Agonist-dependent internalization of D2 receptors: imaging quantification by confocal microscopy

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

Agonist-dependent internalization of D2 receptors: Imaging quantification by confocal microscopy

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

Rat brain serotonin neurones that express neuronal nitric oxide synthase have increased sensitivity to the substituted amphetamine serotonin toxins 3,4-methylenedioxymethamphetamine and p-chloroamphetamine

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphe nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30 mg/kg) or p-chloroamphetamine (2 x 10 mg/kg) was administered to Sprague-Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.