Late-postnatal cannabinoid exposure persistently increases FoxP2 expression within zebra finch striatum

Abstract

Prior work has shown that cannabinoid exposure of zebra finches during sensorimotor stages of vocal development alters song patterns produced in adulthood. We are currently working to identify physiological substrates for this altered song learning. FoxP2 is a transcription factor associated with altered vocal development in both zebra finches and humans. This protein shows a distinct pattern of expression within Area X of striatum that coincides with peak expression of CB(1) cannabinoid receptors during sensorimotor learning. Coincident expression in a brain region essential for song learning led us to test for a potential signaling interaction. We have found that cannabinoid agonists acutely increase expression of FoxP2 throughout striatum. When administered during sensorimotor song learning, cannabinoids increase basal levels of striatal FoxP2 expression in adulthood. Thus, song-altering cannabinoid treatments are associated with persistent increases in basal expression of FoxP2 in zebra finch striatum.

Fig. 1

Montages of 100X micrographs of parasagittal sections of adult male zebra finch telencephalon stained with anti-FoxP2 antibody. Sections shown were reacted together and represent tissue from animals treated with: (A) Vehicle, (B) 0.1 mg/kg, (C) 0.3 mg/kg, (D) 1 mg/kg and, (E) 3 mg/kg WIN. Diffuse labeled nuclei appear throughout striatum. Distinctly higher densities are observed within caudal regions of lateral striatum. Along caudal and caudal-dorsal borders of lateral striatum, characteristic punctate aggregations of labeled nuclei occur. Insets are 1000X DIC images from within striatum as indicated by boxes. Upper high-power panels were taken within regions of lateral striatum, lower panels within regions of Area X of striatum.

Fig 2

Western blot analysis of anti-FoxP2 staining of proteins isolated from the brain of an adult male zebra finch and separated by 10 % PAGE (40 μg protein). The arrow indicates labeling of a primary band of 80.1 kDa, consistent with the mass reported for full-length zebra finch FoxP2 protein (Miller et al., 2008).

Fig 3

Acute administration of the cannabinoid agonist, WIN55212-2 dose-dependently stimulates FoxP2 expression within adult male zebra finch striatum. Animals were perfused 90 min after treatment, and brains removed and processed for anti-FoxP2 immunohistochemistry. Densities of anti-FoxP2 reactive nuclei were determined and transformed to % of maximum stimulation (produced by 3 mg/kg WIN).

Fig. 4

Developmental cannabinoid exposure persistently increases basal densities of FoxP2-reactive nuclei within both Area X and other regions of striatum. Initial daily treatments over 25 days are indicated by first designations (VEH-, WIN-). Later, single acute treatments to stimulate FoxP2 expression in adulthood are indicated second (-VEH, -WIN, see Table 1). (A) Effects of treatments within Area X and; (B) in non-Area X-containing striatum. Treatments were delivered during sensorimotor song learning (from 50-75 days) and measured in adulthood (> 110 days). Basal levels of FoxP2 expression are elevated following repeated WIN exposure during development (compare VEH-VEH to WIN-VEH). Acute responsiveness persists (e.g. VEH-WIN and WIN-WIN). Asterisks indicate differences from VEH-VEH treatment groups (p < 0.05, one-way ANOVA followed by Tukey post-tests). Dagger indicates a difference from WIN-VEH group (p < 0.05, one-way ANOVA followed by Tukey post-test).

Fig. 5

Repeated cannabinoid treatment during adulthood decreases basal densities of FoxP2-reactive nuclei within both Area X and other regions of striatum. Initial daily treatments over 25 days are indicated by first designations (VEH-, WIN-). Later, single acute treatments to stimulate FoxP2 expression are indicated second (-VEH, -WIN, see Table 1). Basal levels of FoxP2 expression are decreased following repeated WIN exposure in adulthood in both (A) Area X and (B) other regions of striatum (compare VEH-VEH to WIN-VEH). Acute responsiveness persists only after repeated cannabinoid treatments (e.g. WIN-VEH and WIN-WIN). Asterisks indicate differences from VEH-VEH treatment groups (p < 0.05, one-way ANOVA followed by Tukey post-tests). Dagger indicates a difference from WIN-VEH group (p < 0.05, one-way ANOVA followed by Tukey post-test).

Fig 6

Developmental WIN treatments administered after the CB₁-selective antagonist SR do not persistently alter basal densities of FoxP2-labeled nuclei or alter acute WIN responsiveness in adulthood. Asterisk indicates difference from VEH+SR-VEH, dagger indicates difference from VEH+SR-VEH (p < 0.05, one-way ANOVA, Tukey post-test).