Spastic Long-Lasting Reflexes of the Chronic Spinal Rat Studied In Vitro

2004 ◽  
Vol 91 (5) ◽  
pp. 2236-2246 ◽  
Author(s):  
Y. Li ◽  
P. J. Harvey ◽  
X. Li ◽  
D. J. Bennett
Keyword(s):  
Spinal Cord ◽  
Adrenergic Receptor ◽  
Dorsal Root ◽  
Low Threshold ◽  
Muscle Spasms ◽  
Ventral Roots ◽  
Acute Spinal Rats ◽  
Chronic Spinal Rats ◽  
Adrenergic Receptor Agonist

Over the months following sacral spinal cord transection in adult rats, a pronounced spasticity syndrome emerges in the affected tail musculature, where long-lasting muscle spasms can be evoked by low-threshold afferent stimulation (termed long-lasting reflex). To develop an in vitro preparation to examine the neuronal mechanisms underlying spasticity, we removed the whole sacrocaudal spinal cord of these spastic chronic spinal rats (>1 mo after S2 sacral spinal transection) and maintained it in artificial cerebral spinal fluid in a recording chamber. The ventral roots were mounted on monopolar recording electrodes in grease, and the reflex responses to dorsal root stimulation were recorded and compared with the reflexes seen in the awake chronic spinal rat. When the dorsal roots were stimulated with a single pulse, a long-lasting reflex occurred in the ventral roots, with identical characteristics to the long-lasting reflex in the awake spastic rat tail. The reflex response was low threshold ( T), short latency, long duration (∼2 s), and enhanced by repeated stimulation. Brief high-frequency stimulation trains (0.5 s, 100 Hz, 1.5 × T) evoked even longer duration responses (5–10 s), with repeated bursts of activity that were similar to the repeated muscle spasms evoked in awake rats with stimulation trains or manual skin stimulation. Stimulation of a given dorsal root evoked long-lasting reflexes in both the ipsilateral and contralateral ventral roots. Long-lasting reflexes did not occur in the sacrocaudal spinal cord of acute spinal rats (S2 transection), which is similar to the areflexia seen in awake acute spinal rats. However, long-lasting reflexes could be made to occur in the acute spinal rat by altering K+ (7 mM) or Mg2+ (0 mM) concentrations, or by application of high doses of the neuromodulators norepinephrine (NE, >20 μM) or serotonin (5-HT, >20 μM). In chronic spinal rats, much lower doses of these neuromodulators (0.1 μM) enhanced the long-lasting reflexes, suggesting a denervation supersensitivity to 5-HT and NE following injury. Higher doses of NE or 5-HT produced a paradoxical inhibition of the long-lasting reflexes. The high dose inhibition by NE was mimicked by the α2-adrenergic receptor agonist clonidine but not the α1-adrenergic receptor agonist methoxamine. In summary, the sacral spinal in vitro preparation offers a new approach to the study of spinal cord injury and analysis of antispastic drugs.

Plateau Potentials in Sacrocaudal Motoneurons of Chronic Spinal Rats, Recorded In Vitro

2001 ◽  
Vol 86 (4) ◽  
pp. 1955-1971 ◽  
Author(s):  
David J. Bennett ◽  
Yunru Li ◽  
Merek Siu
Keyword(s):  
Spinal Cord ◽  
Firing Rate ◽  
Dorsal Root ◽  
Reflex Response ◽  
Plateau Potentials ◽  
Chronic Injury ◽  
Acute Spinal Rats ◽  
Chronic Spinal Rats ◽  
Root Stimulation

