A previous study from this laboratory showed that dopamine (DA) D2 receptors (D2Rs) are involved in the reinforcing effectiveness of different magnitudes of food reinforcement. We attempted to assess the contributions of the three D2-like receptor subtypes using knockout (KO) mice completely lacking DA receptors (D2R, D3R, or D4R KO mice) and their wild-type littermates. These subjects were exposed to a series of fixed-ratio (FR) food-reinforcement schedules in two contexts: an open economy with additional food provided outside the experimental setting and a closed economy with all food earned within the experimental setting. A behavioral-economic model was used to quantify reinforcer effectiveness with food pellets obtained as a function of price (FR schedule value) using an exponential model of demand with the equation logQ=logQ0+k(e-alphaQ0C-1), where alpha represents decline rate of the curve and is inversely related to reinforcer effectiveness, Q represents the number of reinforcers consumed, Q0 represents maximum consumption at zero price, C represents the cost of each reinforcer (i.e. FR value), and k is the y-axis range of the function. With both open and closed economies, as price increased, food consumption decreased, and did so more rapidly in DA D2R KO mice compared to WT littermates. Because an assessment of demand may be influenced by how rapidly responding extinguishes, we studied extinction of responding in two contexts: by eliminating food deliveries and by delivering food independently of responding. A hyperbolic model quantified rates of extinction with an equation of the form R=1/(1+kt), with R representing the response rate as a proportion of control, t representing session number, and k representing the decay parameter, which reflects rate of change (i.e., how quickly the behavior extinguishes). Extinction in DA D2R KO mice occurred less rapidly compared to WT mice in both contexts. Elasticity of food demand was higher in DA D4R KO than WT mice in the open, but not closed, economy. Extinction of responding in DA D4R KO mice was not different from that in WT littermates in either context. No differences in elasticity of food demand or extinction rate were obtained in D3R KO mice and WT littermates. These results indicate that the D2R is the primary DA D2-like receptor subtype mediating the reinforcing effectiveness of food. To identify novel ligands for the D2 dopamine receptor (D2R), we screened small molecule chemical libraries using high throughput screening. This identified a hit compound that was found to selectively activate the D2R in a functionally biased fashion. Chemical optimization resulted in a lead compound (VU207) that exhibits full agonist activity in three different D2R signaling assays: Ca2+ mobilization (Gqi5), inhibition of forskolin-stimulated cAMP accumulation (Gi/o), and β-arrestin recruitment. However, VU207 fails to activate D2R-Gβγ-mediated responses including GIRK channel activation and adenylyl cyclase potentiation. VU207 was also found to exhibit potent D3R antagonism with no functional activity at other dopamine receptors. Further, behavioral paradigms (hypothermia and yawning) indicated that VU207 is CNS-penetrant and acts as a D2R agonist and a D3R antagonist in vivo. Using ex vivo brain slices, we investigated the functional activity of VU207 at D2Rs located on dopaminergic neurons. In these cells, the D2Rs located on the cell bodies and dendrites (somatodendritic D2Rs) activate GIRK channels whereas identical D2Rs located on the nerve terminals inhibit dopamine release through a non-GIRK-mediated mechanism. We found that VU207 failed to activate GIRK currents through somatodendritic D2Rs and, in fact, acted as an antagonist of this D2R response. Notably, in contrast, VU207 acted as an agonist in inhibiting dopamine release via the D2Rs at the nerve terminals. This latter response was absent in tissue from autoD2R knockout mice that lack D2R expression in dopaminergic neurons. These findings suggest that VU207 exhibits a form of spatial or location bias acting as either an agonist or an antagonist depending on where in the neuron the D2R is located. It is expected that more cases of location/spatial bias will be observed upon further analyses of receptor-mediated signaling in cells with complex morphologies, especially neurons.
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