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Previous Article | Next Article
Volume 16, Number 15, Issue of August 1, 1996 pp. 4707-4715
Copyright ©1996 Society for NeuroscienceRegulation of ERK (
xtracellular Signal
egulated
inase), Part of the Neurotrophin Signal Transduction Cascade, in the Rat Mesolimbic Dopamine System by Chronic Exposure to Morphine or Cocaine
Melissa T. Berhow, Noboru Hiroi, and Eric J. Nestler Laboratory of Molecular Psychiatry, Departments of Psychiatry and Pharmacology, Yale University School of Medicine, Connecticut Mental Health Center, New Haven, Connecticut 06508
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 demonstrated 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.
Key words: morphine; cocaine; antisense oligonucleotides; ERK; ventral tegmental area; tyrosine hydroxylase; BDNF; NMDA glutamate receptors
INTRODUCTION
The mesolimbic dopamine system, which comprises dopaminergic neurons in the ventral tegmental area (VTA) and their projections to the nucleus accumbens (NAc) and other forebrain structures, is implicated in the rewarding properties of several drugs of abuse (Bozarth and Wise, 1986; Kuhar et al., 1991
One of the most consistent adaptations elicited by several drugs of abuse, including morphine, cocaine, amphetamine, and ethanol, is induction of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, in the VTA (Beitner-Johnson and Nestler, 1991
The neurotrophin signal transduction cascade involves activation of a series of protein kinases, which leads ultimately to a myriad of effects on cell function (Davis, 1993
AP-kinase and
RK
inase). Various forms of MEK then phosphorylate two members of the MAP kinase family: extracellular signal-regulated kinases (ERKs) and Jun kinase (JNK). ERKs are a family of protein serine/threonine kinases of which the best characterized members are ERK1 (p44) and ERK2 (p42) (Robbins et al., 1993
The objective of the present study was to investigate possible cross-talk between the neurotrophin signal transduction cascade and the biochemical actions of drugs of abuse after chronic exposure. ERK was selected as the primary focus of these studies based on its central role in neurotrophin signaling cascades, the established ability of these cascades to regulate TH expression in cultured cells (Gizang-Ginsberg and Ziff, 1990
MATERIALS AND METHODS
Neurotrophic factor infusions and drug treatments. Male Sprague-Dawley rats (initial weight 260-275 gm) (CAMM, Wayne, NJ) were used in these studies. Neurotrophic factor infusions involved implantation of osmotic minipumps (Alzet Model 2002) that provide a constant infusion of 0.5 µl/hr for 14 d. BDNF and ciliary neurotrophic factor (CNTF), human recombinant growth factors expressed in E. Coli, were provided by Regeneron Pharmaceuticals (Tarrytown, NY). The growth factors were delivered in a solution containing 10 mM sodium phosphate, pH 7.4, 0.9% NaCl, and 1% bovine serum albumin. The doses of BDNF (2.5 µg/d) and CNTF (1.5 µg/d) were based on previous research (Berhow et al., 19955.3 mm anterior-posterior and 8.4 mm dorsal-ventral were used. The osmotic pump was placed subcutaneously between the scapulae and connected to the cannula via PE60 tubing cut to 2.5 cm in length. Each end was sealed with LocTite glue. The cannula was secured in place with dental cement. Control rats were implanted with osmotic pumps containing vehicle solution.
