Dissection of corticotropin-releasing factor system involvement in locomotor sensitivity to methamphetamine

Abstract

Sensitivity to the euphoric and locomotor-activating effects of drugs of abuse may contribute to risk for excessive use and addiction. Repeated administration of psychostimulants such as methamphetamine (MA) can result in neuroadaptive consequences that manifest behaviorally as a progressive escalation of locomotor activation, termed psychomotor sensitization. The present studies addressed the involvement of specific components of the corticotropin-releasing factor (CRF) system in locomotor activation and psychomotor sensitization induced by MA (1, 2 mg/kg) by utilizing pharmacological approaches, as well as a series of genetic knockout (KO) mice, each deficient for a single component of the CRF system: CRF-R1, CRF-R2, CRF, or the CRF-related peptide Urocortin 1 (Ucn1). CRF-R1 KO mice did not differ from wild-type mice in sensitization to MA, and pharmacological blockade of CRF-R1 with CP-154,526 (15, 30 mg/kg) in DBA/2J mice did not selectively attenuate either the acquisition or expression of MA-induced sensitization. Deletion of either of the endogenous ligands of CRF-R1 (CRF, Ucn1) either enhanced or had no effect on MA-induced sensitization, providing further evidence against a role for CRF-R1 signaling. Interestingly, deletion of CRF-R2 attenuated MA-induced locomotor activation, elucidating a novel contribution of the CRF system to MA sensitivity, and suggesting the participation of the endogenous urocortin peptides Ucn2 and Ucn3. Immunohistochemistry for Fos was used to visualize neural activation underlying CRF-R2-dependent sensitivity to MA, identifying the basolateral and central nuclei of the amygdala as neural substrates involved in this response. Our results support further examination of CRF-R2 involvement in neural processes associated with MA addiction.

Figure 1

CRF-R1 KO and WT mice do not differ in MA-induced sensitization. (a) Locomotor activity counts (mean ± SEM) in female and male CRF-R1 KO and WT mice from a 15-minute session following administration of saline (D1, D2, D12) or 1 mg/kg MA (D3, D5, D7, D9, D11, D27). (b) Sexes collapsed for clarity. #: main effect of sex across repeated saline days (p < .05). n = 15–20 per genotype, per sex. The same animals contributed to all scores in panels a and b.

Figure 2

Selective blockade of CRF-R1 in DBA/2J mice does not selectively attenuate MA-induced sensitization. (a) Acquisition experiment. Locomotor activity scores (distance traveled in cm; mean + SEM) from Day 11 (see Methods). (b) Expression experiment. Locomotor activity scores (distance traveled in cm; mean + SEM) from Day 11 (see Methods). (c) Control experiment. Locomotor activity scores (distance traveled in cm; mean + SEM) from Day 11 (see Methods). *: main effect of MA (both p < .001); #: main effect of CP-154,526 (both p < .0005); ‡: 0 mg/kg vs. 30 mg/kg (p < .05) (Tukey’s HSD). Boxed numbers indicate number of animals per group; different animals contributed to separate experiments for panels a, b, and c.

Figure 3

CRF-R2 KO mice display a decreased locomotor response to MA. (a) Locomotor activity counts (mean ± SEM) in female and male CRF-R2 KO and WT mice following administration of saline or 1 mg/kg MA. (b) Sexes collapsed for clarity. *: main effect of genotype across repeated MA trials (p < .05). n = 12–22 per genotype, per sex. The same animals contributed to all scores in panels a and b.

Figure 4

MA-induced sensitization is enhanced in CRF KO mice. (a) Locomotor activity counts (mean ± SEM) in female and male CRF KO and WT mice following administration of saline or 1 mg/kg MA. (b) Sexes collapsed for clarity. †: genotype × sex interaction (p < .05); ‡: day × genotype interaction (p < .05), *: simple main effect of genotype (p < .05), **: simple main effect of genotype (p < .01). n = 8–15, per genotype, per sex. The same animals contributed to all scores in panels a and b.

Figure 5

Ucn1 KO and WT mice do not differ in MA-induced sensitization. (a) Locomotor activity counts (mean ± SEM) in female and male Ucn1 KO and WT mice following administration of saline or 1 mg/kg MA. (b) Sexes collapsed for clarity. ‡: day × sex interaction (p = .001). n = 16–19 per genotype, per sex. The same animals contributed to all scores in panels a and b.

Figure 6

Deletion of CRF-R2 abolishes acute stimulation to MA. (a) Locomotor activity counts (mean ± SEM) in female and male CRF-R2 KO and WT mice from a 15-minute session on Days 1 and 2 following administration of saline, and on Day 3 following administration of either saline or MA (1 mg/kg) (sexes collapsed). (b) Locomotor activity counts from Day 3 (mean + SEM) for mice treated with either saline or MA on Day 3, demonstrating a blunted acute stimulant response to MA in CRF-R2 KO mice (sexes collapsed). *: genotype × treatment interaction (p < .05); ‡: simple main effect of genotype within MA-treated groups (p < .05). Boxed numbers indicate number of animals per group; the same animals contributed to all scores in panels a and b.

Figure 7

MA upregulates Fos-IR within the perioculomotor urocortin-containing neurons (pIIIu) of both CRF-R2 KO and WT mice. (a) Results from cell counts (mean + SEM) in the pIIIu. (b) Representative photomicrographs showing Fos-positive cells within the pIIIu; schematic shows the area delineated for counting. *: main effect of MA treatment (p < .05). Boxed numbers indicate number of animals per group.

Figure 8

Deletion of CRF-R2 results in an overall decrease in Fos-IR within the paraventricular nucleus of the hypothalamus (PVN). Results from cell counts (mean + SEM) in (a) the magnocellular division of the PVN (mPVN) and (b) the parvocellular division of the PVN (pPVN). (c) Representative photomicrographs showing Fos-positive cells in the mPVN and pPVN; the schematic shows areas delineated for counting. #: main effect of CRF-R2 genotype (p < .05). Boxed numbers indicate number of animals per group; the same animals contributed to all values in panels a and b.

Figure 9

Deletion of CRF-R2 attenuates acute MA-induced Fos-IR within the basolateral amygdala (BLA) and the central nucleus of the amygdala (CeA). Shown are the results from cell counts (mean + SEM) in the (a) basolateral and (b) central nuclei of the amygdala. (c) Representative photomicrographs showing Fos-positive cells in the lateral amygdala (LA), BLA and CeA; the schematic shows areas delineated for counting (for CeA, both the capsular and lateral divisions of the nucleus were included). *: CRF-R2 genotype × MA treatment interaction (both p < .05); ‡: simple main effect of genotype within MA-treated groups (both p < .05). Boxed numbers indicate number of animals per group; the same animals contributed to all values in panels a and b.