Role of endogenous release of norepinephrine in muscle spasms after chronic spinal cord injury

Abstract

The recovery of persistent inward currents (PICs) and motoneuron excitability after chronic spinal cord transection is mediated, in part, by the development of supersensitivity to residual serotonin (5HT) below the lesion. The purpose of this paper is to investigate if, like 5HT, endogenous sources of norepinephrine (NE) facilitate motoneuron PICs after chronic spinal transection. Cutaneous-evoked reflex responses in tail muscles of awake chronic spinal rats were measured after increasing presynaptic release of NE by administration of amphetamine. An increase in long-lasting reflexes, known to be mediated by the calcium component of the PIC (CaPIC), was observed even at low doses (0.1-0.2 mg/kg) of amphetamine. These findings were repeated in a reduced S2 in vitro preparation, demonstrating that the increased long-lasting reflexes by amphetamine were neural. Under intracellular voltage clamp, amphetamine application led to a large facilitation of the motoneuron CaPIC. This indicates that the increases in long-lasting reflexes induced by amphetamine in the awake animal were, in part, due to actions directly on the motoneuron. Reflex responses in acutely spinal animals were facilitated by amphetamine similar to chronic animals but only at doses that were ten times greater than that required in chronic animals (0.2 mg/kg chronic vs. 2.0 mg/kg acute), pointing to a development of supersensitivity to endogenous NE in chronic animals. In summary, the increases in long-lasting reflexes and associated motoneuron CaPICs by amphetamine are likely due to an increased release of endogenous NE, which motoneurons become supersensitive to in the chronic stages of spinal cord injury.

FIG. 1

Method for measuring maximum flexion and extension angles in spastic rat tail after standardized stretch/rub maneuver is applied. The tail is viewed from the left side of the animal. A: pure flexion spasm where the tail typically forms a coil. The maximum flexion angle is measured relative to vertical from a tangent line at the tip of the tail; the maximum extension angle here is zero as the tail is showing no extension. In a case of pure flexion coiling, the maximum flexion angle occurs at the tip of the tail (absolute tip angle). B: when flexion and extension both occur, the tail forms an inverted s-shape. To calculate the maximum degree of extension, which occurs at the tip of the tail, the maximum flexion angle is first measured relative to vertical from a tangent drawn from the middle of the tail. The absolute extension angle of the tip of the tail is then measured relative to vertical from a tangent drawn from the tip of the tail. To obtain the maximum extension angle, the maximum flexion angle is then added to the absolute extension tip angle. C: when the tail is at rest, the maximum flexion angle occurs at the tip of the tail and therefore is identical to the absolute tip angle, and the maximum extension value is zero similar to the full flexion coil in A.

FIG. 2

Maximum flexion (A) and extension angles (B) measured in a representative chronic spinal rat before amphetamine after the standardized stretch/rub maneuver applied to the tail. Small diagrams in A show the shape of the tail at the time indicated by the arrow. Solid arrows define when a spasm (>50° increase in flexion or extension angle in 5 s) was counted. Maximum flexion (C) and extension (D) angles measured in the same animal after a low dose (0.1 mg/kg) of amphetamine. Note that after a low dose of amphetamine, the animal showed greater maximum flexion and an increase in the number of spasms (from 5 to 20), whereas the maximum extension values decreased to 0. E: group average maximum flexion angles predrug (open bars), at low (gray bars) and high (solid bars) doses of amphetamine. F: same format as E but maximum extension. *, P < 0.05.

FIG. 3

Low and high doses of amphetamine increased cutaneous reflexes in representative chronic spinal rat. A: segmental tail muscle electromyographic (EMG) response to tip of the tail stimulation, single pulse at 10 times reflex threshold (RT), before amphetamine. Tonic short reflex measured 15–500 ms after stim; tonic long reflex measured 500–5,500 ms after stim. B: magnification of A showing greater detail of the polysynaptic reflex (measured 15–40 ms after stim). C and D: same rat as in as A and B but recorded after administration of a low dose (0.2 mg/kg) of amphetamine. E and F: same rat as in A and B, but recorded after administration of a high (1.0 mg/kg) dose of amphetamine. Time scales are the same for A, C, and E and for B, D, and F. Note background EMG was not controlled for in this example to demonstrate increase in prestim EMG after amphetamine.

