CB(1) cannabinoid receptor activation dose dependently modulates neuronal activity within caudal but not rostral song control regions of adult zebra finch telencephalon

Abstract

Rationale: CB(1) cannabinoid receptors are distinctly expressed at high density within several regions of zebra finch telencephalon, including those known to be involved in song learning (lMAN and Area X) and production (HVC and RA) because (1) exposure to cannabinoid agonists during developmental periods of auditory and sensory-motor song learning alters song patterns produced later in adulthood and (2) densities of song region expression of CB(1) waxes and wanes during song learning. It is becoming clear that CB(1)-receptor-mediated signaling is important to normal processes of vocal development.

Materials and methods: To better understand the mechanisms involved in cannabinoid modulation of vocal behavior, we have investigated the dose-response relationship between systemic cannabinoid exposure and changes in neuronal activity (as indicated by expression of the transcription factor, c-Fos) within telencephalic brain regions, with established involvement in song learning and/or control.

Results: In adults, we have found that low doses (0.1 mg/kg) of the cannabinoid agonist WIN-55212-2 decrease neuronal activity (as indicated by densities of c-fos-expressing nuclei) within vocal motor regions of caudal telencephalon (HVC and RA) while higher doses (3 mg/kg) stimulate activity. Both effects were reversed by pretreatment with the CB(1)-selective antagonist rimonabant. Interestingly, no effects of cannabinoid treatment were observed within the rostral song regions lMAN and Area X, despite distinct and dense CB(1) receptor expression within these areas.

Conclusions: Overall, our results demonstrate that, depending on dosage, CB(1) agonism can both inhibit and stimulate neuronal activity within brain regions controlling adult vocal motor output, implicating involvement of multiple CB(1)-sensitive neuronal circuits.

Figure 1

Parasagittal diagram of song regions with established distinct expression of CB1 receptors (adapted from (Soderstrom and Tian 2006). Rostral is left, dorsal top. Telencephalic regions containingdense CB₁ expression are indicated in black and include telencephalic song regions lateral magnocellular nucleus of the anterior nidopallium (lMAN), Area X within songbird medial striatum (Area X), HVC and the robust nucleus of the arcopallium (RA). Thalamic regions nucleus uvaformis (Uva) and dorsal lateral nucleus of the medial thalamus (DLM) that also distinctly express CB₁ receptors are shown in grey and included to illustrate known interconnections between song regions (indicated by arrows, (Bottjer and Johnson 1997).

Figure 2

Immunohistochemical staining of lMAN and Area X regions of rostral telencephalon with anti-c-Fos antibody as a function of vehicle (A and C) or WIN55212-2 (3 mg/kg, B and D) treatment. Medial parasaggital sections represent planes about 1.5 mm lateral from the midline. Rostral is left, dorsal is top, magnification is 100 X. Dark puncta represent stained nuclei. Note relatively low-level expression in lMAN (indicated by dashed outline in panels A and B) and Area X (outlined in panels C and D) relative to that within the caudal song regions HVC and RA (shown in Fig 3).

Figure 3

Immunohistochemical staining of HVC (indicated by dashed outline in panels A and B) and RA (outlined in panels C and D) regions of caudal telencephalon with anti-c-Fos antibody as a function of vehicle (A and C) or WIN55212-2 (3 mg/kg, B and D) treatment. Medial parasaggital sections represent planes about 1.5 mm lateral from the midline. Rostral is left, dorsal is top, magnification is 100 X. Dark puncta represent stained nuclei. Note relatively high-level expression in HVC and RA relative to that within caudal song regions (shown in Fig 2).

Figure 4

The cannabinoid agonist WIN55212-2 increases c-Fos expression within a subset of telencephalic brain regions known to control song learning and control. Birds were killed 90 min following treatment and perfused for immunohistochemistry. Densities of immunoreactive nuclei within each region (n = 6 animals within each treatment group) are summarized. Mean densities were generated from counts within at least five separate tissue sections from each animal. Two-way ANOVA followed by post-tests revealed no significant density changes within rostral regions lMAN (panel A) and Area X (panel B), while increased densities of c-Fos immunoreactive nuclei were noted within the caudal regions HVC (panel C) and RA (panel D) 90 min following treatment with the cannabinoid agonist WIN55212-2 (3 mg/kg, *p < 0.05).

Figure 5

The CB₁-selective antagonist rimonabant (RIM) reverses both high- (3 mg/kg) and low-dosage (0.3 mg/kg) effects of the cannabinoid agonist WIN55212-2. Densities of anti-c-Fos reactive cells within HVC (panels A and C) or RA (panels B and D) following high- (panels A and B) and low-WIN dosage treatments (panels C and D) are shown as a fraction of vehicle controls (VEH). Significant differences from VEH groups following 2-way ANOVA and Student-Neuman-Keuls post-tests are indicated by asterisks (*p < 0.05). Differences from WIN treatments (3 or 0.3 mg/kg WIN) are indicated by single daggers (^†p < 0.05). The double-dagger in panel B indicates a significant difference from the combined treatment of 6 mg/kg rimonabant and 3 mg/kg WIN55212-2 (^‡p < 0.05).

Figure 6

Double-immunohistochemcial labeling with c-Fos and CB₁ cannabinoid receptor antibodies. c-Fos-labeled nuclei are stained blue-grey, CB₁ receptor staining is rust-brown. The pattern of anti-CB1 staining within HVC and RA consists of diffuse neuropil staining with distinct small and irregularly shaped puncta (indicated with arrows labeled ‘P’). c-Fos-labeled nuclei surrounded by unstained cytoplasm are indicated with arrows labeled ‘S’). Dorsal is top, rostral left. 100 X bars = 200 microns, 1000 X bars = 10 microns.