Intracellular recordings were made from sacrocaudal tail motoneurons of acute and chronic spinal rats to examine whether plateau potentials contribute to spasticity associated with chronic injury. The spinal cord was transected at the S2 level, causing, over time, exaggerated long-lasting reflexes (hyperreflexia) associated with a general spasticity syndrome in the tail muscles of chronic spinal rats (1–5 mo postinjury). The whole sacrocaudal spinal cord of chronic or acute spinal rats was removed and maintained in vitro in normal artificial cerebral spinal fluid (ACSF). Hyperreflexia in chronic spinal rats was verified by recording the long-lasting ventral root responses to dorsal root stimulation in vitro. The intrinsic properties of sacrocaudal motoneurons were studied using intracellular injections of slow triangular current ramps or graded current pulses. In chronic spinal rats, the current injection triggered sustained firing and an associated sustained depolarization ( plateau potential; 34/35 cells; mean, 5.5 mV; duration >5 s; normal ACSF). The threshold for plateau initiation was low and usually corresponded to an acceleration in the membrane potential just before recruitment. After recruitment and plateau activation, the firing rate changed linearly with current during the slow ramps [63% of cells had a linear frequency-current ( F-I) relation] despite the presence of the plateau. The persistent inward current ( I PIC) producing the plateau and sustained firing was estimated to be on average 0.8 nA as determined by the reduction in injected current needed to stop the sustained firing [Δ I = −0.8 ± 0.6 (SD) nA], compared with the current needed to start firing ( I = 1.7 ± 1.5 nA; 47% reduction). In motoneurons of acute spinal rats, plateaus were rarely seen (3/22), although they could be made to occur with bath application of serotonin. In motoneurons of chronic spinal rats there were no significant changes in the mean passive input resistance, rheobase or amplitude of the spike afterhyperpolarization (AHP) as compared with acute spinal rats. However, there were significant increases in AHP duration and initial firing rate at recruitment and decreases in minimum firing rate and F-I slope. We suggest that the higher initial firing rate resulted from the plateau activation at recruitment and the lower F-I slope resulted from an increase in active conductance during firing, due to I PIC. Brief dorsal root stimulation also triggered a plateau and sustained discharge (long-lasting reflexes; 2–5 s) in motoneurons of chronic (but not acute) spinal rats. When the plateau was eliminated by a hyperpolarizing current bias, the reflex response was significantly shortened (to 1 s). Thus plateaus contributed substantially to the long-lasting reflexes in vitro and therefore should contribute significantly to the corresponding exaggerated reflexes and spasticity in awake chronic spinal rats.

scholarly journals Effects of Baclofen on Spinal Reflexes and Persistent Inward Currents in Motoneurons of Chronic Spinal Rats With Spasticity

2004 ◽  
Vol 92 (5) ◽  
pp. 2694-2703 ◽  
Author(s):  
Y. Li ◽  
X. Li ◽  
P. J. Harvey ◽  
D. J. Bennett
Keyword(s):  
Spinal Cord ◽  
Ventral Root ◽  
Spinal Reflexes ◽  
Monosynaptic Reflex ◽  
Persistent Inward Currents ◽  
Inward Currents ◽  
Muscle Spasms ◽  
Acute Spinal Rats ◽  
Chronic Spinal Rats ◽  
Higher Doses

In the months after spinal cord injury, motoneurons develop large voltage-dependent persistent inward currents (PICs) that cause sustained reflexes and associated muscle spasms. These muscle spasms are triggered by any excitatory postsynaptic potential (EPSP) that is long enough to activate the PICs, which take >100 ms to activate. The PICs are composed of a persistent sodium current (Na PIC) and a persistent calcium current (Ca PIC). Considering that Ca PICs have been shown in other neurons to be inhibited by baclofen, we tested whether part of the antispastic action of baclofen was to reduce the motoneuron PICs as opposed to EPSPs. The whole sacrocaudal spinal cord from acute spinal rats and spastic chronic spinal rats (with sacral spinal transection 2 mo previously) was studied in vitro. Ventral root reflexes were recorded in response to dorsal root stimulation. Intracellular recordings were made from motoneurons, and slow voltage ramps were used to measure PICs. Chronic spinal rats exhibited large monosynaptic and long-lasting polysynaptic ventral root reflexes, and motoneurons had associated large EPSPs and PICs. Baclofen inhibited these reflexes at very low doses with a 50% inhibition (EC50) of the mono- and polysynaptic reflexes at 0.26 ± 0.07and 0.25 ± 0.09 (SD) μM, respectively. Baclofen inhibited the monosynaptic reflex in acute spinal rats at even lower doses (EC50 = 0.18 ± 0.02 μM). In chronic (and acute) spinal rats, all reflexes and EPSPs were eliminated with 1 μM baclofen with little change in motoneuron properties (PICs, input resistance, etc), suggesting that baclofen's antispastic action is presynaptic to the motoneuron. Unexpectedly, in chronic spinal rats higher doses of baclofen (20–30 μM) significantly increased the total motoneuron PIC by 31.6 ± 12.4%. However, the Ca PIC component (measured in TTX to block the Na PIC) was significantly reduced by baclofen. Thus baclofen increased the Na PIC and decreased the Ca PIC with a net increase in total PIC. By contrast, when a PIC was induced by 5-HT (10–30 μM) in motoneurons of acute spinal rats, baclofen (20–30 μM) significantly decreased the PIC by 38.8 ± 25.8%, primarily due to a reduction in the Ca PIC (measured in TTX), which dominated the total PIC in these acute spinal neurons. In summary, baclofen does not exert its antispastic action postsynaptically at clinically achievable doses (<1 μM), and at higher doses (10–30 μM), baclofen unexpectedly increases motoneuron excitability (Na PIC) in chronic spinal rats.