FIG. 4

Absolute reflex amplitudes (□ on left of graphs) and respective changes (normalized to predrug values) in response to low ( ) and high (▪, right of graphs) doses of amphetamine for the various reflex responses. Dashed line indicates 100% of predrug values. A: polysynaptic reflex (10–40 ms after stimulus): predrug reflex is 60.9 ± 16.7 μV and shows a significant increase of 194.1 ± 48.6% at low doses of amphetamine and an increase of 145.7 ± 41.5% at high doses. B: tonic short reflex (30–600 ms after stim): predrug reflex is 12.8 ± 1.7 μV and shows significant increase at low (129.7 ± 15.9%) but not high (162.5 ± 20.8%) doses of amphetamine. C: tonic long reflex (600–3,600 ms after stim): predrug reflex is 9.4 ± 3.8 μV, and increases by 429.2 ± 145.6% at low and by 392.3 ± 140.2% at high doses of amphetamine. D: trains reflex (1,100–4,100 s after 500 ms, 100-Hz train stim): predrug reflex is 12.8 ± 3.6 μV and increases significantly by 262.8 ± 54.2% at low dose and 270.0 ± 70.1% at high doses of amphetamine. E: background activity: predrug levels of activity measure 4.4 ± 1.0 μV and increase by 270.4 ± 57.1% at low dose and 330.2 ± 58.6% at high dose amphetamine. * = P < 0.05.

FIG. 5

Ventral root reflex responses to dorsal root stimulation in the in vitro sacrocaudal spinal cord of chronic spinal rats. Low and high doses of amphetamine increase ventral root reflexes of short and long duration. A: ventral root response to a single pulse of dorsal root stimulation at 5 times threshold before application of amphetamine. Tonic short reflex is measured from 30 to 600 ms after stimulation and the tonic long reflex is measured from 600 to 3,600 ms after stimulation. B: magnification of A showing the polysynaptic reflex (10–40 ms) in greater detail. C and D and E and F: same as A and B but at low (0.1 μM) and high (10 μM) doses of amphetamine, respectively.

FIG. 6

Facilitation of ventral root reflexes after administration of low (0.1 μM, gray bar) and high (1–10 μM, solid bar) doses of amphetamine. Polysynaptic, tonic short, and tonic long reflexes were measured at the ventral root after dorsal root stimulation at 5 times threshold (5×T; ~0.05 mA). All reflexes are shown normalized to predrug condition, dashed line in all graphs indicates 100%. A: polysynaptic reflex: showed a significant decrease to 75.7 ± 9.4% at low doses of amphetamine and a significant increase to 129.1 ± 7.7% at high doses of amphetamine. B: tonic short reflex: shows significant increases at both low (151.9 ± 16.7%) and high (415.3 ± 94.1%) doses of amphetamine. C: tonic long reflex: low dose of amphetamine caused a reflex increase of 172.3 ± 32.8% and a high dose caused a reflex amplitude increase of 1158.6 ± 274.5%. D: trains reflex: shows significant reflex amplitude increases at low (146.8 ± 10.9%) and high (183.4 ± 18.9%) doses of amphetamine. *, P < 0.05.

FIG. 7

Example recordings of the calcium component of the persistent inward current (CaPIC) from 2 motoneurons of chronic S₂ spinal rats measured during a slow triangular voltage ramp. Voltage command in top traces, resulting current in bottom traces with leak current shown as the thin triangular line on the current trace. A: response of a motoneuron in TTX, to isolate CaPIC, before amphetamine administration. Solid line on voltage ramp at −50 mV indicates spike threshold. V_on is the onset voltage of the CaPIC, I_on is the corresponding onset current. V_off is the offset voltage of the CaPIC. The size of the initial CaPIC, which is measured from the leak current, is indicated by the length of the solid arrow and the sustained CaPIC on the downward voltage ramp is indicated by the dashed arrow. B: response of the same motoneuron in A after administration of amphetamine. Amphetamine causes significant increases in both the initial CaPIC and sustained CaPIC. C: different motoneuron in TTX, which shows a smaller initial and sustained CaPIC than shown in A. D: response of same motoneuron in C after amphetamine application. The small CaPIC also shows substantial enhancement after application of amphetamine.

