervals between stretches (0.25–5.0 s) were performed on 14 SCI subjects with velocity-dependent, hyperexcitable plantarflexors. Repeated stretch elicited significant increases in plantarflexion torques and electromyographic (EMG) activity from the soleus (SOL) and medial gastrocnemius (MG). At higher velocities (≥ 90 deg s⁻¹), reflex torques declined initially, but subsequently increased to levels exceeding the initial response, while mean EMG responses increased throughout the joint perturbations. At lower velocities (≤ 60 deg s⁻¹), both joint torques and EMGs increased gradually. Throughout a range of angular velocities, reflex responses increased significantly only at intervals ≤ 1 s between stretches and following at least four rotations. Ramp-and-hold perturbations used to elicit tonic stretch reflexes revealed significantly prolonged EMG responses following one or two triangular stretches, as compared to single ramp-and-hold excursions. Post hoc analyses revealed reduced reflex facilitation in subjects using baclofen to control spastic behaviours. Evidence of stretch reflex facilitation post-SCI may reflect changes in underlying neuronal properties and provide insight into the mechanisms underlying spastic reflexes.     

Calcium currents and GABAB receptors in the dorsal sensory cells of the lamprey spinal cord

Patch-clamp studies were performed on the isolated dorsal sensory cells of the spinal cords of three species of lamprey,Ichthyomyzon unicuspis, Petromyzon marinus, andLampetra fluviatilis, to measure changes in the amplitudes of calcium current induced by GABA and its specific antagonists and agonists. The experiments showed that GABA (4 mM) reduced the peak amplitude of the calcium current by 28.5±4.9%, with subsequent recovery to 96.2±9.2% of control (n=45). The GABA_B agonist baclofen had similar effects. The GABA_A agonists glycine and taurine had no effect on the Ca²⁺ current. The inhibitory effect of GABA was blocked by 2-hydroxysaclofen (a GABA_B antagonist), but persisted in the presence of bicuculline (a GABA_A antagonist). These results are evidence that the membranes of dorsal sensory cells contain GABA_B receptors, which significantly increases our under-standing of the mechanisms of presynaptic inhibition in the spinal cords of the cyclostomata. Translated from Rossiiskii Fiziologischeskii Zhurnal imeni I. M. Sechenova, Vol. 83, No. 11-12, pp. 79–91, November–December, 1997.     

Crossed reciprocal inhibition evoked by electrical stimulation of the lamprey spinal cord

Activation of a motoneuron pool is often accompanied by inhibition of the antagonistic pool through a system of reciprocal inhibition between the two parts of the neuronal network controlling the antagonistic pools. In the present study, we describe the activity of such a system in the isolated spinal cord of the lamprey, when a tonic motor output is evoked by extracellular stimulation (0.5-1 s train of pulses, 20 Hz) of either end of the spinal cord. With two electrodes symmetrically positioned in relation to the midline, stimulation with either of them separately elicited prolonged (1-5 s) ipsilateral ventral root activity. Activity could be abolished by stronger, simultaneously applied, stimulation of the contralateral side of the cord, suggesting that reciprocal inhibition between hemisegments operates when a tonic motor output is generated. Simultaneous stimulation of both sides of the spinal cord with a single electrode with a large tip (300-400 microm in diameter), positioned over the anatomical midline, elicited inconsistent right-side, leftside, or bilateral ventral root responses. A minor displacement (10-20 microm) to the left or right from the midline resulted in activation of ipsilateral motoneurons, whereas the contralateral motoneurons were silent. These findings indicate that a small asymmetry in the excitatory drive to the left and right spinal hemisegments can be further amplified by reciprocal inhibition between the hemisegments. Longitudinal splitting of the spinal cord along the midline resulted in reduced reciprocal inhibition between the hemisegments separated by the lesion. The reduction was proportional to the extent of the split. The inhibition was abolished when the split reached nine segments in length. From these experiments, the longitudinal distribution of the commissural axons responsible for inhibition of contralateral motor output could be estimated.     

Physiological and morphological correlates of presynaptic inhibition in primary afferents of the lamprey spinal cord.

