Quantification of D1B(D5) receptors in dopamine D1A receptor-deficient mice.

The unavailability of selective D1A(D1) or D1B(D5) dopamine receptor ligands has prevented the direct localization of binding sites for these receptors. Thus, receptor autoradiography with long exposure times was used to detect minor D1-like binding sites in the brains of D1A null mutants. Coronal brain sections were prepared from the caudal portion of the prefrontal cortex of homozygous or heterozygous D1A knockout mice or wildtype mice, and labeled with the D1 receptor antagonist [3H]-SCH23390. Slides were dried, and apposed to film with polymer-calibrated standards for 90 days to allow visualization of any low abundance binding sites. No binding was detected in most regions of homozygote (-/-) mouse brains that have high densities of D1 binding in wildtype mice (e.g., the striatum, nucleus accumbens, olfactory tubercles or amygdala). Conversely, small, but detectable amounts of D1-binding were measured in the hippocampus, albeit with a density less than the lowest standard (ca. 20 fmol/mg). Saturation binding of [3H]-SCH23390 in hippocampal homogenates from homozygous mice confirmed a B(max) of 12.3 fmol/mg protein with a K(D) of 0.57 nM. The current work demonstrates directly the presence of D1B(D5) receptors in hippocampus, and also shows that the loss of functional D1A gene products almost completely eliminates detectable D1-binding sites in striatum, as well as in some regions (e.g., the amygdala) where a non-adenylyl cyclase coupled D1 receptor has been reported. This indicates that these non-adenylyl cyclase coupled D1-like receptors represent alternate signaling pathways rather than novel gene products(s).

Behavioural assessment of mice lacking D1A dopamine receptors.

Dopamine D1A receptor-deficient mice were assessed in a wide variety of tasks chosen to reflect the diverse roles of this receptor subtype in behavioural regulation. The protocol included examination of exploration and locomotor activity in an open field, a test of sensorimotor orienting, both place and cue learning in the Morris water maze, and assessment of simple associative learning in an olfactory discrimination task. Homozygous mice showed broad-based impairments that were characterized by deficiencies in initiating movement and/or reactivity to external stimuli. Data obtained from flash evoked potentials indicated that these deficits did not reflect gross visual impairments. The partial reduction in D1A receptors in the heterozygous mice did not affect performance in most tasks, although circumscribed deficits in some tasks were observed (e.g., failure to develop a reliable spatial bias in the water maze). These findings extend previous behavioural studies of null mutant mice lacking D1A receptors and provide additional support for the idea that the D1A receptor participates in a wide variety of behavioural functions. The selective impairments of heterozygous mice in a spatial learning task suggest that the hippocampal/cortical dopaminergic system may be uniquely vulnerable to the partial loss of the D1A receptor.

Robustness of G protein changes in cocaine sensitization shown with immunoblotting.

Daily cocaine administration has been shown to alter G proteins in mesolimbic nuclei, and these changes have been implicated in the initiation and expression of behavioral sensitization. To evaluate the robustness of changes in G proteins induced by daily cocaine treatments capable of producing behavioral sensitization, the levels of Gi1 alpha, Gi2 alpha, Go alpha, Gs alpha, and G beta protein were measured by immunoblotting at 1 hr after an acute injection of cocaine or saline given 1 or 14 days following the last injection of daily cocaine or saline. A significant decline in Gi1 alpha was seen in the nucleus accumbens at 14 days following daily cocaine administration regardless of whether they received an acute challenge with cocaine or saline 1 hr prior to decapitation. No alterations were observed in the ventral tegmental area, substantia nigra, dorsolateral striatum, or prefrontal cortex in the levels of Gi1 alpha, Gi2 alpha, or Go alpha. No change in G protein immunoreactivity was measured in the nucleus accumbens or ventral tegmental area of rats decapitated 1 hr after discontinuing daily cocaine. The possibility that a long-term change in Gi1 alpha in the nucleus accumbens may be related to cocaine-induced behavioral sensitization is discussed.

Correlation between behavioral sensitization to cocaine and G protein ADP-ribosylation in the ventral tegmental area.

The ventral tegmental area is a site of action by psychostimulants in the production of behavioral sensitization. Recently, G proteins of the ventral tegmental area have been implicated in behavioral sensitization to cocaine. To further investigate the specific role of G proteins, rats were treated with either 15 or 30 mg/kg, i.p., of cocaine (x 5 days), and at 1, 6 or 24 h after the last injection in vitro pertussis toxin catalyzed adenosine diphosphate (ADP)-ribosylation was used to measure the G proteins in the ventral tegmental area, nucleus accumbens, prefrontal cortex, substantia nigra, and striatum. A significant decline in the ADP-ribosylation of G proteins, specific for the ventral tegmental area, was observed at 1 and/or 6 h but had returned to normal by 24 h. A significant negative correlation was found between the percent of G proteins ADP-ribosylated in the ventral tegmental area and the behavioral activity elicited in sensitized but not acute cocaine-treated animals at 1 h after injection. These data suggest that the G proteins ADP-ribosylated by pertussis toxin may be involved in the sensitized motor response produced by repeated cocaine administration in rats.

Pertussis toxin in the A10 region increases dopamine synthesis and metabolism.

Inhibitory regulation of dopamine neurons is mediated by dopamine autoreceptor and gamma-aminobutyric acidB receptor opening of potassium channels. Increased potassium conductance by either receptor is G protein dependent. To evaluate the role of G proteins in vivo, pertussis toxin (PTX) was microinjected into the A10 dopamine region and changes in dopamine metabolism and synthesis measured. PTX produced an elevation in dopamine metabolism and synthesis in the A10 region and nucleus accumbens for up to 4 days after injection. By day 7 the levels of the dopamine precursor and metabolites had returned to normal. A less consistent increase was also measured in the A9 dopamine region and the prefrontal cortex. Although dopamine synthesis and metabolism had returned to normal by day 7, the in vitro ADP-ribosylation of G proteins in the A10 region by PTX remained depressed by approximately 50% from day 1 to day 14 after administration, returning to normal by day 30. The data suggest that in vivo ribosylation of G proteins may lead to a short-term attenuation of the tonic inhibitory control of dopamine neurons, which can be compensated for by PTX-insensitive mechanisms.

Possible role for G-proteins in behavioral sensitization to cocaine.

The role of G-proteins in behavioral sensitization to cocaine was examined by injecting pertussis toxin (PTX) into the A10 dopamine cell group. The capacity of acute cocaine to increase motor activity and dopamine release in the nucleus accumbens was significantly augmented in rats pretreated 14 days earlier with PTX. These data suggest that injection of PTX into the A10 dopamine cell group produces a long-term alteration in mesolimbic dopamine function, and implicates A10 dopamine neurons and G-proteins in the development of behavioral sensitization.