Changes in nucleus accumbens and neostriatal c-Fos and DARPP-32 immunoreactivity during different stages of food-reinforced instrumental training

Abstract

Nucleus accumbens is involved in several aspects of instrumental behavior, motivation and learning. Recent studies showed that dopamine (DA) release in the accumbens shell was significantly increased on the first day of training on a fixed ratio (FR) 5 schedule (i.e. the transition from FR1 to FR5) compared with those rats that continued FR1 training, even though the rats on their first day of FR5 training received less food reinforcement than rats continuing on the FR1 schedule. Additionally, the second day of FR5 responding was marked by a significant increase in DA release in accumbens core. The present studies employed immunohistochemical methods to characterize the changes in cellular markers of accumbens and neostriatal neural activity that occur during various stages of food-reinforced FR5 training. c-Fos and DARPP-32 immunoreactivity in accumbens shell was significantly increased on the first day of FR5 training, while core c-Fos and DARPP-32 expression showed large increases on the second day of FR5 training. Additional studies showed that c-Fos and DARPP-32 expression in neostriatum increased after more extensive training. Double-labeling studies with immunofluorescence methods indicated that increases in accumbens c-Fos and DARPP-32 expression were primarily seen in substance-P-positive neurons. These increases in accumbens c-Fos and DARPP-32 immunoreactivity seen during the initial phases of FR training may reflect several factors, including novelty, learning, stress or the presentation of a work-related challenge to the organism. Moreover, it appears that the separate subregions of the striatal complex are differentially activated at distinct phases of instrumental training.

Figure 1

Representative schematic showing regions of interest for photomicrographs in the nucleus accumbens core and shell, and the dorsomedial and dorsolateral striatum (for clarity, regions are marked on only one side of the brain). The numbers indicated are relative to bregma for each brain level (Figure is modified from Paxinos and Watson, 1998).

Figure 2

Expression of c-Fos immunoreactivity in nucleus accumbens under control conditions and during the transition from FR1 to FR5 lever pressing. A–C. Photomicrographs from a representative rat in the FR1 group. A. Low power photomicrograph of c-Fos expression showing regions of interests in the core (B) and shell (C) that were quantified. B–C. High power images of the accumbens core (B) and shell (C) showing minimal c-Fos expression. D–F. Photomicrographs from a representative rat in the first day FR5 group. D. Low power photomicrograph of c-Fos expression showing regions of interests in the core (B) and shell (C) that were quantified. E. High power image of the accumbens core showing minimal c-Fos expression. F. High power image showing the robust expression of c-Fos containing neurons in the accumbens shell. G. Quantification of number of c-Fos positive cells in the accumbens shell and core across the different training conditions. *p < 0.05, different from control and FR1 groups #p < 0.05, different from all other groups. Abbreviations: ac= anterior commissure, NAc= nucleus accumbens. Scale bars: A, D = 200 µm; B, C, E, F = 75 µm

Figure 3

Expression of DARPP-32 immunoreactivity in nucleus accumbens under control conditions and during the transition from FR1 to FR5 lever pressing. A–C. Low-power images of the accumbens shell showing DARPP-32 immunoreactivity in a representative animal from the control group (A), the FR1 group (B), and the first day FR5 condition (C). D–E. Higher-power images showing DARPP-32-labeled cells (arrowheads) in more detail. Note that the DARPP-32-labeled cells on the first day of FR5 training appear to be more morphologically reactive in appearance with more ramified processes (E). F. Quantification of number of DARPP-32 positive cells in the accumbens shell and core across the different training conditions. *p < 0.05, different from control and FR1 groups #p < 0.05, different from all other groups. Scale bars: A–C =100µm; D–E= 50µm.

