Inhibition of Protein kinase Mzeta (PKMζ) in the mesolimbic system alters cocaine sensitization in rats

Abstract

Chronic cocaine use produces long-lasting changes in reward circuits that may underlie the transition from casual to compulsive patterns of drug use. Although strong neuroadaptations within the mesocorticolimbic system are known to occur, the specific role of these drug-induced plasticities on sensitization remains to be elucidated. Here we investigate whether PKMζ, a protein involved in maintaining long-term potentiation (LTP), plays a role in these cocaine-induced changes in synaptic strengthening. We performed whole-cell voltage clamp recordings of putative ventral tegmental area (VTA) dopamine (DA) cells 24 hours after five days of 15 mg/kg i.p. cocaine or isovolumetric saline injections. We observed that superfusion of 5µM ZIP (PKMζ inhibitory peptide) decreased AMPA currents and AMPA/NMDA ratios only in cocaine sensitized rats. In vivo ZIP microinfusions (10 nmol) into the VTA after cocaine sensitization decreased locomotor activity on a subsequent cocaine challenge only if given ZIP is given before the withdrawal period. On the other hand, ZIP microinfusions into the nucleus accumbens (NAc) core after a seven days withdrawal period disrupt the expression of locomotor sensitization. The present data provide a potentially relevant region, and time-specific PKMζ-dependent brain mechanism that enables sensitization. Our results support the vision that addiction involves a pathological learning process. They imply that if this synaptic strengthening is reversed, changes in the behavioral response may also be overturned.

Keywords: AMPA/NMDA ratio; LTP; Protein kinase Mzeta; VTA; cocaine sensitization; dopamine.

Figure 1. ZIP decreases AMPA currents and AMPA/NMDA ratio in cocaine-sensitized rats

A. Mean total ambulatory activity (pcc/60 min, ± S.E.M.) was recorded each day for saline (white) and cocaine (black) injected animals. Asterisks (*) denote group significance when compared to Day 1. B Sample traces show AMPA EPSC’s reduction upon ZIP superfusion (5µM). Zip had no effect on saline treated animals (left). Notice the potentiated EPSC in cocaine treated rats, which was diminished by ZIP application (right). C: Time course showing that ZIP superfusion significantly decreases AMPA EPSC amplitudes in slices taken from cocaine treated animals (cocaine/ZIP) (*p < 0.05) and has no effect on saline treated animals (saline/ZIP) (p > 0.05; (Two-way ANOVA, followed by bonferroni post-test). ACSF superfusion has no effect on EPSC amplitude in control experiments (saline/ACSF and cocaine/ACSF) D: Bar graph shows AMPA EPSC’s reduction as percentage of change from basal (EPSC amplitude at 4 min). Continuous ZIP application was able to significantly reduce AMPA mediated transmission by ~20% (* p < 0.01 Student t-test) in cocaine treated animals when compared to basal but failed to do so in saline injected rats. ACSF superfusion had no effect in AMPA mediated currents in cocaine or saline injected animals. Data are percent changes ±SEM. E. Representative traces showing AMPA (black)/NMDA (gray) ratio from brain slices incubated in ZIP (5µM) or ACSF from saline or cocaine treated animals (scale: 50pA, 50 ms). F. Bar graph showing AMPA/NMDA ratios of saline and cocaine treated animals. The AMPA/NMDA ratio increased with cocaine injections. ZIP incubation significantly reduced the AMPA/NMDA ratio from cocaine treated rats but failed to do so in saline injected animals (* p < 0.05 One Way ANOVA Newman Keuls, ratios ± SEM).

