Spinal amino acid release and repeated withdrawal in spinal morphine tolerant rats

Abstract

1. We used spinal microdialysis in awake rats to investigate whether the repeated withdrawal with naloxone during continuous spinal infusion of morphine would lead to a progressively greater spinal glutamate release and a more pronounced intrathecal tolerance. 2. Rats received lumbar intrathecal (IT) infusion of morphine (IT-M: 20 nmol microl(-1) h(-1)) or saline (IT-S: 1 microl h(-1)) continuously for 3 days. Both groups were further subdivided to receive intraperitoneal (i.p.) injection of naloxone (IP-N: 0.6 mg kg(-1)) or saline (IP-S: 3 ml kg(-1)) every 24 h after the beginning of IT infusion. Daily thermal escape latencies, withdrawal signs, the resting basal release of spinal amino acids before IP injection and the release immediately after the injection (evoked) were measured. 3. Rats receiving IT morphine showed a maximum increase in thermal escape latency on day 1, after which this value declined, with the fastest decline observed in IT morphine + IP naloxone group. On day 1, no significant difference was observed among groups in the resting basal release of amino acids. Rats in IT morphine + i.p. naloxone group displayed a progressive increase in this value. The release was not significantly altered in other groups. 4. For the IT-M + IP-N group, basal resting dialysate concentrations of Glu, Asp and Tau rose steadily over the 3-day infusion interval. No change in basal resting release was noted for any other treatment. 5. Evoked release (after i.p. naloxone) in IT-M animals displayed a progressive increase over the three repeated exposures. Evoked release did not change significantly in other treatment groups. 6. The degree of precipitated withdrawal significantly correlated with the increase in glutamate acutely evoked by i.p. injection. 7. The present results show that periodic transient withdrawal of spinal opiate agonist activity leads to a progressive increase in glutamate outflow and withdrawal signs, in a manner consistent with an enhanced development of spinal tolerance.

Figure 1

Daily changes in hot plate response latency in rats treated with continuous intrathecal (IT) infusion of morphine (M) (20 nmol h⁻¹) or saline (S) and daily intraperitoneal (i.p.) injections of naloxone (N) (0.6 mg kg⁻¹) or saline (S). (IT-M+IP-N, rats with continuous IT infusion of morphine for 3 days and daily i.p. injection of naloxone; IT-M+IP-S, rats with continuous IT infusion of morphine for 3 days and daily i.p. injection of saline; IT-S+IP-N, rats with continuous IT infusion of saline and daily i.p. injection of naloxone; IT-S+IP-S, rats with continuous IT infusion of saline and daily i.p. injection of saline). Each test was performed prior to the daily injection of naloxone or saline. Each line represents the mean±s.e.mean of six rats. Both IT-M+IP-N and IT-M+IP-S, the withdrawal latency on day 1 was significantly increased from the baseline and then declined. The development of tolerance was more enhanced in IT-M+IP-N compared with IT-M+IP-S. One-way repeated measures ANOVA (P<0.05). Vertical arrows indicate differences of P<0.05.

Figure 2

Daily release of spinal amino acids expressed as a percentage of day 1 resting basal levels (prior to i.p. naloxone or saline) in four groups of rats (as described in Figure 1). Samples were collected by microdialysis prior to the daily intraperitoneal injection of saline or naloxone. Daily basal spinal glutamate release increased modestly in rats with continuous morphine infusion and daily saline injections. The release was significantly elevated in rats treated with morphine infusion and daily naloxone injections on days 2 and 3. The baseline spinal release of taurine in IT-M+IP-N group was also increased significantly on day 3. Each line represents the mean±s.e.mean of six rats. *P<0.05 versus release in other groups on that day, one way ANOVA, Dunnett's t-test post hoc comparison).

Figure 3

Daily spinal glutamate, aspartate, taurine and serine release in the four groups measured concurrently after the injection of intraperitoneal (i.p.) naloxone (0.6 mg kg⁻¹) or saline (S). Release is expressed as the percentage of the respective daily resting, prior to i.p. naloxone or saline in rats undergoing continuous intrathecal (IT) infusion of morphine (M) (20 nmol h⁻¹) or saline (S). The increases in glutamate, aspartate and taurine from the resting release were significant on day 3 in rats with IT morphine infusion and daily i.p. naloxone injections. There was no significant change in the release of spinal serine in any group. Each line represents the mean±s.e.mean of six rats. *P<0.01 versus release in other groups on that day, one way ANOVA Dunnett's t-test comparison.

Figure 4

Figure shows the daily changes of the prominence of the precipitated withdrawal, as reflected by the withdrawal index.

Figure 5

Figure shows the regression and correlation (r) between changes in the release of glutamate and serine following the daily injection and withdrawal scores in rats from the four experimental treatment groups (see text for further detail).