Role of calcineurin in nicotine-mediated locomotor sensitization

Abstract

Calcineurin is a serine/threonine phosphatase that contributes to the effects of nicotine on calcium signaling in cultured cortical neurons; however, the role of calcineurin in behavioral responses to nicotine in vivo has not been examined. We therefore determined whether calcineurin blockade could alter nicotine-mediated locomotor sensitization in Sprague Dawley rats using systemic or brain region-specific administration of the calcineurin inhibitors cyclosporine or FK506. Systemic cyclosporine administration decreased calcineurin activity in the brain, attenuated nicotine-mediated locomotor sensitization, and blocked the effects of nicotine on DARPP32 (dopamine- and cAMP-regulated phosphoprotein-32) activation in the striatum. Direct infusion of calcineurin inhibitors cyclosporine or FK506 into the ventral tegmental area (VTA) also attenuated nicotine-mediated locomotor sensitization, whereas infusion of rapamycin, which binds to FK-binding protein but does not inhibit calcineurin, did not affect sensitization. Together, the data suggest that activation of calcineurin, particularly in the VTA, is a novel signaling event important for nicotine-mediated behavior and intracellular signaling.

Figure 1.

Chronic cyclosporine administration attenuates nicotine-induced locomotor sensitization. Repeated nicotine administration to Sprague Dawley rats (n = 16 per group) leads to locomotor sensitization that is attenuated by chronic systemic cyclosporine coadministration. A, Locomotor testing paradigm and timeline. B, Total activity during the 30 min habituation period across the 17 d treatment period. Statistical analysis revealed an effect of treatment (F_(3,60) = 3.080; p < 0.05, repeated-measures analysis) as well as differences in control versus cyclosporine or nicotine plus cyclosporine-cotreated subjects (p < 0.05, least significant difference post hoc). C, Total locomotor activity in the initial 15 min period after a subcutaneous injection of saline or nicotine (0.35 mg/kg) revealed a significant effect of testing day (F_(8,480) = 27.064; p < 0.001) and drug treatment (F_(3,60) = 105.361; p < 0.001) with an interaction between test day and drug treatment (F_(8,480) = 27.064; p < 0.001). Coadministration of nicotine and cyclosporine attenuated the locomotor sensitization observed with nicotine administration alone (p < 0.005, Tukey's post hoc analysis). D, Subjects that received subcutaneous saline injections throughout the 17 d testing period showed increased locomotor activity in response to an acute nicotine challenge (Nicotine Chall; 0.35 mg/kg) on day 19 (F_(3,60) = 36.873; p < 0.0001) with no significant difference between groups that were pretreated with vehicle or cyclosporine (F_(3,60) = 0.387; p = 0.536). Data are presented as the mean ± SEM. Cyclo, Cyclosporine; Nic, nicotine; Veh, vehicle.

Figure 2.

Chronic cyclosporine administration increases the phosphorylation state of calcineurin target proteins in the brain. Levels of calcineurin and the calcineurin target synapsin I after nicotine and cyclosporine administration are shown. A, Chronic systemic administration of nicotine (0.35 mg·kg⁻¹·d⁻¹, s.c.) and cyclosporine (15 mg·kg⁻¹·d⁻¹, i.p.) did not alter calcineurin levels in the PFC, NAc, striatum, or hippocampus. B, Chronic systemic cyclosporine (15 mg·kg⁻¹·d⁻¹, i.p.) led to increased levels of phospho-Synapsin S62/S67 in the NAc (F_(3,24) = 54.193; p < 0.001), striatum (F_(3,28) = 16.261; p < 0.001), and hippocampus (F_(3,29) = 12.721; p < 0.005, ANOVA). *p < 0.05. C, Chronic systemic cyclosporine (15 mg·kg⁻¹·d⁻¹, i.p.) did not alter levels of phospho-Synapsin S9 in any of the regions tested. D, Chronic nicotine (0.35 mg·kg⁻¹·d⁻¹, s.c.) administration led to decreased total synapsin in the PFC (F_(1,13) = 0.108; p < 0.05, independent-samples t test). Data are presented as the mean ± SEM. Ct, Control; Nic, nicotine; Cyclo, cyclosporine; Nuc Acc, nucleus accumbens; Hipp, hippocampus.

Figure 3.

