The ability of cannabinoid agonists to stimulate immediately early gene expression has been established since the mid-1990s (Mailleux et al. 1994
). Prior studies of cannabinoid stimulation of c-Fos expression within a subset of rat brain regions have provided important insight into the role of cannabinoid signaling in the mammalian brain: For example, discovery of increased activity within nucleus accumbens is consistent with rewarding properties of these compounds, increased activity within caudate putamen is consistent with locomotor effects, and activity within the paraventricular nucleus of the hypothalamus is consistent with known effects on the hypothalamic-pituitary-adrenal axis and involvement in stress responses (McGregor et al. 1998
; Patel et al. 1998
; Patel and Hillard 2003
Although there has been some indication of the ability of low cannabinoid agonist dosages to inhibit neuronal activity in mammalian systems (c.f. , Patel and Hillard 2003
), clear effects have not been previously reported. Prior lack of appreciation of this phenomenon may be due to differences in basal neuronal activity between our avian species and the rodents previously studied, or perhaps more likely, to differences in anatomy of the brain regions studied. Rather than the laminar arrangement of groups of neurons characteristic of mammalian forebrain, the avian telencephalon is organized in a nuclear manner (e.g. and Reiner 2005
). The resulting discrete aggregations of functionally-related neurons are particularly well-suited to spatial analysis, effectively increasing the signal in our studies and allowing precise measurement of c-Fos expression levels.
Our goal was to characterize changes in neural activity within CB1-expressing telencephalic song regions after systemic cannabinoid agonist exposure. Our hypothesis was that changes in activity within all four cannabinoid-receptor-expressing regions studied; lMAN, Area X, HVC and RA, would be observed following cannabinoid treatments. Therefore the finding of altered activity only within the caudal regions, HVC and RA, was unexpected.
The function of the rostral regions, lMAN and Area X, are critical for successful zebra finch vocal development. Lesions of either of these areas prior to completion of song learning results in impaired vocal development, while adult ablation of these regions in does not alter already-learned song (Bottjer et al. 1984
). This raises the possibility that cannabinoid signaling systems known to be present within these rostral song regions serve a learning-related function that is completed prior to maturation, and accompanied by decreased activity. This hypothesis is supported by a distinct increase in CB1
-receptor densities and changes in expression patterns within these regions during vocal learning that wanes in adulthood (Soderstrom and Tian 2006
). This also suggests that altered vocal learning produced by exogenous cannabinoid exposure during late-postnatal development (Soderstrom and Johnson 2003
; Soderstrom and Tian 2004
) may be attributable to a premature reduction in cannabinoid-sensitive activity within rostral song regions, a possibility that merits further study.
The caudal telencephalic song regions, HVC and RA are critical for vocal motor output of adult song (Nottebohm et al. 1976
). The ability of systemic WIN55212-2 to alter activity within these motor regions is consistent with results of behavioral experiments demonstrating cannabinoid agonist inhibition of adult song production (Soderstrom and Johnson 2001
) and the well-established effects of cannabinoid agonists to reduce locomotor activity in other vertebrate species (including amphibians, e.g. Soderstrom et al. 2000
and reviewed by Chaperon and Thiebot 1999
). These results also suggest that low cannabinoid dosages may produce behavioral effects that oppose those of higher dosages. In the case of song production, low doses tend to increase output, while higher dosages inhibit it (see , Soderstrom and Johnson 2001
Distinct dose-dependent effects of WIN55212-2 are particularly interesting, and suggest presence of multiple cannabinoid-sensitive systems within the vocal motor regions HVC and RA. In mammalian species, evidence supports a presynaptic modulatory role for CB1
signaling (Elphick and Egertova 2001
) that involves a reduced probability of neurotransmitter release following inhibition of calcium- and activation of potassium-channels (Mackie and Hille 1992
; Mackie et al. 1995
). Through this mechanism, accumulating evidence suggests that cannabinoid signaling is an essential component of “depolarization-induced suppression of inhibition” or DSI (Wilson and Nicoll 2002
). DSI is a retrograde process wherein postsynaptic activity promotes presynaptic inhibition of transmitter release. From this it seems likely that the altered neuronal activity measured in our avian system may be attributable to reduced transmitter release within the rostral vocal motor song regions, HVC and RA. From this it follows that low dosage effects, characterized by reduced neuronal activity (0.3 mg/kg WIN55212-2, see ) are likely attributable to reduced excitatory neurotransmitter release, while higher dosage effects (3 mg/kg WIN55212-2) follow reduced inhibitory input. This hypothesis suggests that: (1) vocal motor song regions contain both excitatory and inhibitory input; (2) the excitatory input is more sensitive to cannabinoid agonists than the inhibitory and; (3) an inhibitory tone predominates within vocal motor regions of zebra finch telencephalon. Pending detailed studies of the neuroanatomy of HVC and RA, similar to the one recently completed for the striatal region Area X (Reiner et al. 2004
) it is difficult to predict which neurotransmitter systems are likely involved in the biphasic cannabinoid effects observed.
