Apamin-Sensitive Calcium-Activated Potassium Currents (SK) Are Activated by Persistent Calcium Currents in Rat Motoneurons

Low voltage–activated persistent inward calcium currents (Ca PICs) occur in rat motoneurons and are mediated by Cav1.3 L-type calcium channels (L-Ca current). The objectives of this paper were to determine whether this L-Ca current activates a sustained calcium-activated potassium current (SK current) and examine how such SK currents change with spinal injury. For comparison, the SK current that produces the postspike afterhyperpolarization (mAHP) was also quantified. Intracellular recordings were made from motoneurons of adult acute and chronic spinal rats while the whole sacrocaudal spinal cord was maintained in vitro. Spikes/AHPs were evoked with current injection or ventral root stimulation. Application of the SK channel blocker apamin completely eliminated the mAHP, which was not significantly different in chronic and acute spinal rats. The Ca PICs were measured with slow voltage ramps (or steps) with TTX to block sodium currents. In chronic spinal rats, the PICs were activated at –58.6 ± 6.0 mV and were 2.2 ± 1.2 nA in amplitude, significantly larger than in acute spinal rats. Apamin significantly increased the PIC, indicating that there was an SK current activated by L-Ca currents (SKL current), which ultimately reduced the net PIC. This SKL current was not different in acute and chronic spinal rats. The SKAHP and the SKL currents were activated by different calcium currents because the mAHP/SKAHP was blocked by the N, P-type calcium channel blocker ω-conotoxin MVIIC and was resistant to the L-type calcium channel blocker nimodipine, whereas the L-Ca and SKL currents were blocked by nimodipine. Furthermore, the SKAHP current activated within 10 ms of the spike, whereas the SKL current was delayed ∼100 ms after the onset of the L-Ca current, suggesting that the SKL currents were not as spatially close to the L-Ca currents. Finally, the SKL and the L-Ca currents were poorly space clamped, with oscillations at their onset and hysteresis in their activation and deactivation voltages, consistent with currents of dendritic origin. The impact of these dendritic currents was especially pronounced in 15% of motoneurons, where apamin led to uncontrollable L-Ca currents that could not be deactivated, even with large hyperpolarizations of the soma. Thus, although the SKL currents are fairly small, they play a critical role in terminating the dendritic L-Ca currents.

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

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.

Spastic Tail Muscles Recover From Myofiber Atrophy and Myosin Heavy Chain Transformations in Chronic Spinal Rats

Without intervention after spinal cord injury (SCI), paralyzed skeletal muscles undergo myofiber atrophy and slow-to-fast myofiber type transformations. We hypothesized that chronic spasticity-associated neuromuscular activity after SCI would promote recovery from such deleterious changes. We examined segmental tail muscles of chronic spinal rats with long-standing tail spasticity (7 mo after sacral spinal cord transection; older chronic spinals), chronic spinal rats that experienced less spasticity early after injury (young chronic spinals), and rats without spasticity after transection and bilateral deafferentation (spinal isolated). These were compared with tail muscles of age-matched normal rats. Using immunohistochemistry, we observed myofiber distributions of 15.9 ± 3.5% type I, 18.7 ± 10.7% type IIA, 60.8 ± 12.6% type IID(X), and 2.3 ± 1.3% type IIB (means ± SD) in young normals, which were not different in older normals. Young chronic spinals demonstrated transformations toward faster myofiber types with decreased type I and increased type IID(X) paralleled by atrophy of all myofiber types compared with young normals. Spinal isolated rats also demonstrated decreased type I myofiber proportions and increased type II myofiber proportions, and severe myofiber atrophy. After 4 mo of complete spasticity (older chronic spinals), myofiber type transformations were reversed, with no significant differences in type I, IIA, IID(X), or IIB proportions compared with age-matched normals. Moreover, after this prolonged spasticity, type I, IIA, and IIB myofibers recovered from atrophy, and type IID(X) myofibers partially recovered. Our results indicate that early after transection or after long-term spinal isolation, relatively inactive tail myofibers atrophy and transform toward faster myofiber types. However, long-term spasticity apparently produces neuromuscular activity that promotes recovery of myofiber types and myofiber sizes.

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

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.

Persistent Sodium Currents and Repetitive Firing in Motoneurons of the Sacrocaudal Spinal Cord of Adult 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.

Endogenous Monoamine Receptor Activation Is Essential for Enabling Persistent Sodium Currents and Repetitive Firing in Rat Spinal Motoneurons

The spinal cord and spinal motoneurons are densely innervated by terminals of serotonin (5-HT) and norepinephrine (NE) neurons arising mostly from the brain stem, but also from intrinsic spinal neurons. Even after long-term spinal transection (chronic spinal), significant amounts (10%) of 5-HT and NE (monoamines) remain caudal to the injury. To determine the role of such endogenous monoamines, we blocked their action with monoamine receptor antagonists and measured changes in the sodium currents and firing in motoneurons. We focused on persistent sodium currents (Na PIC) and sodium spike properties because they are critical for enabling repetitive firing in motoneurons and are facilitated by monoamines. Intracellular recordings were made from motoneurons in the sacrocaudal spinal cord of normal and chronic spinal rats (2 mo postsacral transection) with the whole sacrocaudal cord acutely removed and maintained in vitro (cords from normal rats termed acute spinal). Acute and chronic spinal rats had TTX-sensitive Na PICs that were respectively 0.62 ± 0.76 and 1.60 ± 1.04 nA, with mean onset voltages of −63.0 ± 5.6 and −64.1 ± 5.4 mV, measured with slow voltage ramps. Application of 5-HT2A, 5-HT2C, and α1-NE receptor antagonists (ketanserin, RS 102221, and WB 4101, respectively) significantly reduced the Na PICs, and a combined application of these three monoamine antagonists completely eliminated the Na PIC, in both acute and chronic spinal rats. Likewise, reduction of presynaptic transmitter release (including 5-HT and NE) with long-term application of cadmium also eliminated the Na PIC. Associated with the elimination of the Na PIC in monoamine antagonists, the motoneurons lost their ability to fire during slow current ramps. At this point, the spike evoked by antidromic stimulation was not affected, suggesting that activation of the transient sodium current was not impaired. However, the spike evoked after a slow ramp depolarization was slightly reduced in height and rate-of-rise, suggesting decreased sodium channel availability as a result of increased channel inactivation. These results suggest that endogenous monoamine receptor activation is critical for enabling the Na PIC and decreasing sodium channel inactivation, ultimately enabling steady repetitive firing in both normal and chronic spinal rats.