FIG. 8

Progression of CaPIC from the time of amphetamine application to the time where peak effectiveness is observed (15–20 min); same recording conditions as in Fig. 7. Solid line on voltage ramp at −50 mV indicates spike threshold. A: response of motoneuron in TTX and before amphetamine application. The sustained CaPIC (solid arrow) is modest prior to amphetamine application. B: same motoneuron as in A 8 min after amphetamine application. The sustained CaPIC already shows amplification but still returns to baseline levels at the end of the voltage ramp. C: same motoneuron as in A and B, 15 min after amphetamine application. Sustained CaPIC is increased substantially and could not be terminated by the downward voltage ramp. The current does not return to control levels when the voltage ramp is completed.

FIG. 9

Similar format as Fig. 2 with example kinematic data from an acute spinal rat for predrug maximum flexion (A), predrug maximum extension (B), high dose (0.6 mg/kg) maximum flexion (C), and high dose maximum extension (D). Note that the amount of maximum flexion and the number of spasms counted increased with a high dose of amphetamine. E: group data: average maximum flexion predrug is 37.2 ± 3.4° and increases significantly by 163.2% at high doses of amphetamine. A trend for increased maximum flexion is evident, but not significant, at low doses (0.2 mg/kg) of amphetamine. F: same format as E but maximum extension. Maximum extension values are negligible prior to amphetamine, and do not show any significant increases with subsequent doses (note the scale). * = P < 0.05.

FIG. 10

Development of amphetamine supersensitivity in chronic S₂ spinal rats as seen by changes in cutaneous reflexes recorded in the segmental tail muscles evoked by train stimulation (1.5×RT) applied to the tip of the tail. A: surface EMG response in acute spinal rat before amphetamine administration. B: same rat as in A but recorded after administration of a low dose of amphetamine (0.2 mg/kg). C: same rat as in A and B but recorded after a high dose of amphetamine (2.0 mg/kg). D: response of a chronic spinal rat to train stimulation prior to amphetamine administration. Reflex response is already much larger than in the acute spinal rat. E: same rat as in D but after a low dose of amphetamine (0.2 mg/kg). F: same rat as in D and E but after administration of a high dose of amphetamine (1.0 mg/kg). Despite doses 10 times that of the chronic spinal rat, the reflexes recorded in the acute spinal rat never reach similar increases in amplitude, pointing toward the development of a supersensitivity to endogenous NE present below the transection.

FIG. 11

Similar format as Fig. 4. A: polysynaptic reflex: predrug reflex in the acute spinal rat is 3.33 ± 1.6 μV compared with the chronic spinal rat 60.9 ± 16.7 μV (left). At low doses of amphetamine (0.2 mg/kg), the polysynaptic reflex in acute spinal animals increases by 318.0 ± 167.0%, but only significantly at high doses (0.6–2.0 mg/kg) to 679.0 ± 255.0% (right). B: tonic short reflex: predrug reflex 4.1 ± 2.1 μV acute, 12.8 ± 1.7 μV chronic. At a low dose of amphetamine, the reflex increases only slightly to 109.7 ± 29.3%, whereas the increase of 481.9 ± 235.8% is significant after a high dose. C: tonic long reflex: predrug reflex acute: 1.1 ± 0.5 μV, chronic 9.4 ± 3.7 μV in predrug. A low dose of amphetamine increases the reflex by 154.2 ± 37.1%, and a high dose increases the reflex significantly to 433.0 ± 133.9%. D: train reflex: 1.8 ± 1.3 μV acute, 12.7 ± 3.6 μV chronic in predrug. Low doses of amphetamine increase the reflex to 167.7 ± 67.8% and high doses cause a significant increase to 461.6 ± 182.8%. E: background: predrug 2.1 ± 0.9 μV acute, 4.4 ± 0.9 μV chronic. The background activity shows a modest increase of 101.4 ± 37.2% at low doses of amphetamine and a significant increase to 227.2 ± 60.2% at high doses of amphetamine. * = P < 0.05.