The studies done with the antagonist rimonabant were essential to demonstrate involvement of CB1
receptors in the agonist effects we measured. WIN55212-2 is an effective agonist of both CB1
receptor subtypes, while rimonabant (referred to prior to clinical development as SR141716A) is CB1
selective (Pertwee 1997
). In the case of WIN55212-2-altered activity within HVC and RA, both the inhibitory low-dose and stimulatory higher dose effects were reversed by rimonabant and therefore are both attributable to CB1
receptor activation (). Although 6 mg/kg rimonabant was effective in reversing effects of the agonist administered alone (), within RA delivery of 6 mg/kg rimonabant alone resulted in significantly higher levels of c-Fos-reactive cells than did a combination of 6 mg/kg rimonabant with 3 mg/kg WIN55212-2. This effect may be attributable to incomplete displacement of agonist-receptor complexes within RA, resulting in an effectively reduced agonist dosage to levels associated with inhibitory effects on activity (similar to those produced by 0.3 mg/kg WIN55212-2 alone, see ). Rimonabant is a problematic antagonist. In some systems (and in most behavioral systems) it appears to function as a true CB1
-selective antagonist (reviewed by Fowler 2007
). In other systems, particularly those in vitro, it clearly has the ability to function as an inverse agonist, possibly through promoting functional coupling to Gs (Glass and Felder 1997). Therefore we cannot be sure if effects measured following administration of rimonabant alone (those presented in ) are attributable to inverse agonism, or to antagonism of endocannabinoid tone within RA.
Double labeling experiments allowed us to assess the relative patterns of expression of CB1
receptors and c-Fos-expressing nuclei within the caudal telencephalic song regions HVC and RA (see ). Within these regions, the distinct expression of CB1
within neuropil suggests a generalized expression throughout neuronal processes that surround the cell bodies of c-Fos-expressing neurons. Densely stained puncta, some of which also surround c-Fos-expressing cell bodies, are consistent in size and appearance with pre-synaptic densities, such as those previously described to express aromatase in zebra finch telencephalon (Peterson et al. 2005
). This dual pattern of CB1 receptor expression may provide additional insight into the distinct efficacies of low- and high- cannabinoid agonist dosages described above. For example, signaling coupled to the dense CB1
-expressing puncta is likely to be more sensitive to receptor activation than that within the more diffusely-expressing neuropil. Because CB1 signaling is most clearly associated with presynaptic inhibition of synaptic release, (Elphick and Egertova 2001
) our hypothesis suggests that at low agonist dosages, CB1 expression within puncta likely functions to reduce excitatory input to c-Fos expressing neurons, decreasing their activity from basal levels. As agonist dosages increase, effective activation of the lower density, but more wide-spread population of neuropil receptors may become more significant. Because higher agonist dosages are associated with increased neural activity (as indicated by increased c-Fos expression) this suggests that neuropil expression may function to mitigate inhibitory neural input. These interesting possibilities will be the subject of further experimentation.
Overall, results reported herein demonstrate for the first time dose-dependent effects of cannabinoid receptor activation to both inhibit and stimulate neuronal activity within a subset of CB1 receptor-expressing brain regions. This knowledge will be helpful for interpretation of the complex neuromodulary effects of cannabinoids in other systems, and improves our understanding of the nature and function of cannabinoid signaling within the vertebrate brain.