Acute neurotrophin administration involved similar preoperative techniques as described above. The tip of a Hamilton syringe needle (25 gauge) containing vehicle or BDNF solution was lowered to
Chronic morphine was administered via implantation of one morphine pellet (containing 75 mg of morphine base) [National Institute on Drug Abuse (NIDA)] subcutaneously while rats were under light halothane anesthesia daily for 5 d. Animals were killed on the morning of day 6 via decapitation, and tissue samples were obtained. Control rats underwent sham surgery or no treatment; the two controls yielded equivalent results. Acute morphine was administered 2 hr before the animal was killed. The one-time intraperitoneal injection contained 20 mg/kg morphine sulfate (NIDA) prepared in a saline solution. Concomitant morphine/naltrexone treatments involved administering naltrexone before each morphine implantation as described by Guitart and Nestler (1989)
Immunolabeling of proteins. Brains were removed from decapitated rats and cooled in ice-cold physiological buffer. The VTA, substantia nigra, NAc, and frontal cortex were obtained as 12-15 gauge punches of coronal cross-sections of brain as described previously (for review, see Beitner-Johnson et al., 1992
(PLC-
Proteins were then immunolabeled with the following antibodies: anti-TH (diluted 1:5000, John Haycock, Louisiana State University), ERK (diluted 1:5000, Transduction Laboratories, Lexington, KY; diluted 1:2000, Santa Cruz), phospho-ERK (diluted 1:500, New England Biolabs, Beverly, MA), anti-GFAP and anti-NF200 (diluted 1:10000, Sigma), anti-NF160 and anti-NF68 (diluted 1:5000, Sigma), anti-JAK2, anti-PLC-
ERK activity assay. Samples were prepared as described above with 50 µg of protein loaded per lane for VTA, NAc, substantia nigra, and frontal cortex. Proteins were resolved by SDS/polyacrylamide gel electrophoresis on 8% resolving gels containing 0.1 mg/ml of myelin basic protein (MBP), added before separation. Protein kinases in the gels were assayed by the method of Kameshita and Fujisawa (1989) and modified by Ortiz et al. (1995b)
2 test.
In vivo antisense oligonucleotide infusions. Oligonucleotides were infused via osmotic minipumps as described above for BDNF. Oligonucleotides were obtained from Midland Certified Reagent Company (Midland, TX). All sequences were partially modified; that is, modified with phosphorothioate moieties on the 5
and 3
In most studies, animals received intra-VTA infusions of antisense, sense, or scrambled oligonucleotides or vehicle solutions for a 7 d period, at which time the animals were killed. In some cases, on the seventh day of oligonucleotide infusion, animals were anesthetized and the osmotic pump containing the antisense oligonucleotide was replaced with a pump containing vehicle solution. After an additional 5 d, animals were killed and brains removed. In some experiments, animals were treated concomitantly with chronic morphine, as described above, with morphine pellet implantations beginning on day 3 of oligonucleotide infusion. To confirm cannula placement, as well as lack of damage after the antisense oligonucleotide infusions, Nissl staining was performed on a set of animals (see below).
Infusion of NMDA receptor antagonists. NMDA receptor antagonists were infused into the VTA as described above for BDNF. D-AP-5, a potent and selective NMDA receptor antagonist, was obtained from RBI (Natick, MA). L-AP-5, which exhibits close to 100-fold lower potency as an NMDA antagonist (Olverman et al., 1988
Histological analysis. Rats that received chronic intra-VTA infusions of ERK sense or antisense oligonucleotide were anesthetized with 120 mg/kg sodium pentobarbital and perfused with 0.9% saline followed by 4% paraformaldehyde. Brains were removed and kept for 1-2 hr in 4% paraformaldehyde and then in 20% glycerol overnight. Sections were cut at 30 µm. We used Nissl staining and TH immunohistochemistry to study the effect of chronic oligonucleotide infusions on the cytoarchitecture of the midbrain. For Nissl staining, a standard protocol with 0.25% cresyl violet was used. For TH immunohistochemistry, sections were treated with 5% H2O2/5% normal goat serum and incubated in PBS containing a rabbit polyclonal anti-TH antibody (1:2000) (Eugene Tech, Allendale, NJ) overnight at 4°C. Sections were then incubated for 1 hr in biotinylated goat anti-rabbit IgG (1:500) (Vector Labs) and for 1 hr in avidin-biotin complex (Vector Labs). Immunoreactivity was detected with DAB. The histological analyses were performed ``blind'' with respect to treatment conditions.
