Amphetamine alters Ras-guanine nucleotide-releasing factor expression in the rat striatum in vivo

Abstract

Ras-guanine nucleotide-releasing factors (Ras-GRFs) are densely expressed in neurons of the mammalian brain. As a Ras-specific activator predominantly concentrated at synaptic sites, Ras-GRFs activate the Ras-mitogen-activated protein kinase (Ras-MAPK) cascade in response to changing synaptic inputs, thereby modifying a variety of cellular and synaptic activities. While the Ras-MAPK cascade in the limbic reward circuit is well-known to be sensitive to dopamine inputs, the sensitivity of its upstream activator (Ras-GRFs) to dopamine remains to be investigated. In this study, the response of Ras-GRFs in their protein expression to dopamine stimulation was evaluated in the rat striatum in vivo. A single systemic injection of the psychostimulant amphetamine produced an increase in Ras-GRF1 protein levels in both the dorsal (caudoputamen) and ventral (nucleus accumbens) striatum. The increase in Ras-GRF1 proteins was dose-dependent. The reliable increase was seen 2.5 h after drug injection and returned to normal levels by 6 h. In contrast to Ras-GRF1, protein levels of Ras-GRF2 in the striatum were not altered by amphetamine. In addition to the striatum, the medial prefrontal cortex is another forebrain site where amphetamine induced a parallel increase in Ras-GRF1 but not Ras-GRF2. No significant change in Ras-GRF1/2 proteins was observed in the hippocampus. These data demonstrate that Ras-GRF1 is a susceptible and selective target of amphetamine in striatal and cortical neurons. Its protein expression is subject to the modulation by acute exposure of amphetamine.

Introduction

Mitogen-activated protein kinases (MAPKs) refer to a large family of serine/threonine protein kinases that function as a key signaling cascade to mediate cellular growth and differentiation in mammalian proliferative cells (Volmat and Pouyssequr, 2001). In postmitotic neurons of adult mammalian brain, MAPKs are also densely expressed. They are highly sensitive to diverse synaptic signals, and are vigorously involved in the transcription-dependent regulation of synaptic plasticity (Wang et al., 2007). The typical MAPK cascade involves a consecutive and sequential activation of four levels of signaling proteins: small GTPases (such as Ras), MAPK kinase kinases (Raf or MEKKs), MAPK kinases (MEKs), and MAPKs. The initial Ras proteins localize to the inner surface of the plasma membrane. They are activated when converted from the GDP-bound to the GTP-bound state. This conversion is catalyzed by a class of Ras-guanine nucleotide exchange factors (Ras-GEFs), including Ras-guanine nucleotide-releasing factors (Ras-GRFs). Ras-GRFs are expressed in neurons, but not glial cells, in the central nervous system of adult animals (Zippel et al., 1997). The two subtypes of Ras-GRFs, Ras-GRF1/CDC25^Mm and Ras-GRF2 (Cen et al., 1992, Martegani et al., 1992, Shou et al., 1992), have been found to be predominantly enriched at synaptic sites (Sturani et al., 1997). These synaptic Ras-GRFs are sensitive to cytosolic Ca²⁺ signals. Through Ca²⁺/calmodulin binding to their N-terminal IQ motifs (Shou et al., 1992, Farnsworth et al., 1995, Fam et al., 1997), Ca²⁺ signals, derived from Ca²⁺ influx through either ligand or voltage-operated Ca²⁺ channels including NMDA receptors or from intracellular Ca²⁺ release following G-protein-coupled receptor activation, activate Ras-GRFs (Farnsworth et al., 1995). Active Ras-GRFs subsequently promote the activation of their specific effector Ras, which leads to the activation of the central signaling cascade, i.e., the MAPK cascade. Through regulating the strength and efficacy of excitatory synapses, activated MAPKs are involved in normal neural activities and the development of various enduring neuropsychiatric illnesses (reviewed in Sweatt, 2004, Thomas and Huganir, 2004, Wang et al., 2007).

A number of recent studies have demonstrated that dopamine stimulation with psychostimulants activates MAPKs in the forebrain in vivo. Acute injection of the psychostimulant cocaine increased phosphorylation of extracellular signal-regulated kinases (ERKs), a subclass of MAPKs, in the striatum (Valjent et al., 2000, Valjent et al., 2005, Valjent et al., 2006, Zhang et al., 2004, Jenab et al., 2005). Acute injection of the psychostimulant amphetamine also increased ERK phosphorylation in the striatum (Choe et al., 2002, Choe and Wang, 2002, Valjent et al., 2004, Valjent et al., 2005, Valjent et al., 2006). The amphetamine-stimulated ERK phosphorylation requires the activation of group I metabotropic glutamate receptors and Ca²⁺/calmodulin-dependent protein kinases (CaMKs) since the inhibitors selective for these receptors or CaMKs blocked ERK responses to amphetamine (Choe et al., 2002, Choe and Wang, 2002). Together, these data indicate that the Ras-GRF-dependent MAPK cascade in striatal neurons is sensitive to psychostimulants. The drug-regulated MAPK activity likely plays a critical role in plastic changes in the limbic reward circuit essential for the addictive properties of drugs of abuse (Valjent et al., 2000, Wang et al., 2007). However, to date, no attempt has been made to unravel the influence of amphetamine over Ras-GRF expression in the forebrain.

This study was then designed to investigate the possible regulation of Ras-GRF expression in striatal neurons by dopamine inputs in vivo. A single systemic injection of amphetamine was given to adult rats. Alterations in basal levels of both Ras-GRF1 and Ras-GRF2 protein abundance in the striatum, including both the dorsal striatum/caudate putamen (CPu) and the ventral striatum/nucleus accumbens (NAc), and other forebrain structures were examined after drug injection.