The PKC inhibitor Ro31-8220 blocks acute amphetamine-induced dopamine overflow in the nucleus accumbens

Abstract

Acute administration of the psychostimulant amphetamine increases extracellular levels of dopamine (DA) by reversing the DA transporter on ascending midbrain DA neurons. In vitro studies using striatal synaptosomal, slice and nucleus accumbens (NAcc) tissue preparations have implicated protein kinase C (PKC) in this effect. The present study further examined this effect in vivo by assessing the ability of the PKC inhibitor, Ro31-8220 (10 μM), to inhibit acute amphetamine-induced DA overflow when applied with this drug to the NAcc via reverse dialysis. Amphetamine was applied at a concentration of 30 μM and the core and shell subregions of the NAcc were assayed separately in freely moving rats. These brain regions play a role in the acute locomotor-activating and motivational effects of amphetamine. Consistent with the findings of previous in vitro experiments, reverse dialysis of Ro31-8220 with amphetamine robustly attenuated the ability of this drug to increase extracellular levels of dopamine in both the core and shell subregions of the NAcc. These results confirm that amphetamine stimulates dopamine overflow via a PKC-dependent mechanism.Keywords dopamine overflow; nucleus accumbens; PKC; amphetamine; microdialysis Psychomotor stimulant drugs like amphetamine (AMPH) increase locomotion and support self-administration [29,30,31]. There is wide-spread acceptance that AMPH produces these effects through an action-potential independent mechanism by interacting with the dopamine transporter (DAT) to increase extracellular levels of dopamine (DA) in the nucleus accumbens (NAcc), the major subcortical projection field of midbrain DA neurons [18,21,26]. The outward-facing, membrane-bound form of DAT can bind AMPH, transport the drug into the nerve terminal, release the AMPH in exchange for DA, and transport the neurotransmitter out of the neuron, releasing it into the extracellular space [5,15,26]. In addition to this exchange diffusion mechanism, a number of studies have demonstrated that additional intracellular signal transduction mechanisms may also play a role in AMPH-induced DA release.There is evidence that the cytoplasmic serine/threonine protein kinase C (PKC) contributes importantly to AMPH-stimulated DA release. The PKC activator phorbol ester 12-0-*, Correspondence: Jessica Loweth, 5841 South Maryland Avenue, MC 3077, Chicago, IL 60637, TEL: (773) 702-2891, FAX: (773) 702-0857, E-MAIL: E-mail: jloweth@uchicago.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Together, the above findings indicate that PKC activity contributes to AMPH-stimulated DA re...

Phospho-specific antibodies targeting the amino terminus of the human dopamine transporter

“…Here we report the development and characterization of phospho-specific antibodies that can detect phosphorylation of hDAT at positions Ser7 and Ser12. Consistent with previous studies, our data show that Ser7 and Ser12 are major targets for PKC and CaMKII, both of which have been shown to phosphorylate the DAT N terminus (Cervinski et al, 2005; Fog et al, 2006; Foster et al, 2006; Foster et al, 2002; Gorentla et al, 2009; Granas et al, 2003; Lin et al, 2003; Moritz et al, 2013) and to play a role in DA efflux in cells and rodent brain slice (Chen et al, 2009; Gnegy, 2003; Johnson et al, 2005; Loweth et al, 2009). The precise functional relationship between the two kinases remains to be determined.…”

Cocaine-seeking is associated with PKC-dependent reduction of excitatory signaling in accumbens shell D2 dopamine receptor-expressing neurons

“…R(+)-SKF-81297 hydrobromide (1.0 μg/0.5 μl) and (−)-quinpirole hydrochloride (3.0 μg/0.5 μl) were purchased from Sigma/RBI (St. Louis, MO) and dissolved in sterile 0.9% saline. Doses for each of the aforementioned pharmacological compounds were based on the following rat intra-cranial microinjection experiments: Ro 31-8220 mesylate (Loweth et al, 2009; Schmidt et al, 2014; Schmidt et al, 2013), chelerythrine chloride (Li et al, 2011; Narita et al, 2004; Schmidt et al, 2014; Schmidt et al, 2013), R(+)-SKF-81297 hydrobromide (Schmidt et al, 2006; Schmidt and Pierce, 2006a) and (−)-quinpirole hydrochloride(Schmidt et al, 2006; Schmidt and Pierce, 2006a). While the doses of chelerythrine chloride and Ro 31-8220 have been shown to have behavioral and biochemical effects specific to PKC inhibition in the rat brain, it is possible that these compounds could be influencing activity of other signaling molecules in our experiments.…”

A Role for the PKC Signaling System in the Pathophysiology and Treatment of Mood Disorders: Involvement of a Functional Imbalance?

“…Two other PKC inhibitors, H-7 and Ro31-8220, administered respectively in the ventral tegmental area or in the nucleus accumbens, also reduced psychostimulant-induced hyperactivity [50,51]. This effect of PKC inhibitors could be, at least in part, dopaminedependent, because it is known that PKC regulates psychostimulant-mediated dopamine release probably via the regulation of dopamine transporter activity [52,53]. A recent study showing that PKCβ knockout (KO) mice exhibited impaired locomotor responsiveness to amphetamine provided evidence that the β isoform may be more specifically included in this regulation [54].…”