Regulation of ERK (extracellular signal regulated kinase), part of the neurotrophin signal transduction cascade, in the rat mesolimbic dopamine system by chronic exposure to morphine or cocaine

Abstract

Local infusion of brain-derived neurotrophic factor (BDNF) into the ventral tegmental area (VTA) can prevent and reverse the ability of chronic morphine or cocaine exposure to induce tyrosine hydroxylase (TH) in this brain region. The present study examined a possible role for extracellular signal regulated kinases (ERKs), the major effector for BDNF and related neurotrophins, in morphine and cocaine action in the VTA. Chronic, but not acute, administration of morphine or cocaine increased ERK catalytic activity specifically in the VTA. This increase in ERK activity reflected an increase in the state of phosphorylation of ERK, with no change in levels of total ERK immunoreactivity. Chronic infusions of BDNF into the VTA reduced total ERK immunoreactivity with no change in ERK activity, and also blocked the morphine-induced increase in ERK activity. These results suggest that chronic BDNF elicits a compensatory increase in the phosphorylation of the remaining ERK molecules and thereby prevents any additional increase in response to drug exposure. Such a role for ERK in morphine action was demnostrated directly by chronically infusing antisense oligonucleotides to ERK1 into the VTA. This treatment selectively reduced levels of ERK1 immunoreactivity in a sequence-specific manner without detectable toxicity. Intra-VTA infusion of ERK1 antisense oligonucleotides mimicked the effects of chronic BDNF infusions on ERK immunoreactivity, ERK activity, and TH immunoreactivity in the VTA under both control and morphine-treated conditions. The chronic morphine-induced increases in ERK activity and TH expression in the VTA also were blocked by local infusion of NMDA glutamate receptor antagonists, suggesting a role for glutamate in mediating these drug effects. Together, these findings support a scheme whereby chronic, systemic administration of morphine or cocaine leads to a sustained increase in ERK phosphorylation state and activity in the VTA, which, in turn, contributes to drug-induced increases in TH, and perhaps other drug-induced adaptations, elicited selectively in this brain region.

Fig. 1.

A, Effect of chronic morphine treatment and chronic intra-VTA BDNF infusions on ERK immunoreactivity and ERK activity in the VTA. B, Effect of acute intra-VTA BDNF infusions on ERK activity in the VTA. After the various treatments, VTA extracts were analyzed for total ERK immunoreactivity by immunoblotting and for ERK activity measured with an in-gel ERK activity assay. Note that the relative levels of ERK1 and ERK2 shown do not represent an accurate measure of the absolute amounts of these proteins present in the VTA, because the antibody used (Santa Cruz) (see Materials and Methods) shows greater relative reactivity for ERK2 than ERK1. Indeed, ERK1 is the predominant form of the enzyme present in the VTA (for review, see Ortiz et al., 1995b).

Fig. 2.

Effect of chronic morphine and cocaine treatments on ERK activity in selected brain regions. A, Graph quantifying effect of chronic morphine (M) and cocaine (C) treatments on ERK activity as measured with an in-gel ERK activity assay. Regions analyzed included VTA, substantia nigra (SN), frontal cortex (FC), and nucleus accumbens (NAc). Data are expressed as mean ± SEM (n = 8 in each treatment group) (*p < 0.05 vs sham by χ² test). Inset, Graph illustrating linearity of ERK activity with sample protein content.B, Representative autoradiograms illustrating regulation of ERK (44 kDa) activity by chronic morphine treatment, but not of other unidentified bands of different molecular weights from the same gel.

Fig. 3.

Effect of chronic morphine treatment on total ERK immunoreactivity and phospho-ERK immunoreactivity in the VTA.A, Graph quantifying effect of chronic morphine treatment on total ERK immunoreactivity (ERK) and phospho-ERK immunoreactivity (P-ERK). Data are expressed as mean ± SEM (*p < 0.05 vs sham by χ² test). The number of animals used for total ERK immunoreactivity was 15 and for phospho-ERK immunoreactivity was 6. B, Representative autoradiograms of ERK and P-ERK immunoblots are shown. The specificity of the anti-P-ERK antibody for phospho-ERK was demonstrated by analysis of purified dephospho- and phospho-ERK (data not shown).

Fig. 4.

Effect of intra-VTA infusions of ERK1 antisense oligonucleotides on ERK1 immunoreactivity in the VTA. Represented are infusions of vehicle, 10 μg/d sense oligonucleotides, 10 μg/d scrambled oligonucleotides, and 5, 10, and 20 μg/d antisense (AS) oligonucleotide as well as 10 μg/d antisense infusions followed by 5 d of vehicle infusions (reversal). Data are expressed as mean ± SEM (*p < 0.05 vs vehicle by χ² test). The number of animals used was 6 (vehicle), 7 (sense), 8 (scrambled), 7 (AS-5), 15 (AS-10), 7 (AS-20), and 8 (reversal). Inset, Representative autoradiogram of ERK immunoreactivity without (−) and with (+) ERK1 antisense oligonucleotide (10 μg/d) infusion. Note that the ratio of ERK1 to ERK2 is much greater than that shown in Figures 1 and 3. This is because the anti-ERK antibody used in this experiment (from Transduction Labs) exhibits greater relative reactivity for ERK1 than ERK2 compared with the antibody (from Santa Cruz) used in other experiments (for review, see Ortiz et al., 1995b).

Fig. 5.

Sections of midbrain from rats after intra-VTA infusion of ERK1 sense (A) or antisense (B) oligonucleotide analyzed by TH immunohistochemistry. Rats received intra-VTA infusions of oligonucleotides for 7 d at a rate of 10 μg/d, after which 30-μm-thick coronal sections of brain were subjected to TH immunohistochemistry as described in Materials and Methods.

Fig. 6.

Scheme illustrating a possible mechanism by which chronic morphine or cocaine exposure increases ERK activity in the VTA. The neurotrophins, e.g., BDNF, regulate neuronal function via activation of Trk receptors, which leads to the activation of Ras and a protein kinase cascade involving Raf, MEK, and ERK. Activation of ERK then leads to the direct phosphorylation of effector proteins (one example of which is TH), as well as of transcription factors and other protein kinases, which results in the regulation of many additional effector proteins. Chronic morphine and cocaine treatments have been shown to increase levels of specific glutamate receptor subunits (NMDAR1 and GluR1) selectively in the VTA. This increase could account for the increased firing rate of VTA dopamine neurons demonstrated under drug-treated conditions which, in turn, would be expected to increase intracellular Ca²⁺ levels. Increased Ca²⁺ levels would then lead to activation of the ERK cascade, as has been demonstrated in cultured cells, although the exact mechanisms remain unknown. The resulting increase in ERK activity would then result in a multitude of downstream effects, including increases in TH expression, as has also been observed in cultured cells.