RESULTS
Regulation of ERK activity by morphine, cocaine, and BDNF treatments in the VTA
An in-gel assay of ERK catalytic activity was used to study the effect of morphine and cocaine on the enzyme in the VTA. In this assay, MBP is phosphorylated in the presence of [
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
[View Larger Version of this Image (27K GIF file)]
Animals treated with chronic morphine or cocaine demonstrated a 54 and 37% increase in ERK activity, respectively, compared with control (Figs. 1A, 2). This increase was seen only in the VTA and not in other brain regions examined, which included the substantia nigra, frontal cortex, and NAc (Fig. 2). In contrast, chronic morphine and cocaine treatments did not affect the activity of other protein kinases detected by this in-gel assay (see Fig. 2).
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
[View Larger Version of this Image (30K GIF file)]
Acute intra-VTA infusions of BDNF, as expected, also produced a significant increase in ERK activity (153 ± 10% of control, n = 4, p < 0.05) (Fig. 1B). In contrast, chronic infusion of BDNF into the VTA did not result in any change in ERK activity in this brain region (105 ± 9% of control, n = 5) (Fig. 1A). Interestingly, though, when morphine was administered concomitantly with chronic intra-VTA BDNF infusions, the expected increase in VTA ERK activity associated with morphine did not occur (99 ± 15% of control, n = 4).
To ascertain whether drug regulation of ERK activity in the VTA required chronic drug exposure, acute morphine and cocaine studies were undertaken. There was no increase in ERK activity in the VTA in response to a single acute exposure to morphine (112 ± 15% of control, n = 4) or cocaine (105 ± 7% of control, n = 4). Additionally, concomitant administration of naltrexone, an opioid receptor antagonist, along with morphine, under conditions that block morphine tolerance and dependence (see Materials and Methods), prevented the chronic morphine-induced increase in ERK activity (101 ± 5% of control, n = 4); this treatment also prevented the characteristic morphine-induced increase in TH (data not shown).
Regulation of ERK immunoreactivity and phosphorylation state by morphine, cocaine, and BDNF treatments in the VTA
The morphine- and cocaine-induced increases in ERK activity in the VTA could be attributable to increases in levels of ERK protein, increases in the phosphorylation state of ERK, or a combination of the two. To study these possibilities, we measured levels of total ERK and phospho-ERK immunoreactivity by immunoblotting. We did not detect any difference in total ERK immunoreactivity after chronic morphine or chronic cocaine treatment (see Table 1, Figs. 1A, 3). This lack of effect of morphine on total ERK immunoreactivity in the VTA is consistent with our previous findings (Ortiz et al., 1995b
Table 1. Effect of ERK1 antisense and sense oligonucleotide infusions (10 µg/d) on levels of ERK1, ERK2, and TH immunoactivity, and of ERK activity, with and without concomitant chronic morphine treatment
Morphine ERK1 immunoreactivity ERK2 immunoreactivity ERK activity TH immunoreactivity Vehicle 100 ± 9 (15) 100 ± 11 (15) 100 ± 8 (10) 100 ± 7 (7) + 106 ± 14 (6) 95 ± 11 (6) 154 ± 12* (10) 158*a (8) Sense (10 µg/d) 96 ± 14 (6) 109 ± 12 (6) n.a. 106 ± 10 (6) + 95 ± 6 (5) 92 ± 8 (5) n.a. 130 ± 11* (6) Antisense (10 µg/d) 65 ± 16* (15) 93 ± 13 (15) 96 ± 6 (9) 98 ± 10 (6) + 59 ± 18* (6) 104 ± 8 (6) 98 ± 9 (9) 103 ± 8 (5) a Originally published in Berhow et al. (1995) *p < 0.05 versus vehicle ( 
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
[View Larger Version of this Image (28K GIF file)]
In contrast to morphine and cocaine, chronic BDNF infusions significantly decreased ERK immunoreactivity in the VTA (78 ± 11 of control, n = 8, p < 0.05) (Fig. 1A). The finding of a decrease in total ERK protein with no change in ERK activity suggests that a higher fraction of the ERK protein present is phosphorylated after chronic BDNF infusions. Intra-VTA infusion of CNTF, a member of a distinct neurotrophic factor family that acts via different signaling pathways and that does not attenuate morphine and cocaine actions in the VTA (Berhow et al., 1995
Effect of ERK1 antisense oligonucleotides on ERK immunoreactivity in the VTA