scholarly journals Serotonin Facilitates a Persistent Calcium Current in Motoneurons of Rats With and Without Chronic Spinal Cord Injury

2007 ◽  
Vol 97 (2) ◽  
pp. 1236-1246 ◽  
Author(s):  
X. Li ◽  
K. Murray ◽  
P. J. Harvey ◽  
E. W. Ballou ◽  
D. J. Bennett
Keyword(s):  
Spinal Cord ◽  
Calcium Currents ◽  
High Dose ◽  
Chronic Injury ◽  
Persistent Inward Currents ◽  
Cord Injury ◽  
Inward Currents ◽  
Acute Spinal Rats ◽  
Chronic Spinal Rats

In the months after spinal cord transection, motoneurons in the rat spinal cord develop large persistent inward currents (PICs) that are responsible for muscle spasticity. These PICs are mediated by low-threshold TTX-sensitive sodium currents (Na PIC) and L-type calcium currents (Ca PIC). Recently, the Na PIC was shown to become supersensitive to serotonin (5-HT) after chronic injury. In the present paper, a similar change in the sensitivity of the Ca PIC to 5-HT was investigated after injury. The whole sacrocaudal spinal cord from acute spinal rats and spastic chronic spinal rats (S2 level transection 2 mo previously) was studied in vitro. Intracellular recordings were made from motoneurons and slow voltages ramps were applied to measure PICs. TTX was used to block the Na PIC. For motoneurons of chronic spinal rats, a low dose of 5-HT (1 μM) significantly lowered the threshold of the Ca PIC from −56.7 ± 6.0 to −63.1 ± 7.1 mV and increased the amplitude of the Ca PIC from 2.4 ± 1.0 to 3.0 ± 0.73 nA. Higher doses of 5-HT acted similarly. For motoneurons of acute spinal rats, low doses of 5-HT had no significant effects, whereas a high dose (about 30 μM) significantly lowered the threshold of the L-Ca PIC from −58.5 ± 14.8 to −62.5 ± 3.6 mV and increased the amplitude of the Ca PIC from 0.69 ± 1.05 to 1.27 ± 1.1 nA. Thus Ca PICs in motoneurons are about 30-fold supersensitive to 5-HT in chronic spinal rats. The 5-HT–induced facilitation of the Ca PIC was blocked by nimodipine, not by the Ih current blocker Cs+ (3 mM) or the SK current blocker apamin (0.15 μM), and it lasted for hours after the removal of 5-HT from the nCSF, even increasing initially after removing 5-HT. The effects of 5-HT make motoneurons more excitable and ultimately lead to larger, more easily activated plateaus and self-sustained firing. The supersensitivity to 5-HT suggests the small amounts of endogenous 5-HT below the injury in a chronic spinal rat may act on supersensitive receptors to produce large Ca PICs and ultimately enable muscle spasms.

scholarly journals 5-HT2 Receptor Activation Facilitates a Persistent Sodium Current and Repetitive Firing in Spinal Motoneurons of Rats With and Without Chronic Spinal Cord Injury

2006 ◽  
Vol 96 (3) ◽  
pp. 1158-1170 ◽  
Author(s):  
P. J. Harvey ◽  
X. Li ◽  
Y. Li ◽  
D. J. Bennett
Keyword(s):  
Spinal Cord ◽  
Sodium Current ◽  
Receptor Activation ◽  
Repetitive Firing ◽  
Persistent Sodium Current ◽  
Spinal Motoneurons ◽  
Spinal Transection ◽  
Acute Spinal Rats ◽  
Chronic Spinal Rats