Figure 2. Intra-VTA ZIP microinfusions’s effect on behavioral sensitization

A: Diagram showing the cocaine sensitization protocol with the time point for intra VTA ZIP microinfusion. B: Mean total ambulatory activity (pcc/60 min, ± S.E.M.) was recorded each day for every group. Asterisks (*) and diamonds (♦) denote group significance when compared to Day 1 for they respective group. One day after ZIP treatment (Day 6) ZIP/cocaine treated animals showed a significant decrease (#) in locomotor activity compared to Day 5 (p < 0.01). Locomotion was also significantly different (+) from that of Vehicle/Cocaine animals on Day 6. On day 14, ZIP/cocaine pretreated animals showed a sensitized response. No statistical differences between ZIP/cocaine and vehicle/cocaine treated animals were observed on day 14 (p > 0.05). There was no change between ZIP/saline and vehicle/saline controls throughout the experiment (p > 0.05). C: Time course of locomotor responses after one (grey) and five (black) days of cocaine (15 mg/kg, i.p.) (filled) or saline (unfilled). Cocaine sensitization was manifested as an increase in locomotor activity primarily during the first 10–40 min (25–55 min) after cocaine injection. Significant differences (p < 0.05) are indicated by asterisks (*) for vehicle/cocaine and numeral (#) for ZIP/cocaine. Each time point represents the mean ± SEM. D: Time course for the locomotor response on days 5 (black) and 6 (grey). ZIP/cocaine injected animals (filled grey squares) show decreased locomotor activity (# p < 0.05) compared to Day 5. Asterisks (*) denote significance between ZIP/cocaine and vehicle/cocaine animals on day 6. Each point represents the mean ± SEM. E: Diagram showing the cocaine sensitization protocol with the time point for intra VTA ZIP microinfusion after a withdrawal period. F: On day 5 cocaine treated animals were sensitized to cocaine. One day after ZIP treatment (Day 12) ZIP/cocaine treated animals showed no significant change in locomotor activity compared to Day 5 or to vehicle/cocaine animals on Day 12 (p > 0.05). There was no change between ZIP/saline and vehicle/saline controls throughout the experiment (p > 0.05, Two-way ANOVA, followed by bonferroni post-test for all comparisons). G,H: Injection sites within the VTA for saline (grey circles) and cocaine (black circles) treated animals.

Figure 3. Intra NAc ZIP microinfusions decrease the expression of cocaine sensitization

A: Diagram shows cocaine sensitization protocol with time points for intra NAc ZIP microinfusions. B: Mean total ambulatory activity (pcc/60 min, ± S.E.M.) was recorded for each group. Asterisks (*) denote significance of the group compared to corresponding Day 1 (p < 0.01). Numeral (#) denotes statistical significance between groups on Day 6. On Day 5 cocaine treated animals show sensitization to cocaine. On Day 13 ZIP/cocaine treated animals did not express sensitization (p > 0.05). Instead, they showed a significant decrease (Day 13) in locomotor activity when compared to Vehicle/cocaine animals (p < 0.05). There was no significance between ZIP/saline and vehicle/saline controls throughout the experiment (p > 0.05). C: Time course of locomotor responses after one (grey) and five (black) days of cocaine (15 mg/kg, i.p.) (filled) or saline (unfilled) administration. Cocaine sensitization was manifested as an increase in locomotor activity in all time points. Significant differences between day 1 and day 5 (p < 0.05) are indicated by asterisk (*) for vehicle/cocaine and # for ZIP/cocaine. Each time point represents the mean ± SEM. D: Time course for the locomotor response on Days 5 (black) and 13 (grey). ZIP/cocaine injected animals (filled grey squares) show decreased locomotor activity (* p < 0.05) compared to Day 5 (#). Asterisks (*) denote significance between ZIP/cocaine and vehicle/cocaine animals on day 13. Each point represents the mean ± SEM. (Two-way ANOVA, followed by bonferroni post-test for all comparisons). E: Injection sites within the NAc for saline (grey circles) and cocaine (black circles) treated animals.

Figure 3. Intra NAc ZIP microinfusions decrease the expression of cocaine sensitization

A: Diagram shows cocaine sensitization protocol with time points for intra NAc ZIP microinfusions. B: Mean total ambulatory activity (pcc/60 min, ± S.E.M.) was recorded for each group. Asterisks (*) denote significance of the group compared to corresponding Day 1 (p < 0.01). Numeral (#) denotes statistical significance between groups on Day 6. On Day 5 cocaine treated animals show sensitization to cocaine. On Day 13 ZIP/cocaine treated animals did not express sensitization (p > 0.05). Instead, they showed a significant decrease (Day 13) in locomotor activity when compared to Vehicle/cocaine animals (p < 0.05). There was no significance between ZIP/saline and vehicle/saline controls throughout the experiment (p > 0.05). C: Time course of locomotor responses after one (grey) and five (black) days of cocaine (15 mg/kg, i.p.) (filled) or saline (unfilled) administration. Cocaine sensitization was manifested as an increase in locomotor activity in all time points. Significant differences between day 1 and day 5 (p < 0.05) are indicated by asterisk (*) for vehicle/cocaine and # for ZIP/cocaine. Each time point represents the mean ± SEM. D: Time course for the locomotor response on Days 5 (black) and 13 (grey). ZIP/cocaine injected animals (filled grey squares) show decreased locomotor activity (* p < 0.05) compared to Day 5 (#). Asterisks (*) denote significance between ZIP/cocaine and vehicle/cocaine animals on day 13. Each point represents the mean ± SEM. (Two-way ANOVA, followed by bonferroni post-test for all comparisons). E: Injection sites within the NAc for saline (grey circles) and cocaine (black circles) treated animals.