We found that AMPH increases locomotor activity and sensitization becomes evident with repeated administration. There was a significant increase in the total distance traveled between the first and the last test session (difference: +1018 ± 182 cm, t = −5.594, df = 30, p < 0.001) for animals administered AMPH repeatedly, whereas locomotor activity in the vehicle treated group was not different between the first and the last sessions (difference: +85 ± 52 cm, t = −1.641, df = 30, p = 0.116).
Verification of FG injection sites
We placed the retrograde tracer, FG, into target regions of mPFC output neurons. Only animals with minimal or no spread of FG deposit beyond the intended region (NAc, BLA, LH) were included in the final analyses. illustrate typical FG injection sites within the NAc, BLA, and LH. The injection sites had minimal tissue damage. Injections of FG into the NAc were centered in the medial portion of the nucleus, including both core and shell areas (), and targeted the intermediate to caudal NAc. In the BLA, FG deposition encompassed the entire complex () and was centered in the anterior BLA, previously shown to receive the densest projections from the mPFC (Sesack et al. 1989
; Vertes 2004
). In the LH, injection sites were typically centered lateral to the fornix and included the majority of the LH (). The injections were central to the rostrocaudal extent of the LH.
Figure 1 Representative micrographs showing typical FG injection sites for the (a) NAc, (b) BLA, and (c) LH. Diagrams representing the corresponding ipsilateral areal distributions of neurons (black dots) in the mPFC retrogradely labeled from the (d) NAc, (e) (more ...)
Retrograde transport of FG
Pyramidal neurons, retrogradely labeled with FG, typically showed a granular, blue-black cytoplasm with no label over the nucleus (). are diagrammatic representations of the distribution of FG immunolabeled neurons from a representative caudal section of the mPFC (from bregma: +2.7) for each target region. Because axons of mPFC projection neurons rarely collateralize (Gabbott et al. 2005
; Pinto and Sesack 2000
), the retrogradely labeled cells predominantly represent single output pathways. Injections of FG into the NAc and LH retrogradely labeled more neurons in the deep layers of the IL and PrL cortices than those placed in the BLA (; ). Injections targeting the NAc retrogradely labeled pyramidal neurons primarily in the deep layers (III-VI) of the IL and PrL cortices (). These FG injections also labeled a small population in the anterior cingulate (AC) cortex, especially if the injection site was situated more dorsally (one case). In addition, labeled cells were seen in the claustrum and the anterior insular, lateral and ventral orbital, dorsal penduncular, and piriform cortices. Injections in the BLA retrogradely labeled cells in layers II and V of the IL and PrL cortices, with a few neurons in the AC (). The anterior insular, dorsal peduncular, and ventral orbital cortices also contained a few labeled cells. Tracer injection in the LH retrogradely labeled cells in the deep layers, especially layer V of the IL and PrL (). A few labeled cells were also found in the AC, anterior insular, and dorsal peduncular cortices and even fewer were located in the ventral and lateral orbital cortices.
Figure 2 Representative micrographs of deep layers of the mPFC showing FG-filled neurons (DAB-Ni) at (a) 20X and (b) 60X, and neurons dually immunolabeled for Fos (DAB) and FG (Vector SG) at (c) 20X and (d) 60X. Dually immunolabeled neurons (arrows) have an amber (more ...)
We analyzed the density of Fos immunoreactivity in the IL and PrL areas of the mPFC and their major projection targets, i.e. the NAc shell and core, BLA, and LH (). The statistical analysis demonstrated significant differences between treatment groups (F1,42 = 169.4, p < 0.001) and between regions (F5,42 = 10.0, p < 0.001). A significant interaction was also found between regions and treatment groups (F5,42 = 4.1, p = 0.004), indicating that the density of Fosimmunoreactive cells for each region was dependent on treatment. Post-hoc analyses revealed that all regions except the NAc shell (p = 0.052, nonsignificant trend), showed a significant increase in the density of Fos-immunoreactive cells after repeated AMPH treatment compared to vehicle (P < 0.001; ). Between regions, the Fos-immunoreactive cell density in the IL, PrL, NAc core, and LH was significantly greater than the density in the NAc shell (p < 0.001), the density of Fos in the PrL, NAc core, and LH was also significantly greater than the density in the BLA (p < 0.001), and the density in the NAc core was greater than in the IL (p < 0.001) following repeated AMPH treatment. Comparisons between regions also revealed that within the repeated AMPH group, the IL, PrL, NAc core, and BLA had the greatest increases in Fosimmunoreactive cell densities, all of which were significant. Finally, we found no significant differences in Fos-immunoreactive cell density between the vehicle groups for all regions.
Figure 3 Bar graphs showing the density of Fos immunolabeled cells after repeated AMPH or vehicle administration in the IL and PrL areas and their major target regions (NAc shell and core, BLA, and LH); filled bars, repeated AMPH administration (n = 5) and open (more ...)
Pathway-specific Fos expression
Overall, Fos immunoreactivity in the mPFC is elevated following repeated AMPH treatment, and Fos is differentially activated in mPFC efferent pathways. In order to evaluate pathway-specific Fos expression, we counted cells that co-localized FG and Fos in the deep layers of the IL and PrL and found that the dually labeled cells appeared in both cortical areas for all the projection targets studied (, ). There was no difference in the relative density of FG-immunolabeled neurons in the single and dual-labeled preparations (compare ). Quantitative analysis revealed that neither the mPFC-NAc nor the mPFC-BLA efferent neurons differed in their Fos expression between repeated AMPH- and vehicle-treated groups (). Specifically, there was no difference between groups for the mPFC-NAc projection neurons in the IL (p = 0.249) or the PrL (p = 0.983). There was also no difference between groups for the mPFC-BLA pyramidal cells in the IL (p = 0.413) or the PrL (p = 0.256). There was, however, a significant increase in Fos expression in neurons of the mPFC-LH pathway in both the IL and PrL areas after repeated AMPH treatment when compared to vehicle-treated controls (). Finally, there was no difference in Fos induction in both the PrL and IL areas of the mPFC for animals that had sham surgeries as compared to those with Fluoro-Gold injections in one of the three targets described above (Student's t-test, not significant).
Figure 4 Bar graphs showing pathway-specific Fos induction after repeated AMPH or vehicle administration. The proportion of colocalized FG- and Fos- immunolabeled neurons in the IL or PrL areas of the mPFC is shown following FG deposition in the (a) NAc (n = 5 (more ...)
Given the increase in Fos in the mPFC-LH pathway and the induction of Fos in the LH, we dually labeled LH neurons for Fos and orexin immunohistochemistry. Comparing AMPH (n = 4) to vehicle (n = 3) treatments, we found that the orexin-immunoreactive neurons co-localized with Fos (). Indeed, the proportion of orexin neurons that expressed Fos significantly increased following repeated AMPH treatment when compared to vehicle (61.0 ± 4.9% and 17.4 ± 2.6% respectively; p < 0.001).
Figure 5 Representative micrographs of the LH. (a) Dually immunolabeled neurons (arrows) with an amber nucleus (Fos-immunoreactive cell) surrounded by blue cytoplasm (orexin-immunoreactive neuron) after AMPH treatment. (b) Orexin-immunoreactive neuron (singly (more ...)