We examined the modulation of persistent inward currents (PICs) by serotonin (5-HT) in spinal motoneurons of normal and chronic spinal rats. PICs are composed of both a TTX-sensitive persistent sodium current (Na PIC) and a nimodipine-sensitive persistent calcium current (Ca PIC), and we focused on quantifying the Na PIC (and its action on the total PIC), which is known to be critical in enabling repetitive firing. Intracellular recordings were made from motoneurons of the whole sacrocaudal spinal cord of normal adult rats after the cord was acutely transected at the S2 spinal level (acute spinal rat condition), removed from the animal, and then maintained in vitro. In vitro motoneuron recordings were likewise made from rats that had a sacral spinal transection 2 mo previously (chronic spinal rats). In motoneurons from acute spinal rats, moderately high doses of 5-HT (≥10 μM), or the 5-HT2 receptor agonist DOI (≥30 μM), significantly increased the total PIC, hyperpolarized the PIC onset voltage, and hyperpolarized the spike threshold, whereas lower doses had no effect. Both 5-HT and DOI specifically increased the Na PIC portion of the total PIC (tested with nimodipine blocking the Ca PIC). Additionally, 5-HT, but not DOI, depolarized the resting membrane potential ( Vm) and increased the input resistance ( Rm) in a dose-dependent manner. Therefore 5-HT2 receptor activation facilitated the Na PIC, whereas other 5-HT receptors modulated Vm and Rm. Motoneurons of chronic spinal rats responded to 5-HT and DOI in the same way, but with larger responses and at much lower doses (0.3–1 μM), thus exhibiting a 30-fold supersensitivity to 5-HT. Specifically the Na PIC was supersensitive to 5-HT2 receptor activation with DOI. Also, Rm and Vm were supersensitive to 5-HT. Consistent with the known critical role of the Na PIC in repetitive firing, enhancement of the Na PIC by DOI or 5-HT facilitated the repetitive firing evoked by steady current injection and enabled repetitive firing in a subpopulation of motoneurons of acute spinal rats that were initially unable to produce sustained repetitive firing. We suggest that after spinal transection, residual endogenous spinal sources of 5-HT help facilitate the Na PIC and repetitive firing. With chronic injury, the developed 5-HT supersensitivity more than compensates for lost brain stem 5-HT, so that the Na PIC is large and motoneurons are very excitable, thus contributing to spasticity.

scholarly journals Persistent Sodium Currents and Repetitive Firing in Motoneurons of the Sacrocaudal Spinal Cord of Adult Rats

2006 ◽  
Vol 96 (3) ◽  
pp. 1141-1157 ◽  
Author(s):  
P. J. Harvey ◽  
Y. Li ◽  
X. Li ◽  
D. J. Bennett
Keyword(s):  
Spinal Cord ◽  
Input Resistance ◽  
Calcium Currents ◽  
Repetitive Firing ◽  
Resting Membrane Potential ◽  
Sodium Currents ◽  
Adult Rats ◽  
Acute Spinal Rats ◽  
Chronic Spinal Rats

Months after sacral spinal transection in rats (chronic spinal rats), motoneurons below the injury exhibit large, low-threshold persistent inward currents (PICs), composed of persistent sodium currents (Na PICs) and persistent calcium currents (Ca PICs). Here, we studied whether motoneurons of normal adult rats also exhibited Na and Ca PICs when the spinal cord was acutely transected at the sacral level (acute spinal rats) and examined the role of the Na PIC in firing behavior. Intracellular recordings were obtained from motoneurons of acute and chronic spinal rats while the whole sacrocaudal spinal cord was maintained in vitro. Compared with chronic spinal rats, motoneurons of acute spinal rats were more difficult to activate because the input resistance was 22% lower and resting membrane potential was hyperpolarized 4.1 mV further below firing threshold (−50.9 ± 6.2 mV). In acute spinal rats, during a slow voltage ramp, a PIC was activated subthreshold to the spike (at −57.2 ± 5.0 mV) and reached a peak current of 1.11 ± 1.21 nA. This PIC was less than one-half the size of that in chronic spinal rats (2.79 ± 0.94 nA) and usually was not large enough to produce bistable behavior (plateau potentials and self-sustained firing not present), unlike in chronic spinal rats. The PIC was composed of two components: a TTX-sensitive Na PIC (0.44 ± 0.36 nA) and a nimodipine-sensitive Ca PIC (0.78 ± 0.82 nA). Both were smaller than in chronic spinal rats (but with similar Na/Ca ratio). The presence of the Na PIC was critical for normal repetitive firing, because no detectable Na PIC was found in the few motoneurons that could not fire repetitively during a slow ramp current injection and motoneurons that had large Na PICs more readily produced repetitive firing and had lower minimum firing rates compared with neurons with small Na PICs. Furthermore, when the Na PIC was selectively blocked with riluzole, steady repetitive firing was eliminated, even though transient firing could be evoked on a rapid current step and the spike itself was unaffected. In summary, only small Ca and Na PICs occur in acute spinal motoneurons, but the Na PIC is essential for steady repetitive firing. We discuss how availability of monoamines may explain the variability in Na PICs and firing in the normal and spinal animals.