To study the functional significance of the regulation of ERK activity by chronic morphine and cocaine treatments, we used antisense oligonucleotides to ERK1 to directly alter ERK levels in the VTA. The targeting of ERK1, as opposed to ERK2, is based on the finding that ERK1 is the more abundant form of the enzyme in the VTA (Ortiz et al., 1995bERK1 antisense oligonucleotides were infused into the VTA for 7 days at a dose of 5, 10, or 20 µg/d. Levels of ERK1 immunoreactivity were significantly reduced at both the 10 and 20 µg/d doses but not at the 5 µg/d dose (Fig. 4). To assess the degree of diffusion of the antisense oligonucleotides, we analyzed the substantia nigra, a region that is in close anatomical proximity to the VTA. After a chronic intra-VTA infusion of 10 µg/d of ERK1 antisense oligonucleotides, there was no change in levels of ERK1 immunoreactivity in the substantia nigra (89 ± 9% of control). The decrease in ERK1 immunoreactivity in the VTA after infusion of ERK1 antisense oligonucleotides at a 10 µg/d dose was not apparent after infusion of sense or scrambled oligonucleotides (Fig. 4).
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
[View Larger Version of this Image (20K GIF file)]
To assess further the specificity of the ERK1 antisense oligonucleotide effects, several other proteins were examined. ERK2 has the overall closest homology to ERK1 in terms of nucleotide sequence. The region of ERK1 selected for the antisense probe has the least homology (<59%) to ERK2 of any region that also includes the AUG translation start site. There was no change in ERK2 immunoreactivity at any of the doses of ERK1 antisense used (Fig. 4, inset, Table 1). Levels in the VTA of other signaling proteins (phosphotidylinositol 3-kinase, phospholipase C-
Table 2. Proteins unchanged by ERK1 antisense oligonucleotide intra-VTA infusions
% of sham GFAP 102 ± 12 NF 68 92 ± 11 NF 160 112 ± 9 NF 200 106 ± 8 PI3-K 94 ± 11 PLC- 103 ± 8 JAK2 110 ± 12 Lack of effect of ERK1 antisense oligonucleotide infusions (10 µg/d) on cellular signaling proteins. Proteins analyzed include glial fibrillary acidic protein (GFAP); neurofilament proteins (NF 68, 160, and 200); phosphatidylinositol 3-kinase (PI3-K); phospholipase C- To provide still further support for the interpretation that the antisense oligonucleotide-induced decrease in ERK1 is attributable to a selective action on ERK1 and not to generalized neurotoxicity, we examined the reversibility of this effect as well as the histological integrity of the VTA. To study the reversibility of the antisense oligonucleotide effects, osmotic minipumps containing the oligonucleotides were replaced with pumps containing vehicle solution for an additional 5 d (for review, see Widnell et al., 1996
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.
[View Larger Version of this Image (86K GIF file)]
Effect of ERK1 antisense oligonucleotides on morphine regulation of ERK activity and TH immunoreactivity in the VTA
Next, we combined the intra-VTA ERK1 antisense oligonucleotide infusions with systemic chronic morphine treatments. In this paradigm, animals received 7 d of antisense oligonucleotide infusions (10 µg/d) with morphine treatments beginning on day 3 and extending until day 7, at which time the animals were used. There was no difference in ERK immunoreactivity between the antisense oligonucleotide-treated animals that received morphine and those that had not; both showed a significant decrease in total ERK1 immunoreactivity relative to controls (Table 1). In the same VTA samples, however, antisense oligonucleotide infusions alone did not alter levels of ERK activity, but completely blocked the ability of morphine to increase ERK activity. The finding that ERK antisense oligonucleotide infusions did not alter ERK activity even though they reduced total ERK immunoreactivity suggests that, like chronic BDNF infusions, ERK antisense oligonucleotide infusions increased the fraction of the remaining ERK molecules that are in the phosphorylated-activated state (see Discussion).Regulation of TH immunoreactivity generally paralleled that of ERK activity (Table 1). Animals that received the antisense oligonucleotide infusions alone showed no difference in levels of TH immunoreactivity in the VTA compared with control. Animals that were treated concomitantly with the antisense oligonucleotides plus morphine also showed no significant change in TH levels. Thus, the ERK1 antisense oligonucleotide infusions blocked the ability of morphine to increase TH expression.
