Administration of the calcineurin inhibitor cyclosporine modulates cocaine-induced locomotor activity in rats

Abstract

Rationale: Cocaine administration in rats increases locomotor activity as a result of underlying changes in neurotransmitter dynamics and intracellular signaling. The serine/ threonine phosphatase, calcineurin, is known to modulate several signaling proteins that can influence behavioral responses to cocaine.

Objective: This study aimed to determine whether calcineurin plays a role in locomotor responses associated with acute and repeated cocaine exposure. Second, we examined cocaine-mediated changes in intracellular signaling to identify potential mechanism underlying the ability of calcineurin to influence cocaine-mediated behavior.

Methods: Locomotor activity was assessed over 17 days in male Sprague-Dawley rats (n = 48) that received daily administration of cocaine (15 mg/kg, s.c.) or saline in the presence or absence of the calcineurin inhibitor, cyclosporine (15 mg/kg, i.p.). Non-cocaine-treated animals from this initial experiment (n = 24) also received an acute cocaine challenge on day 18 of testing.

Results: Daily cyclosporine administration potentiated the locomotor response to repeated cocaine 5 min after cocaine injection and attenuated the sustained locomotor response 15 to 40 min after cocaine. Furthermore, cyclosporine pretreatment for 17 days augmented the acute locomotor response to acute cocaine 5 to 30 min after cocaine injection. Finally, repeated exposure to either cocaine or cyclosporine for 22 days increased synapsin I phosphorylation at the calcineurin-sensitive Ser 62/67 site, demonstrating a common downstream target for both calcineurin and cocaine.

Conclusion: Our results suggest that calcineurin inhibition augments locomotor responses to cocaine and mimics cocaine-mediated phosphorylation of synapsin I.

Figure 1. Repeated cyclosporine administration leads to biphasic modulation of cocaine-mediated locomotor sensitization

(A) Schematic of the locomotor testing paradigm for male Sprague-Dawley rats (n=48). (B) Chronic systemic cyclosporine administration did not alter baseline habituation activity during the 30 mins before cocaine administration. (C) Daily administration of cocaine at 15 mg/kg (s.c.) led to a sensitized and sustained locomotor response (main effect of cocaine, p < 0.001) that was attenuated by daily co-administration of cyclosporine (15 mg/kg, i.p, cocaine X cyclosporine interaction, p < 0.05). (D) Repeated cyclosporine administration (15 mg/kg, i.p.) led to peak LM activity 5 mins after injection and potentiated the locomotor response to repeated cocaine (p < 0.05, independent samples t-test with a Bonferroni correction) 5 mins after injection on day 11 of testing. In contrast, repeated cyclosporine (15 mg/kg, i.p.) administration attenuated the sustained response to repeated cocaine as revealed by a cocaine X cyclosporine interaction on test days 9, 13 and 17 (p < 0.05). All data are displayed as the mean ± SEM.

Figure 2. Chronic cyclosporine, but not rapamycin, pretreatment leads to a sensitized locomotor response to acute cocaine administration

(A) Schematic of locomotor testing paradigm for male Sprague-Dawley rats (n=24). (B) Acute cocaine administration on day 18 (15 mg/kg, s.c.) led to increased locomotor activity (p < 0.005, paired samples t-test), while animals pretreated with chronic cyclosporine showed potentiated locomotor activation in response to cocaine (p < 0.001, independent samples t-test) compared to saline vehicle pretreated animals. (C) Chronic cyclosporine pre-treatment led to a potentiated locomotor response to cocaine (15 mg/kg, s.c.) in the first 30 mins after injection (p < 0.05, independent samples t-test with a Bonferroni correction for multiple comparisons). (D) Chronic pre-treatment with 5 or 20 mg/kg cyclosporine or the immunophilin rapamycin (15 mg/kg) did not affect the locomotor response to acute cocaine challenge. In contrast, animals pretreated with 15 mg/kg cyclosporine for 17 days showed a potentiated response to an acute 15 mg/kg cocaine challenge on day 18 (p < 0.05, independent samples t-test), consistent with results in the previous experiment (2B)Cyclo cyclosporine; Rap: rapamycin. All data are presented as the mean± SEM.

Figure 3. Chronic administration of cyclosporine or cocaine regulates synapsin I phosphorylation in subcortical brain structures

(A) Calcineurin levels in the PFC, NAc, striatum and VTA were not altered by chronic administration of cocaine (15 mg/kg, s.c.) or cyclosporine (15 mg/kg, i.p.). (B) Analysis of pSynapsin I levels in the NAc, ST and VTA revealed a main effect of cyclosporine administration as well as a main effect of cocaine administration (p < 0.05). In addition, animals that received either cyclosporine alone or co-administration of cocaine and cyclosporine showed increased pSynI (in the NAc, ST and VTA) compared to control treated animals (p < 0.05, independent samples t-test). (C) Chronic cocaine and cyclosporine administration did not alter total synapsin I levels in the NAc, striatum or VTA. (D and E) Acute cocaine administration (15 mg/kg, s.c.) did not alter pSynI (S62/67) or total synapsin I in the PFC, NAc, striatum or VTA. All data are presented as mean± SEM.

Figure 4. Model of a potential mechanism underlying calcineurin and cocaine effects on readily releasable pool

Calcineurin activity leads to dephosphorylation of the pre-synaptic protein, synapsin I at the calcineurin sensitive S62/67 site (Jovanovic et al. 2001). In its dephosphorylated form, synapsin I binds to both synaptic vesicles and the actin cytoskeleton (Bahler and Greengard 1987; Huttner et al. 1983). In the current work, we demonstrate that repeated cocaine exposure leads to increased synapsin I phosphorylation (Fig 3B), which in turn, would be expected to decrease its affinity for both actin and synaptic vesicles, thus releasing these vesicles from the actin cytoskeleton and allowing them to move to the readily releasable pool (Bahler and Greengard 1987; Greengard et al. 1993; Huttner et al. 1983).