Patch-clamp recordings in a whole-cell mode were performed on dorsal sensory cells enzymatically isolated from the spinal cord of two lamprey species, Ichthyomyzon unicuspis and Lampetra fluviatilis. The voltage-activated currents through calcium channels were analysed. GABA and the specific GABA(B) receptor agonist baclofen reduced the peak amplitude of inward Ba2+ current, as a robust alternate charge carrier through voltage-dependent Ca2+ channels. These effects were dose-dependent and reversible. GABA(B) receptor antagonists, 2-hydroxysaclofen and delta-amino-n-valeric acid, blocked the reduction of Ba2+ currents by GABA and baclofen, while bicuculline, a GABA(A) receptor antagonist, had no blocking action. GABA and baclofen did not modify the dorsal sensory cell membrane conductance, indicating that they did not activate ligand-gated channels. However, GABA, but not baclofen, considerably increased membrane conductance and induced Cl- currents in isolated multipolar neurons (presumably interneurons and/or motoneurons). These findings suggest that GABA and baclofen action on lamprey dorsal sensory cells is mediated by GABA(B) receptors. We concluded that GABA-mediated presynaptic inhibition of lamprey dorsal sensory cell fibers results from GABA(B) receptor activation followed by a decrease of inward voltage-activated calcium currents. Appositions of GABA-immunoreactive boutons to horseradish peroxidase-labeled fibers from the dorsal root were observed at the ultrastructural level in the dorsal column using postembedding immunogold cytochemistry. It seems likely that these appositions represent the morphological substrate of dorsal sensory cell fiber presynaptic inhibition. In very rare cases, ultrastructural features were observed which could be interpreted as synaptic specializations between the GABA-immunoreactive boutons and the primary afferent fibers. The extrasynaptic action of GABA as a basis of presynaptic inhibition of this population of primary afferent neurons is discussed.     

Transient facilitation and inhibition of sexual reflexes in female hamsters

Artificial stimuli were used to study transient changes in responsiveness of female hamsters in natural estrus to tactile stimuli occurring during mating. Tactile stimulation just lateral to the vagina produces a transient facilitation of responses to stimulation of the contralateral side, while tactile stimulation of the midline produces a short-term inhibition of lateral movement in response to tactile stimulation. These short-term changes in responsiveness are probably an integral part of the basic sexual responses of the female hamster that contribute to insertion during copulatory attempts.     

Slow dorsal-ventral rhythm generator in the lamprey spinal cord

In the isolated lamprey spinal cord, a very slow rhythm (0.03-0.11 Hz), superimposed on fast N-methyl-D-aspartate (NMDA)-induced locomotor activity (0.26-2.98 Hz), could be induced by a blockade of GABA(A) or glycine receptors or by administration of (1 s, 3 s)-l-aminocyclopentane-1,3-dicarboxylic acid a metabotropic glutamate receptor agonist. Ventral root branches supplying dorsal and ventral myotomes were exposed bilaterally to study the motor pattern in detail. The slow rhythm was expressed in two main forms: 1) a dorsal-ventral reciprocal pattern was the most common (18 of 24 preparations), in which bilateral dorsal branches were synchronous and alternated with the ventral branches, in two additional cases a diagonal dorsal-ventral reciprocal pattern with alternation between the left (or right) dorsal and the right (or left) ventral branches was observed; 2) synchronous bursting in all branches was encountered in four cases. In contrast, the fast locomotor rhythm occurred always in a left-right reciprocal pattern. Thus when the slow rhythm appeared in a dorsal-ventral reciprocal pattern, fast rhythms would simultaneously display left-right alternation. A longitudinal midline section of the spinal cord during ongoing slow bursting abolished the reciprocal pattern between ipsilateral dorsal and ventral branches but a synchronous burst activity could still remain. The fast swimming rhythm did not recover after the midline section. These results suggest that in addition to the network generating the swimming rhythm in the lamprey spinal cord, there is also a network providing slow reciprocal alternation between dorsal and ventral parts of the myotome. During steering, a selective activation of dorsal and ventral myotomes is required and the neural network generating the slow rhythm may represent activity in the spinal machinery used for steering.     

The effects of baclofen on calcium channel currents in dorsal sensory cells of the spinal cord in the lamprey