Effect of NMDA glutamate receptor antagonists on morphine regulation of ERK activity and TH immunoreactivity in the VTA
As a first step in studying the mechanism by which chronic morphine administration might result in a sustained increase in ERK activity, we tested the effect of intra-VTA infusions of NMDA glutamate receptor antagonists. This is based on the reported ability of such antagonists to oppose many of the effects of morphine and other drugs of abuse on mesolimbic dopamine function and on recent evidence that glutamatergic function is increased in the VTA under chronic drug-treated conditions (see Discussion). As shown in Table 3, chronic infusion of D-AP-5, a specific and potent antagonist of NMDA receptors, into the VTA blocked the ability of morphine to increase ERK activity in this brain region. D-AP-5 infusions alone failed to alter ERK activity. Intra-VTA infusion of D-AP-7, another potent NMDA receptor antagonist, also prevented the morphine-induced increase in ERK activity, whereas infusion of the L-stereoisomer of AP-5, which exhibits close to a 100-fold lower potency as an NMDA receptor antagonist compared with D-AP-5, failed to produce this effect.
Table 3. Effect of glutamate receptor antagonist infusions on levels of ERK activity, ERK immunoreactivity, and TH immunoreactivity, with and without concomitant chronic morphine treatment
Morphine ERK activity ERK immunoreactivity TH immunoreactivity Vehiclea 100 100 100 + 154 101 158 D-AP-5 109 ± 13 97 ± 9 98 ± 6 + 114 ± 16 102 ± 8 97 ± 8 L-AP-5 + 142 ± 17 104 ± 11 137 ±12 D-AP-7 + 108 ± 9 96 ± 13 104 ± 14 a Data from Table 1. *p < 0.05 versus vehicle ( These NMDA receptor antagonists produced parallel effects on TH expression (Table 3). Intra-VTA infusions of D-AP-5 or D-AP-7 prevented the morphine-induced increase in TH levels in this brain region, without altering TH levels when given alone. In contrast, infusion of L-AP-5 failed to influence morphine regulation of TH levels.
DISCUSSION
Previous research has shown a pharmacological interaction between neurotrophins and the biochemical adaptations in the VTA that are seen with chronic morphine and cocaine exposure. Specifically, intra-VTA infusions of BDNF or NT4 have been shown to prevent as well as reverse the morphine- and cocaine-induced increase in TH and certain other biochemical adaptations in this brain region (Berhow et al., 1995We also found that acute injection of BDNF into the VTA increased levels of ERK activity, without a change in total ERK levels, in this brain region. This increase in ERK activity would be expected based on the known ability of BDNF and other neurotrophins to activate the ERK signaling cascade in cultured cells (for review, see Davis, 1993
Together, these findings support a hypothesis whereby chronic BDNF infusions attenuate morphine and cocaine regulation of TH and other biochemical endpoints in the VTA by reducing the adaptive capacity of the VTA to respond to drug exposure with increased levels of ERK activity. That is, BDNF, by leading to lower levels of more highly phosphorylated-activated ERK, prevents the ability of morphine or cocaine to further activate the enzyme by phosphorylation. An attractive feature of this model is that it can account for the lack of effect of BDNF infusions alone, as well as the ability of the neurotrophin to completely obliterate morphine and cocaine regulation of TH and other target proteins.