Figure 4

Double labeling of c-Fos and substance P immunoreactivity in nucleus accumbens on the first day of FR5 training. A. A low-power image of the shell in which many cells are c-Fos positive (green). B. A low-power image of many cells showing substance P immunoreactivity (red). C. A low-power image of substance P cells (red) showing pyknotic DAPI-labeled nuclei (blue); note the substance P cells with clear nuclei (arrowheads). D. Higher-power image showing numerous double-labeled cells expressing both substance P and c-Fos imunoreactivity (yellow, from the merging of the two separate images). An intact cell clearly demonstrates co-localization (arrowhead). D. Inset, A high power image shows the double-labeled cells. E. Low-power image of the accumbens core showing few c-Fos expressing cells (arrowheads). F. Low-power image of the accumbens core showing few substance P-positive neurons (arrowheads). G. A higher power image showing co-localization of c-Fos expression in substance P containing neurons (arrowheads) in the accumbens core. Abbreviations: ac, anterior commissure, Scale bars: A-C. 100 µm; D. 50 µm (inset), 25 µm; E–F. 100 µm; G. 75µm.

Figure 5

Double labeling of DARPP-32 and substance P immunoreactivity in nucleus accumbens on the first day of FR5 training. A–B. The shell of the nucleus accumbens showing a large number of DARPP-32-positive cells (A; green) and substance P containing cells (B; red). C. Merged low-power image showing the co-localization of DARPP-32 and substance P containing neurons (yellow). D. High-power image showing numerous double-labeled cells with both substance P and DARPP-32 immunoreactivity; some neurons appear to have DARPP-32, but not substance P immunoreactivity in the dendrites (arrows). E–G. Photomicrograph of nucleus accumbens core, showing a few DARPP-32 positive cells (E; green) and substance P positive cells (F; red) that are co-localized (G; yellow). H–J. Low-power image showing cells in the accumbens shell containing c-Fos (H; green) and DARPP-32 (I; red) immunoreactivity, and their co-localization in the merged image (J; yellow). K–M. Low-power image showing cells in the accumbens core containing some c-Fos (K; red) and DARPP-32 (L; red) immunoreactivity, and their co-localization in the merged image (M; yellow). Abbreviations: ac, anterior commissure, Scale bars: A–C. 100 µm; D. 50 µm; 25 µm, (inset); E–F. 75 µm; G. 50 µm; H–M. 100 µm.

Figure 6

Double labeling of c-Fos and DARPP-32 with enkephalin immunoreactivity on the first day of FR5 training. A–B. Photomicrographs of accumbens shell, showing c-Fos (A; green) and enkephalin (B; red) immunoreactivity. C. High-power view of the merged image, showing a separate localization of c-Fos expression (green, arrowheads) and enkephalin (red, arrows) immunoreactivity. D–F. Low-power photomicrograph of the accumbens core, showing c-Fos (D; green) and enkephalin (E; red) immunoreactivity; merged image (F) shows separate labeling of cells (i.e., separate red and green cells). G–H. Photomicrographs of accumbens shell, depicting DARPP-32 (G; green) and enkephalin (H; red) immunoreactivity. I. Merged high-power image shows separate DARPP-32 (green) and enkephalin (red) immunoreactive cells in the shell. J–L. DARPP-32 immunoreactivity (J; green), and enkephalin immunoreactivity (K; red), and their separate localization (L; separate green and red labeled cells) in the accumbens core. Abbreviations: ac, anterior commissure, Scale bars: A–B, 100 µm; C, 50 µm D–F, 100 µm; G–H, 100 µm; I, 50 µm, J–L, 100 µm.

Figure 7

Expression of c-Fos immunoreactivity in neostriatum under control conditions and during the transition from FR1 to FR5 lever pressing. A–B, Quantification of c-Fos density in the dorsomedial neostriatum (A; DMS) and dorsolateral neostriatum (B; DLS) after control and the different FR lever pressing training days (means ± SEM). *p< 0.05, different from control and FR1 training groups, collapsed across both DMS and DLS

Figure 8

Expression of DARPP-32 immunoreactivity in neostriatum under control conditions and during the transition from FR1 to FR5 lever pressing. A–B, Quantification of DARPP-32-labeled cells in the dorsomedial neostriatum (A; DMS) and dorsolateral neostriatum (B; DLS) after control and the different FR lever pressing training days (means ± SEM). *p< 0.05, different from control and FR1 training groups, collapsed across both DMS and DLS