Long-term changes in reward-seeking following morphine withdrawal are associated with altered N-methyl-D-aspartate receptor 1 splice variants in the amygdala

Abstract

The NR1 subunit of the NMDA receptor can be alternatively spliced by the insertion or removal of the N1, C1, C2, or C2' regions. Morphine dependence and withdrawal were previously demonstrated to lower N1 and C2' in the accumbens and lower N1, C1, and C2' in the amygdala (AMY). Withdrawal has also been demonstrated to increase motivational and anxiety/stress behaviors in rats. We tested the hypothesis that NR1 splicing would be associated with these behaviors during an extended withdrawal period of 2 months. Motivation was measured using an operant orofacial assay at non-aversive temperatures (37°C) while anxiety and stress were measured by examining this behavior at aversive temperatures (46°C). Lower C1 and C2 expression levels were observed in the AMY in a subset of the population of withdrawn rats even after 2 months of morphine withdrawal. These subsets were associated with a hypersensitivity to adverse conditions which may reflect long-term alterations in the withdrawn population.

Figure 1. Weights

A. No differences in weight were observed at baseline or during morphine administration. B. Morphine injected rats had significantly lower weights during the acute withdrawal phase. C. No differences in weight were observed during extended withdrawal. For all graphs: saline N=8, morphine N=32.

Figure 2. Morphine 37°C behavior

A. Morphine treated rats had significantly higher time per contact values throughout the administration period. Post-hoc tests were significant for the 10, 20, and 40 mg/kg doses (p<0.001, p<0.001, and p<0.05). B. Morphine also increased the facial contact times during the administration period especially at the 10 and 40 mg/kg doses (post-hoc tests, p<0.05). For both graphs: saline N=8, morphine N=32.

Figure 3. Behavioral differences between acute versus extended withdrawal at 37°C and 46°C

A. Morphine treated rats had significantly higher time per contact values at 37°C during acute withdrawal, but not extended withdrawal. B. No differences were observed in the facial contact times for acute or extended withdrawal at 37°C. C. No differences were observed in the time per contact values for acute or extended withdrawal at 46°C. D. No differences were observed for morphine treated rats during acute withdrawal at 46°C for facial contact times. During extended withdrawal however, significantly lower facial contact times were observed for morphine treated rats. For all graphs: saline N=8, morphine N=32.

Figure 4. AMY C1 changes are associated with long-term responses to aversive stimuli

A. No differences in the means were observed in the C1 cassette of withdrawn animals and controls but more variance is found in the withdrawn group. B. One Gaussian population explains some of the data. C. Two Gaussian populations best explain the data. D. Significantly lower facial contact times were observed for the altered group during the extended withdrawal period at 46°C. E. Representative blot of the C1 levels of three saline, high C1, and low C1 rats in the AMY. All bands illustrated are from the same gel and blot. For A: Saline N=8, Morphine N=39. For E: Saline N=8, High C1 N=23, Low C1 N=8.

Figure 5. AMY C2 changes are associated with long-term motivation and responses to aversive stimuli

A. C2 expression levels in the withdrawn animals were not significantly different from controls but more variance is found in the withdrawn group. B. One Gaussian population explains some of the data. C. Two Gaussian populations best explain the data. D. Low C2 rats had lower facial contact times at 46°C over time. E. Representative blot of the C2 levels of three saline, high C2, and low C2 rats in the AMY. All bands illustrated are from the same gel and blot. For A: Saline N=8, Morphine N=31. For D: Saline N=8, Low C2 N=21, High C2 N=10.

Figure 6. AMY C2 and C1 levels approach significant a significant correlation

Decreased C1 and C2 levels may be found in the same rats although these populations are not exactly the same.

Figure 7. AMY N1 changes are not associated with long-term motivation and responses to aversive stimuli

A. N1 expression levels in the withdrawn animals were not significantly different from controls but more variance is found in the withdrawn group. B. One Gaussian population explains some of the data. C. Two Gaussian populations best explain the data. D. No differences were observed for lower facial contact times at 46°C over time between the groups. For A: Saline N=8, Morphine N=31. For D: Saline N=8, Low N1 N=23, High N1 N=8.

Figure 8. NACC C1 changes are not associated with long-term motivation and responses to aversive stimuli

A. NACC C1 expression levels in the withdrawn animals were not significantly different from controls but more variance is found in the withdrawn group. B. One Gaussian population explains some of the data. C. Two Gaussian populations best explain the data. D. No differences were observed for lower facial contact times at 46°C over time between the groups. For A: Saline N=8, Morphine N=31. For D: Saline N=8, Low N1 N=22, High N1 N=9.