Cannabinoid exposure during zebra finch sensorimotor vocal learning persistently alters expression of endocannabinoid signaling elements and acute agonist responsiveness

Abstract

Background: Previously we have found that cannabinoid treatment of zebra finches during sensorimotor stages of vocal development alters song patterns produced in adulthood. Such persistently altered behavior must be attributable to changes in physiological substrates responsible for song. We are currently working to identify the nature of such physiological changes, and to understand how they contribute to altered vocal learning. One possibility is that developmental agonist exposure results in altered expression of elements of endocannabinoid signaling systems. To test this hypothesis we have studied effects of the potent cannabinoid receptor agonist WIN55212-2 (WIN) on endocannabinoid levels and densities of CB1 immunostaining in zebra finch brain.

Results: We found that late postnatal WIN treatment caused a long-term global disregulation of both levels of the endocannabinoid, 2-arachidonyl glycerol (2-AG) and densities of CB1 immunostaining across brain regions, while repeated cannabinoid treatment in adults produced few long-term changes in the endogenous cannabinoid system.

Conclusions: Our findings indicate that the zebra finch endocannabinoid system is particularly sensitive to exogenous agonist exposure during the critical period of song learning and provide insight into susceptible brain areas.

Figure 1

Developmental cannabinoid exposure alters CB₁ immunostaining within various zebra finch brain regions. Initial daily treatments over 25 days are indicated by first designations (VEH-, WIN-). Later, single acute treatments in adulthood are indicated second (-VEH, -WIN, see Table 1). Treatments were delivered during sensorimotor song learning (from 50-75 days) and measured in adulthood (> 100 days). Basal levels of staining are decreased following repeated WIN exposure during development in all regions but DLM (compare VEH-VEH to WIN-VEH). Acute responsiveness is increased in all regions (compare VEH-WIN to WIN-WIN). Asterisks indicate differences from VEH-VEH treatment groups (p < 0.05, one-way ANOVA followed by SNK post-tests). Daggers indicate differences from VEH-WIN groups, double-daggers indicate differences from WIN-VEH.

Figure 2

Repeated cannabinoid treatment during adulthood alters CB₁ immunostaining primarily within vocal-motor-related regions of zebra finch brain (HVC, RA, and DLM). Initial daily treatments over 25 days are indicated by first designations (VEH-, WIN-). Later, single acute treatments are indicated second (-VEH, -WIN, see Table 1). Basal staining levels are increased following repeated WIN exposure in adulthood in (B) HVC, (D) RA, and (F) DLM (compare VEH-VEH to WIN-VEH in these panels). Acute responsiveness is not modified following repeated treatments (compare VEH-WIN and WIN-WIN). Chronic treatment did increase responsiveness within the molecular layer of the cerebellum (double-dagger, panel G) Asterisks indicate differences from VEH-VEH treatment groups (p < 0.05, one-way ANOVA followed by SNK post-tests).

Figure 3

Effects of chronic, developmental, cannabinoid treatments on endogenous 2-AG levels in zebra finch brain. Brains were rapidly dissected into rostral (Rostral Tel.) and caudal (Caudal Tel.) telencephalon, midbrain and cerebellum. Lipids were extracted, spiked with deuterium-labeled internal standards and subjected to LC-ESI-MS-MS for quantitative analysis of 2-arachidonyl glycerol (2-AG) content. (A): ANOVA indicates a significant relationship between brain region and 2-AG content (p < 0.05). Post-hoc analysis reveals a significant increase in 2-AG content in rostral telencephalon (which contains the song regions lMAN and Area X). Significant decreases in endocannabinoid content within cerebellum were observed following developmental WIN (*p < 0.05). (B): No effect of repeated WIN treatments given during adulthood on 2-AG levels were found.

Figure 4

Representative CB1 immunostaining. (A) Medial parasagittal sections contain rostral song regions lMAN and Area X, thalamic regions DLM and Ov, and cerebellum. (B) More lateral parasagittal sections capture HVC and RA. Dorsal and caudal are indicated by arrows, the bars = 1 mm. (C) A tracing of the micrograph in panel A serves as a diagram summarizing relative locations of song regions studied. Regions present in panel A are represented in black, other regions are diagrammatically represented in grey (HVC, RA and nXII). Established interconnections between song regions are indicated with arrows. Grey arrows indicate rostral forebrain circuitry essential for vocal learning. Black arrows indicate caudal vocal-motor circuitry.