Dorsal sensory cells isolated from the spinal cord of the lamprey speciesIchthyomyzon unicuspis andLampetra fluviatilis were used for whole-cell patch-clamp studies of the effects of baclofen on calcium channel currents, evoked in conditions in which Na⁺, K⁺ currents were blocked, by depolarizing membranes from constant holding potentials of −100 or −80 mV to +30 mV. Ba ions were used as carriers of currents through calcium channels. These studies demonstrated that baclofen (0.5 mM) decreased the peak amplitude of the Ba²⁺ current by an average of 22.5±4.2% (n=12) in dorsal sensory cells of the lampreyIchthyomyzon unicuspis and by 28.4±3.3% in the dorsal sensory cells ofLampetra fluviatilis (n=25). The conductivity of dorsal sensory cell membranes in the presence of baclofen (and GABA) did not change. The blocking action of baclofen persisted in the presence of bicuculline (100 μM) and was lifted by addition of δ-aminovaleric acid and 2-hydroxysaclofen to the perfusing solution. These results are interpreted as evidence for the presence of GABA_B receptors in dorsal sensory cell membranes. The data were compared with published results, and the question of the functional significance of GABA_B receptors in the dorsal sensory cells (primary afferent cells) of cyclostomata is discussed. Translated from Rossiiskii Fiziologischeskii Zhurnal imeni I. M. Sechenova, Vol. 83, No. 11-12, pp. 92–104, November–December, 1997.     

Sensory cells in the spinal cord of the sea lamprey

1. Intracellular recordings were made from dorsal cells in the spinal cord of the sea lamprey.2. Dorsal cells were excited by mechanical stimulation of the ipsilateral skin and were established as first-order sensory cells using a variety of physiological criteria. Receptive fields were mapped.3. Dorsal cells were subdivided into three functional types on the basis of their responses to mechanical stimulation of the skin. Touch (T) cells gave rapidly adapting responses to indentation of the skin. Pressure (P) cells gave slowly adapting responses to indentation of the skin. Nociceptive (N) cells gave slowly adapting responses to severe (often destructive) indentation of the skin. The three types also differed in their spontaneous activity and in their response to repeated stimulation.4. Pressure and nociceptive cells were excited by heating the skin over the receptive field. The amount of heat necessary to excite nociceptive cells was greater than that necessary for pressure cells and often left a permanent burn mark on the skin.5. The three types of dorsal cell also showed differences in resting potential, membrane time constant, spike threshold, and response to sustained depolarization.     

Modulation of cellular and synaptic variability in the lamprey spinal cord

Variability is increasingly recognized as a characteristic feature of cellular, synaptic, and network properties. While studies have traditionally focused on mean values, significant effects can result from changes in variance. This study has examined cellular and synaptic variability in the lamprey spinal cord and its modulation by the neuropeptide substance P. Cellular and synaptic variability differed in different types of cell and synapse. Substance P reduced the variability of subthreshold locomotor-related depolarizations and spiking in motor neurons during network activity. These effects were associated with a reduction in the variability of spiking in glutamatergic excitatory network interneurons and with a reduction in the variance of excitatory interneuron-evoked excitatory postsynaptic potentials (EPSPs). Substance P also reduced the variance of postsynpatic potentials (PSPs) from crossing inhibitory and excitatory interneurons, but it increased the variance of inhibitory postsynpatic potentials (IPSPs) from ipsilateral inhibitory interneurons. The effects on the variance of different PSPs could occur with or without changes in the PSP amplitude. The reduction in the variance of excitatory interneuron-evoked EPSPs was protein kinase A, calcium, and N-methyl-d-aspartate (NMDA) dependent. The NMDA dependence suggested that substance P was acting postsynaptically. This was supported by the reduced variability of postsynaptic responses to glutamate by substance P. However, ultrastructural analyses suggested that there may also be a presynaptic component to the modulation, because substance P reduced the variability of synaptic vesicle diameters in putative glutamatergic terminals. These results suggest that cellular and synaptic variability can be targeted for modulation, making it an additional source of spinal cord plasticity.     

Distribution of serotonergic neurons and processes in the lamprey spinal cord

The distribution of serotonin in the spinal cord in two species of lamprey, Ichthyomyzon unicuspis and Petromyzon marinus, was studied by indirect immunofluorescence techniques. Multipolar cell bodies containing serotonin-like immunoreactivity were found along the length of the spinal cord, along the midline and slightly ventral to the central canal. These cell bodies send a diffuse projection of processes throughout the spinal cord, including: (1) a dense projection to the ventral surface; (2) a strong projection to the ventromedial longitudinal fiber tracts; (3) a less intense projection to the dorsal longitudinal fiber tracts; and (4) a weak projection to the lateral fiber tracts. Lesion experiments showed that processes descending from the brain or rostral spinal cord provide a major projection to the lateral fiber tracts and smaller contributions to the dorsal and ventromedial fiber tracts. Fluorescent processes were also observed in the dorsal roots and serotonergic peripheral cell bodies were seen adjacent to the dorsal roots.Our results suggest that the serotonergic innervation of the lamprey spinal cord arises from three sources: spinal interneurons, descending tracts and peripheral (possibly sensory) input. This provides an anatomical substrate for our recent finding^12,13 that serotonin modulates the central pattern generator for locomotion in the lamprey spinal cord.     

