Repeated amphetamine administration outside the home cage enhances drug-induced Fos expression in rat nucleus accumbens

Abstract

Induction of the immediate early gene protein product Fos has been used extensively to assess neural activation in the striatum after repeated amphetamine administration to rats in their home cages. However, this technique has not been used to examine striatal activation after repeated administration outside the home cage, an environment where repeated drug administration produces more robust psychomotor sensitization. We determined the dose-response relationship for amphetamine-induced psychomotor activity and Fos expression in nucleus accumbens and caudate-putamen 1 week after repeated administration of amphetamine or saline in locomotor activity chambers. Repeated administration of amphetamine enhanced amphetamine-induced locomotor activity and stereotypy and Fos expression in nucleus accumbens, but not in caudate-putamen. In comparison, levels of Fos expression induced by 1mg/kg amphetamine were not altered in nucleus accumbens or caudate-putamen by repeated amphetamine administration in the home cage. Double-labeling of Fos protein and enkephalin mRNA indicates that Fos is expressed in approximately equal numbers of enkephalin-negative and enkephalin-positive neurons in nucleus accumbens and caudate-putamen following injections outside the home cage. Furthermore, repeated amphetamine administration increased drug-induced Fos expression in enkephalin-positive, but not enkephalin-negative, neurons in nucleus accumbens. We conclude that repeated amphetamine administration outside the home cage recruits the activation of enkephalin-containing nucleus accumbens neurons during sensitized amphetamine-induced psychomotor activity.

Figure 1

(A) Locomotor activity and stereotyped behavior are sensitized after repeated amphetamine administration in the locomotor activity chamber. Values for locomotor activity represent mean±SEM (n=7–8 per group) of distance traveled during 2 hours following challenge injections of different doses of amphetamine after repeated administration of amphetamine or saline. (B) Values for stereotyped behavior represent mean±SEM (n=7–8 per group) assessed during the 30-second interval 20 minutes following challenge injections. * indicates significantly more amphetamine-induced locomotor activity or stereotyped behavior for each challenge dose after repeated amphetamine versus repeated saline administration.

Figure 2

(A) Schematic representation of brain regions captured for image analysis. Images were captured from coronal sections approximately 2 and 1 mm anterior to Bregma for nucleus accumbens and approximately 2, 1, and 0 mm anterior to Bregma for caudate-putamen. Fos-IR nuclei were quantified from images of the regions indicated with gray rectangles. The size of each image was 1.76 × 1.36 mm with a total area of 2.39 mm². Drawings of coronal section and coordinates were obtained from Paxinos and Watson (1998). Photomicrographs (with 5X objective) of amphetamine-induced Fos-IR nuclei in (B) caudate-putamen and (D) nucleus accumbens following repeated amphetamine administration. The large round area of white matter in the left-hand side of the nucleus accumbens image is the anterior commissure. The two rectangles in the nucleus accumbens image indicate core and shell subregions quantified and shown in Figure 4; the size of each rectangle was 0.62 × 0.41 mm with a total area of 0.25 mm². (C) Higher magnification photomicrograph (with 40X objective) of nucleus accumbens.

Figure 3

Amphetamine-induced Fos expression after repeated drug administration in the locomotor activity chamber. Graphs indicate the number of Fos-IR nuclei in nucleus accumbens (top row) and caudate-putamen (bottom row) at 2, 1, and 0 mm anterior to Bregma after repeated administration of amphetamine or saline. Values represent mean±SEM (n=6–8 per group). * indicates significantly more amphetamine-induced Fos-IR for each challenge dose after repeated administration of amphetamine versus saline.

Figure 4

Amphetamine-induced Fos expression is enhanced in core and shell subregions after repeated amphetamine administration in the locomotor activity chamber. Graphs indicate the number of Fos-IR nuclei in core (A) and shell (B) 2 mm anterior to Bregma. Values represent mean±SEM (n=6–8 per group). * indicates significantly more amphetamine-induced Fos-IR for each challenge dose after repeated administration of amphetamine versus saline.

Figure 5

Representative images of Fos-immunoreactive nuclei expressed in enkephalin-positive and enkephalin-negative neurons in the caudate-putamen (B,C) and nucleus accumbens (D,E) after repeated amphetamine administration in the locomotor activity chamber. (A) Schematic representation of brain regions captured for image analysis. Images were captured from areas indicated with gray rectangles from coronal sections approximately 2 mm anterior to Bregma. The size of each image was 0.4 × 0.34 mm with a total area of 0.14 mm². Drawings of coronal section and coordinates were obtained from Paxinos and Watson (1998). (B,D) Photomicrographs (400X) of sections double-labeled for Fos-immunoreactive nuclei (brown, oval objects) and enkephalin mRNA (clusters of black silver grains). (C,E) Photomicrographs (400X) of same sections after reduction of silver grains with ferricyanide. Arrows indicate enkephalin-positive Fos-immunoreactive neurons. Asterisks indicate enkephalin-negative Fos-immunoreactive neurons. Cross signs indicate enkephalin-positive Fos negative neurons.

Figure 6

Levels of Fos expression in enkephalin-positive and enkephalin-negative neurons in the nucleus accumbens and caudate-putamen after repeated amphetamine administration in the locomotor activity chamber. For nucleus accumbens (A,B), asterisk indicates significantly more amphetamine-induced Fos-IR in enkephalin-positive neurons after repeated administration of amphetamine versus saline. Pound sign indicates that amphetamine challenge injections produced a significant decrease of Fos-IR in enkephalin-positive neurons after repeated saline administration. Cross sign indicates that amphetamine challenge injections significantly increased Fos-IR in enkephalin-negative neurons. For caudate-putamen (C,D), asterisks indicate significantly more Fos-IR after repeated administration of amphetamine versus saline. Cross signs indicate that amphetamine challenge injections significantly increased overall Fos-IR. Values represent mean±SEM (n=8 per group for amphetamine challenge injections; n=4 per group for saline challenge injections).

Figure 7

Amphetamine-induced Fos expression after repeated drug administration in the home cage. Graphs indicate the number of Fos-IR nuclei in nucleus accumbens (top row) and caudate-putamen (bottom row) at 2, 1, and 0 mm anterior to Bregma following challenge injections of 1 mg/kg amphetamine or saline after repeated administration of amphetamine or saline. Values represent mean±SEM (n=6–8 per group). * indicates significantly more Fos-IR induced by challenge injections of amphetamine; no significant differences between repeated administration of amphetamine and saline.