Chronic cyclosporine administration blocks the ability of chronic nicotine to activate DARPP32. DARPP32 levels after chronic or acute nicotine and cyclosporine administration are shown. A, Chronic nicotine (0.35 mg·kg⁻¹·d⁻¹, s.c.) leads to increased pDARPP32 T34 in the striatum (F_(3,30) = 4.106; p = 0.05, ANOVA), whereas cyclosporine (15 mg·kg⁻¹·d⁻¹, i.p.) leads to decreased pDARPP32 T34 in the striatum (F_(3,30) = 4.113; p = 0.05, ANOVA). Coadministration of nicotine (0.35 mg·kg⁻¹·d⁻¹, s.c) and cyclosporine (15 mg·kg⁻¹·d⁻¹, i.p.) decreases pDARPP32 T34 levels in the striatum compared with nicotine (0.35 mg·kg⁻¹·d⁻¹, s.c.)-treated subjects (p < 0.05, independent-samples t test). Cyclosporine administration (15 mg·kg⁻¹·d⁻¹, i.p.) also increased levels of pDARPP32 T75 in the NAc (F_(3,30) = 5.134; p < 0.05, ANOVA). *p < 0.05. B, Chronic drug treatment did not alter DARPP32 levels in the NAc or striatum. C, D, Acute administration of nicotine (0.35 mg/kg, s.c.) and cyclosporine (15 mg/kg, i.p.) did not alter levels of pDARPP32 T34, pDARPP32 T75, or total DARPP32 in the NAc or striatum. Data are presented as the mean ± SEM. Ct, Control; Nic, nicotine; Cyclo, cyclosporine; Nuc Acc, nucleus accumbens.

Figure 4.

Intra-VTA cyclosporine administration decreases nicotine-induced locomotor sensitization. Repeated systemic nicotine administration leads to locomotor sensitization in Sprague Dawley rats that is attenuated by chronic cyclosporine infusion into the VTA (n = 9–12 per group). A, Locomotor testing paradigm and timeline. B, Total activity during the 30 min habituation period before saline or nicotine (0.35 mg/kg, s.c.) administration during testing days 1–16 revealed no effect of treatment on prechallenge lomocotor activity. C, Total locomotor activity during a 15 min period after a subcutaneous injection of saline vehicle or nicotine (0.35 mg/kg) revealed a significant effect of testing day (F_(7,273) = 4.955; p < 0.001) and drug treatment (F_(3,39) = 74.109; p < 0.001) as well as a significant interaction between day and treatment (F_(21,273) = 2.228; p < 0.005). Coadministration of systemic nicotine (0.35 mg·kg⁻¹·d⁻¹) with cyclosporine infusion into the VTA (1 μg per side per day) attenuated the locomotor sensitization observed with nicotine administration alone (p < 0.05, Tukey's post hoc analysis). D, Subjects that received daily saline administration for 16 d showed increased locomotor activity in response to an acute nicotine challenge (Nicotine Chall) on day 17 (F_(1,38) = 42.569; p < 0.001, ANOVA) with no significant difference between those that had had received chronic cyclosporine (1 μg per side per day) or vehicle infusions into the VTA. Data are presented as the mean ± SEM. Veh, Vehicle; Nic, nicotine; Cyclo, cyclosporine.

Figure 5.

VTA FK506 decreases, whereas VTA rapamycin and NAc cyclosporine do not alter, nicotine-induced locomotor sensitization. Repeated, systemic nicotine administration leads to locomotor sensitization in Sprague Dawley rats that is attenuated by chronic FK506, but not rapamycin, infusion into the VTA (n = 4–8 per group) and is not altered by chronic cyclosporine infusion into the NAc (n = 4–8 per group. A, FK506 into VTA. Total activity during the 30 min habituation period before saline or nicotine (0.35 mg/kg, s.c.) administration on each day of testing demonstrated no effect of treatment. B, FK506 into VTA. Total locomotor activity during the initial 15 min period after saline vehicle or nicotine (0.35 mg/kg) administration revealed locomotor sensitization in nicotine-treated animals (F_(3,45) = 5.335; p < 0.005). FK506 (1 μg per side per day) infusion into the VTA significantly attenuated this locomotor sensitization (F_(1,15) = 5.555; p < 0.05). C, Rapamycin into VTA. Total activity during the habituation period before nicotine or saline administration on each day of testing revealed no effect of treatment. D, Rapamycin into VTA. Total locomotor activity in the 15 min after saline or nicotine (0.35 mg/kg) revealed locomotor sensitization in nicotine-treated animals (F_(3,27) = 2.944; p = 0.05) with no effect of rapamycin coadministration. E, Cyclosporine into NAc. Total activity during the 30 min habituation period before saline or nicotine (0.35 mg/kg, s.c.) administration on each day of testing demonstrated no effect of treatment. F, Cyclosporine into NAc. Total locomotor activity in the 15 min period after subcutaneous injection of saline vehicle or nicotine revealed a significant effect of testing day (F_(7,112) = 2.978; p < 0.01) and drug treatment (F_(3,16) = 68.533; p < 0.001) along with a significant interaction between day and treatment (F_(21,112) = 2.417; p < 0.005). In addition, nicotine administration led to locomotor sensitization (p < 0.005 vs control) that was not altered by intra-NAc infusion of cyclosporine (1 μg per side per day). Nic, nicotine; Cyclo, cyclosporine; Nuc Acc, nucleus accumbens; Veh, vehicle.