Transmitter phenotypes of commissural interneurons in the lamprey spinal cord

The fundamental network for locomotion in all vertebrates contains a central pattern generator or CPG that produces the required motor output in the spinal cord. In the lamprey spinal cord different classes of interneuron's forming the core CPG circuitry have been characterized based on their morphological and electrophysiological features. The commissural interneuron's (C-INs) represent one essential component of CPG that have been implicated in controlling left–right alternation of the motor activity during swimming. However, it is still unclear if the C-INs displays a homogenous neurotransmitter phenotype and how they are distributed. In this paper we investigated the segmental distribution of glycine, glutamate and GABA-immunoreactive (ir) C-INs by combining retrograde Neurobiotin tracing with specific antibodies for these transmitters. The C-INs were more abundant in caudal and rostral segments adjacent to the injection site and their number gradually decreased in more distal segments, suggesting that these interneurons project over a short distance. The glycine-ir neurons represented around 50% of the total C-INs, while glutamate-ir neurons represented only 29%. Both types of C-INs were homogenously distributed over different segments along the spinal cord. Finally, no Neurobiotin labeled C-INs displayed GABA-ir, although many interneurons were ir to GABA, suggesting that GABAergic interneurons are not directly responsible for controlling left–right alternation of activity during locomotion in lamprey. Overall, these results show that the C-INs display a gradual rostrocaudal distribution and consist of both glycine- and glutamate-ir neurons. The difference in the proportion of inhibitory and excitatory C-INs represents an anatomical substrate that can ensure the predominance of alternating activity during locomotion.     

Cholinergic modulation of the locomotor network in the lamprey spinal cord

Acetylcholine (ACh) was found here to be a strong modulator of swimming activity in the isolated spinal cord preparation of the adult lamprey (Ichthyomyzon unicuspis). During fictive swimming induced with either D-glutamate or N-methyl-D-aspartate, addition of ACh (200 microM) significantly reduced the cycle period of ventral root bursts to 54%, intersegmental phase lag to 32%, and ventral root burst proportion to 80% of control levels. Effects of ACh were apparent at concentrations as low as 1 microM. Both nicotinic and muscarinic receptors are involved, in that application of either nicotinic or muscarinic agonists alone significantly reduced cycle period. There is sufficient endogenous ACh in the spinal cord to modulate ongoing fictive swimming, as shown by application of the cholinesterase inhibitor eserine (physostigmine). Eserine (20 microM) significantly reduced the cycle period to 78% and phase lag to 58% of control levels, and these effects were reversed with the addition of cholinergic blockers. Addition of only a nicotinic or muscarinic antagonist, mecamylamine (10 microM) or scopolamine (20 microM), respectively, to the spinal cord during fictive swimming produced significant increases in cycle period and phase lag, suggesting that both types of cholinergic receptors participate in endogenous cholinergic modulation. It is concluded that ACh is an endogenous modulator of the locomotor network in the lamprey spinal cord and that ACh may take part in the regulation of cycle period, intersegmental coupling, and ventral root burst duration.     

GABA- and glycine-immunoreactive terminals contacting motoneurons in lamprey spinal cord

Double postembedding GABA- and glycine-immunostaining was performed on the lamprey (Lampetra fluviatilis) spinal cord after previous HRP labeling of motoneurons. Immunopositive boutons contacting motoneurons were counted and distinguished as GABA (39%), glycine (30%) and both GABA+glycine-immunopositive (31%). Densely-packed, flattened synaptic vesicles were only observed in glycine-immunopositive boutons while GABA-immunoreactive and GABA+glycine-immunoreactive boutons contained rounded or oval synaptic vesicles. Dense-core vesicles of different diameters were associated with conventional synaptic vesicles in 74% of GABA-only-immunopositive boutons, 50% of double GABA+glycine-immunopositive boutons, but were only observed in 9% of glycine-only-immunopositive boutons. The presence of terminals immunoreactive to either GABA or glycine contacting the motoneurons suggests that there is a morphological substrate for both GABAergic and glycinergic postsynaptic inhibition of motoneurons in the lamprey spinal cord.     

