Using an event-triggered recording system, the quantity of daily song bout production was measured weekly in male zebra finches (Taeniopygia guttata) during sensory-motor learning and at one year of age. Our aim was to ask whether the development of a stereotyped vocal pattern involves a practice-driven component. If so, we hypothesized that juvenile males learning song should sing more often than adults reciting a vocal pattern they had already learned, and that greater levels of juvenile singing should be associated with improvement in the quality of the adult song. Across the period measured (36-365 days of age), subjects showed an inverted U-shaped pattern of daily song bout production. Song bout production was lowest during subsong, with increased production associated with plastic song and song crystallization, although individual differences were large. Daily song bout production decreased in adulthood. Higher levels of song bout production during plastic song correlated with fewer sequencing errors in adult song patterns (r 2 =0.77). In contrast, quantity of singing during song crystallization showed no relationship to vocal stereotypy (r 2 =0.002). Our data suggest a sensitive period for vocal practice during zebra finch sensory-motor learning with consequences for the note-sequence fidelity of the adult vocal pattern.

Singing by adult male zebra finches is a learned behavior important for courtship, kin recognition, and nest defense (Zann, 1996) and is inhibited by both brief periods of limited food availability and systemic injection of cannabinoids. These similar effects on singing, combined with increasing evidence for endocannabinoid involvement in feeding behavior, led us to evaluate a possible shared mechanism. We found that limited food availability both reduces singing in a cannabinoid antagonist-reversible manner and increases levels of the endocannabinoid 2-arachidonyl glycerol in various brain regions including the caudal telencephalon, an area that contains auditory telencephalon including the L2 subfield of L (L2) and caudal medial nidopallium (NCM). Development and use of an anti-zebra finch cannabinoid receptor type 1 (CB 1 ) antibody demonstrates distinct, dense cannabinoid receptor expression within song regions including Area X, lMAN (lateral magnocellular nucleus of anterior nidopallium), HVC, RA (robust nucleus of arcopallium), and L2. NCM receives L2 projections and is implicated in integration of auditory information. Activity in this area, determined through expression of the transcription factor ZENK, is increased after exposure to unfamiliar song. Because previous work has shown that these novel songstimulated increases in NCM activity are mitigated by cannabinoid exposure, we tested and found that similar effects on ZENK expression are produced by limiting food. Limited food-related reductions in the activity of NCM neurons were reversed by the cannabinoid antagonist SR141716A (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide), implicating CB 1 cannabinoid receptor involvement. Taken together, these experiments indicate a link between feeding state and gene expression related to auditory perception that is mediated by endocannabinoid signaling.

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Zebra finches learn song during distinct developmental stages, making them an important species for studying mechanisms underlying vocal development. Distinct interconnected forebrain regions have been identified as important to specific features of zebra finch vocal learning and production. Because prior experiments have demonstrated that late postnatal exposure to cannabinoid agonists alters zebra finch song learning, we have sought to identify brain regions likely involved in it. By using an affinity-purified polyclonal antibody directed against the zebra finch CB(1) cannabinoid receptor, we have studied staining patterns in groups of males at 25, 50, 75, and >100 days of age (adults). A general waxing and waning of staining intensity were observed over this developmental period. Distinct staining of song-related brain regions was also noted. Early establishment of staining patterns within rostral telencephalic song regions [area X and lateral magnocellular nucleus of the anterior nidopallium (lMAN)] suggests a role in auditory learning. Later establishment and maintenance in adulthood of small somata and neuropil staining within regions of rostral telencephalon [HVC and robust nucleus of the arcopallium (RA)] are consistent with a vocal motor role for cannabinoid signaling. Our results provide insight into brain regions likely responsible for cannabinoid-altered vocal learning and add to accumulating evidence supporting an important role for cannabinoid signaling in CNS development.

Investigation of cannabinoid pharmacology in a vertebrate with a phylogenetic history distinct from that of mammals may allow better understanding of the physiological significance of cannabinoid neurochemistry. Taricha granulosa, the roughskin newt, was used here to characterize an amphibian cannabinoid receptor. Behavioral experiments demonstrated that the cannabinoid agonist levonantradol inhibits both newt spontaneous locomotor activity and courtship clasping behavior. Inhibition of clasping was dose-dependent and potent (IC 50 ϭ 1.2 g per animal). Radioligand binding studies using [ 3 H]CP-55940 allowed identification of a specific binding site (K D ϭ 6.5 nM, B max ϭ 1,853 fmol/mg of protein) in brain membranes. Rank order of affinity of several ligands was consistent with that reported for mammalian species (K D , nM): CP-55940 (3.8) Ͼ levonantradol (13.0) Ͼ WIN55212-2 (25.7) Ͼ Ͼ anandamide (1,665) Ϸ anandamide ϩ 100 M phenylmethylsulfonyl fluoride (2,398). The cDNA encoding the newt CB1 cannabinoid receptor was cloned, and the corresponding mRNA of 5.9 kb was found to be highly expressed in brain. A nonclonal Chinese hamster ovary cell line stably expressing the newt CB1 cannabinoid receptor was prepared that allowed demonstration of cannabinoid-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity. This inhibition was dose-dependent and occurred at concentrations consistent with affinities determined through radioligand binding experiments. The behavioral, pharmacological, and molecular cloning results demonstrate that a CB1 cannabinoid receptor is expressed in the CNS of the roughskin newt. This amphibian CB1 is very similar in density, ligand binding affinity, ligand binding specificity, and amino acid sequence to mammalian CB1. The high degree of evolutionary conservation of cannabinoid signaling systems implies an important physiological role in vertebrate brain function. Key Words: CannabinoidAmphibian-G protein-coupled receptor-Adenylate cyclase -Locomotor behavior-Sex behavior. J. Neurochem. 75, 413-423 (2000).The active constituents of Cannabis sativa have been used for centuries for their medicinal and psychoactive properties. Despite this long history of cannabinoid use an understanding of cannabinoid pharmacology has only recently begun to emerge. This progress was initiated with evidence that cannabinoids inhibit neuronal adenylate cyclase activity through activation of G proteins (Howlett and Fleming, 1984;Howlett, 1985). Cannabinoid activation of G proteins with an appropriate pharmacological signature implied the presence of specific cannabinoid receptors. Two subtypes of specific cannabinoid receptors have been identified: CB1, CNS-associated [although peripheral expression has been detected (Devane et al., 1988;Matsuda et al., 1990)], and CB2, which may be exclusively expressed within tissues of the immune system (Munro et al., 1993).Both cannabinoid receptor subtypes alter cellular activity through activation of G proteins of the G i/o subtype, although reports sugges...