Small-caliber afferent inputs produce a heterosynaptic facilitation of the synaptic responses evoked by primary afferent A-fibers in the neonatal rat spinal cord in vitro

1993 ◽  
Vol 69 (6) ◽  
pp. 2116-2128 ◽  
Author(s):  
S. W. Thompson ◽  
C. J. Woolf ◽  
L. G. Sivilotti
Keyword(s):  
Spinal Cord ◽  
Dorsal Root ◽  
Fiber Strength ◽  
Conditioning Stimulus ◽  
Neonatal Rat ◽  
Primary Afferent ◽  
Synaptic Potentials ◽  
C Fiber ◽  
Afferent Inputs

1. The effect of brief primary afferent inputs on the amplitude and duration of the synaptic potentials evoked in ventral horn (VH) neurons by the activation of other unconditioned primary afferents was studied by current-clamp intracellular recording in the neonatal rat hemisected spinal cord in vitro. Low-frequency (1 Hz) trains of stimulation were applied to a lumbar dorsal root (Conditioning root) for 20-30 s. Test excitatory synaptic potentials (EPSPs) were evoked by single electrical shocks applied to an adjacent Test dorsal root. 2. Test and Conditioning inputs were generated at stimulation strengths sufficient to activate A beta-, A delta- and C-afferent fibers successively. At A delta- and C-fiber strength the EPSPs lasted for 4-6 s, and, during the repetitive Conditioning inputs, these summated to produce a progressively incrementing cumulative depolarization that slowly decayed back to the control Vm over tens of seconds. 3. Dorsal root conditioning produced heterosynaptic facilitation, defined as an enhancement of Test EPSPs above their DC matched controls, in 7 out of 20 neurons. To facilitate the unconditioned afferent input, the intensity of conditioning stimulation had to exceed the threshold for the activation of thin myelinated (A delta) afferents: conditioning at A beta-fiber strength had no effect, whereas A delta- and C-fiber strength conditioning were equally effective. 4. Heterosynaptic facilitation of only A beta- or A delta-fiber-evoked Test EPSPs was observed, no enhancement of C-fiber strength Test EPSPs could be demonstrated. The facilitation manifested as increases in the EPSP peak amplitude, area or the number of action potentials evoked. 5. Conditioning trials that produced heterosynaptic facilitation generated cumulative depolarizations larger than those produced by ineffective conditioning trials (9.1 +/- 3.1 vs. 3.3 +/- 0.5 mV after 20 s conditioning at resting Vm, mean +/- SE, n = 6 and 13, respectively; P < 0.05). The slope of the Vm trajectory during the summation of the conditioning EPSPs was higher in trials resulting in heterosynaptic facilitation, at 0.31 +/- 0.10 mV/s in neurons with heterosynaptic facilitation and 0.06 +/- 0.02 mV/s in cells without heterosynaptic facilitation (P < 0.05). 5. Four of the 20 VH neurons in our sample responded to A delta/C-fiber conditioning with action-potential windup: all 4 also displayed heterosynaptic facilitation. 6. Heterosynaptic facilitation decayed after the completion of the conditioning stimulus with a time course that was parallel to but not superimposable on that of the slow Vm depolarization evoked by the conditioning.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Fictive Rhythmic Motor Patterns Induced by NMDA in an In Vitro Brain Stem–Spinal Cord Preparation From an Adult Urodele

1999 ◽  
Vol 82 (2) ◽  
pp. 1074-1077 ◽  
Author(s):  
Isabelle Delvolvé ◽  
Pascal Branchereau ◽  
Réjean Dubuc ◽  
Jean-Marie Cabelguen
Keyword(s):  
Spinal Cord ◽  
Brain Stem ◽  
Rhythm Generation ◽  
Motor Patterns ◽  
Rhythmic Motor ◽  
Bath Application ◽  
The Neural Networks ◽  
Ventral Roots ◽  
Pleurodeles Waltl

An in vitro brain stem–spinal cord preparation from an adult urodele ( Pleurodeles waltl) was developed in which two fictive rhythmic motor patterns were evoked by bath application of N-methyl-d-aspartate (NMDA; 2.5–10 μM) with d-serine (10 μM). Both motor patterns displayed left-right alternation. The first pattern was characterized by cycle periods ranging between 2.4 and 9.0 s (4.9 ± 1.2 s, mean ± SD) and a rostrocaudal propagation of the activity in consecutive ventral roots. The second pattern displayed longer cycle periods (8.1–28.3 s; 14.2 ± 3.6 s) with a caudorostral propagation. The two patterns were inducible after a spinal transection at the first segment. Preliminary experiments on small pieces of spinal cord further suggested that the ability for rhythm generation is distributed along the spinal cord of this preparation. This study shows that the in vitro brain stem–spinal cord preparation from Pleurodeles waltl may be a useful model to study the mechanisms underlying the different axial motor patterns and the flexibility of the neural networks involved.

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