Topographic organization of monosynaptic reflexes in the cat spinal cord

The topographic organization of monosynaptic reflexes in the cat spinal cord has been studied by comparing the amplitude of reflex discharges recorded from ventral roots consequent to stimulation of dorsal roots entering the cord at different spinal segments. The results indicate that up to 80% of the potentiated monosynaptic reflex discharge recorded from a ventral root can be attributed to afferent input entering the spinal cord at the same segmental level. Moreover, within the same segment, afferents with a more rostral cord entry level exert a stronger synaptic effect on the more rostral portion of the corresponding ventral root.     

Developmental differences in neuromodulation and synaptic properties in the lamprey spinal cord

Functional properties in the spinal cord change during development to adapt motor outputs to differing behavioral requirements. Here, we have examined whether there are also developmental differences in spinal cord plasticity by comparing the neuromodulatory effects of substance P in the larval lamprey spinal cord with its previously characterized effects in premigratory adults. The premigratory adult effects of substance P were all significantly reduced in larvae. As the adult effects of substance P depend on the N-methyl-d-aspartate (NMDA)-dependent potentiation of glutamatergic synaptic transmission, we examined if the developmental differences in neuromodulation were associated with differences in synaptic properties. We found that the amplitude, rise time, and half-width of excitatory postsynaptic potentials (EPSPs) from excitatory network interneurons were all significantly reduced in larvae compared with adults. These differences were associated with a reduction in the NMDA component of larval EPSPs, an effect that could have contributed to the reduced modulatory effects of substance P in larvae. In contrast to glutamatergic inputs, the amplitude, rise time, and half-width of inhibitory postsynaptic potentials (IPSPs) from ipsilateral inhibitory interneurons were all significantly increased in larvae compared with adults. Substance P also potentiated larval IPSP amplitudes, an effect not seen in adults. This increase in inhibition contributed to the reduced effects of substance P in larvae, as premigratory adult-like modulation could be evoked when inhibition was blocked with strychnine. These results suggest that opposite developmental changes in excitatory and inhibitory synaptic transmission and their modulation are associated with developmental differences in spinal cord neuromodulation.     

AMPA receptor-PDZ interactions in facilitation of spinal sensory synapses.

Silent synapses form between some primary sensory afferents and dorsal horn neurons in the spinal cord. Molecular mechanisms for activation or conversion of silent synapses to conducting synapses are unknown. Serotonin can trigger activation of silent synapses in dorsal horn neurons by recruiting AMPA receptors. AMPA-receptor subunits GluR2 and GluR3 interact via their cytoplasmic C termini with PDZ-domain-containing proteins such as GRIP (glutamate receptor interacting protein), but the functional significance of these interactions is unclear. Here we demonstrate that protein interactions involving the GluR2/3 C terminus are important for serotonin-induced activation of silent synapses in the spinal cord. Furthermore, PKC is a necessary and sufficient trigger for this activation. These results implicate AMPA receptor-PDZ interactions in mechanisms underlying sensory synaptic potentiation and provide insights into the pathogenesis of chronic pain.     

Synaptic regeneration and glial reactions in the transected spinal cord of the lamprey

Summary We have examined axonal growth and synaptic regeneration in identified giant neurons of the transected lamprey spinal cord using intracellular injection of horseradish peroxidase. Wholemounts together with serial section light and electron microscopy, show that axons from identified Müller and Mauthner reticulospinal neurons grow across the lesion and regenerate new synaptic contacts. Relatively normal swimming returns in these animals by 3–4 weeks after spinal transection. This occurs despite the formation of regenerated synapses in regions of the cord that are not usually occupied by these neurons.The regenerating axons branch profusely in contrast to their unbranched state in the normal animal. In addition to showing the two synaptic configurations found normally, synapses may be formed by slender sprouts from the growing giant axon. These sprout type synaptic contacts appear unique to the regenerating neuron. Only regenerated chemical synapses were seen; the morphologically mixed chemical and electrical (gap junction) synaptic complex common in the normal animal was not observed at regenerated synapses.The site of spinal transection in the functionally recovered animal shows an increase in the number of ependymal and glial cells. Ependymal-like cells appear in regions away from the central canal. The expanded ependymal and glial processes covering the peripheral surface of the injured cord become convoluted, in contrast to their normal smooth configuration. There is no collagen within the cord at the site of transection but a considerable deposition is seen external to the cord surface.Axonal growth across a spinal lesion and subsequent synaptic regeneration can be examined in single identifiable giant interneurons in the spinal cord of the larval lamprey. This preparation may be used as an assay to investigate factors that could contribute to functional recovery following central nervous system injury in the higher vertebrates.