This hypothesis was tested by use of antisense oligonucleotides directed against ERK1 to directly reduce ERK levels selectively in the VTA in vivo. The use of antisense oligonucleotides was necessitated by the lack of specific ERK inhibitors. We found that the infusion of partially phosphorothioate-modified ERK1 antisense oligonucleotides into the VTA resulted in a selective and dose-dependent reduction in ERK1 levels in this brain region. Although the use of intracerebrally administered antisense oligonucleotides should be viewed with caution, the specificity of the ERK1 antisense oligonucleotides was demonstrated by several control experiments (for review, see Stein and Cheng, 1994; Widnell et al., 1996
Chronic intra-VTA infusion of ERK1 antisense oligonucleotides mimicked the effects of chronic intra-VTA infusion of BDNF in several ways. The antisense oligonucleotides decreased levels of total ERK1, with no net effect on ERK activity. It would appear that a larger percentage of the remaining ERK molecules are phosphorylated to compensate for the oligonucleotide-induced decrease in total ERK levels. Interestingly, ERK1 antisense oligonucleotide infusions also blocked the ability of morphine to increase ERK activity and to increase TH levels in the VTA. These data support the hypothesis stated above that morphine regulation of TH may be mediated in part by drug regulation of ERK activity, and that prolonged BDNF treatment prevents these effects by reducing the adaptive capacity of the ERK system. More specifically, the antisense oligonucleotide experiments demonstrate that primary changes in ERK per se are sufficient to mimic BDNF interactions with morphine. Whether such changes in ERK are also necessary for these interactions will require additional investigations.
We also provide evidence in this study that chronic morphine and cocaine treatments result in a sustained increase in ERK activity via a glutamate-dependent mechanism. Intra-VTA infusion of specific NMDA glutamate receptor antagonists prevented the ability of chronic morphine to increase both ERK activity and TH expression. This finding is consistent with the reported ability of NMDA and other glutamate receptor antagonists, given systemically as well as locally within the VTA, to block many of the effects of opiates and cocaine on mesolimbic dopamine function (for review, see Karler et al., 1991
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.
[View Larger Version of this Image (17K GIF file)]
An alternative mechanism by which chronic morphine or cocaine exposure could conceivably increase ERK activity in the VTA is through regulation of other steps in the neurotrophin signal transduction cascade. However, attempts to demonstrate chronic morphine- or chronic cocaine-induced adaptations at points in these cascades proximal to ERK have to date yielded negative results. For example, preliminary studies indicate that chronic morphine or cocaine exposure does not alter levels of BDNF or TrkB (the predominant form of Trk in the VTA) in the VTA (Russell et al., 1994
Clearly, additional work is needed to study the mechanisms by which chronic morphine and cocaine exposure lead to changes in ERK activity selectively in the VTA. In addition, it will be essential in future studies to further evaluate the role played by drug regulation of ERK on TH expression and other biochemical actions of the drugs in this brain region as well as on the profound behavioral effects that these drugs elicit via the mesolimbic dopamine system. It should also be emphasized that morphine and cocaine regulation of ERK is likely just one of several mechanisms by which chronic drug exposure leads to long-term biochemical adaptations in the VTA. As just one example, we have found recently that chronic cocaine treatment increases levels of JAK2, a cytoplasmic protein tyrosine kinase regulated by CNTF, specifically in the VTA (Berhow et al., 1996
FOOTNOTES
Received March 26, 1996; revised May 6, 1996; accepted May 9, 1996.
This work was supported by U.S. Public Health Service Grants DA07359, DA08227, DA10160, and DA00203; the Abraham Ribicoff Research Facilities; and the Yale University MD/PhD Program. We thank John Haycock, Ronald Duman, Moses Chao, Melanie Cobb, David Russell, and Ronald Lindsay for their input regarding these studies and manuscript preparation.
Correspondence should be addressed to Dr. Eric J. Nestler, Laboratory of Molecular Psychiatry, Departments of Psychiatry and Pharmacology, Yale University School of Medicine, Connecticut Mental Health Center, 34 Park Street, New Haven, CT 06508.
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