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Published as doi: 10.1096/fj.07-099135.
 (The FASEB Journal. 2008;22:2393-2404.)
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Dynorphin is a downstream effector of striatal BDNF regulation of ethanol intake

Marian L. Logrip*,{dagger}, Patricia H. Janak*,{dagger},{ddagger} and Dorit Ron*,{dagger},{ddagger},1

* The Gallo Research Center,

{dagger} Neuroscience Graduate Program, and

{ddagger} Department of Neurology, University of California, San Francisco, Emeryville, California, USA

1Correspondence: Ernest Gallo Research Center, 5858 Horton St. Ste. 200, Emeryville, CA 94608, USA. E-mail: dorit.ron{at}ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently identified brain-derived neurotrophic factor (BDNF) in the dorsal striatum to be a major component of a homeostatic pathway controlling ethanol consumption (1 , 2) . We hypothesized that ethanol-mediated activation of the BDNF signaling cascade is required for the ethanol-related function of the neurotrophic factor. Here, we demonstrate that exposure of striatal neurons to ethanol results in the activation of the BDNF receptor TrkB, leading to the activation of the mitogen-activated protein kinase (MAP kinase) signaling pathway and the subsequent increase in the expression of preprodynorphin (Pdyn) via BDNF. Finally, we show that activation of the dynorphin receptor, the kappa opioid receptor (KOR), is required for the BDNF-mediated decrease in ethanol intake, illustrating a function of dynorphin in BDNF’s homeostatic control of ethanol consumption. Taken together, these results demonstrate that BDNF regulates ethanol intake by initiation of MAP kinase signaling and the ensuing production of downstream gene products, including Pdyn.—Logrip, M. L., Janak, P. H., Ron, D. Dynorphin is a downstream effector of striatal BDNF regulation of ethanol intake.


Key Words: MAP kinase • alcohol • addiction • TrkB • ERK


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ALCOHOLISM IS A DEVASTATING DISEASE, with 8.5% of the American population—over 17 million adults—categorized as alcohol abusing or dependent (3) . Yet 60 to 70% of the population has admitted to consuming alcohol in self-report surveys taken annually over the past 65 years (Gallup Poll, surveys conducted annually since 1939; ref. 4 ). The discrepancy between these two statistics suggests the existence of mechanisms that curb excessive ethanol intake such that the majority of social drinkers do not develop dependence.

The neurotrophic factor BDNF is a likely candidate for inhibiting the transition to addiction, as in humans, both chromosomal markers flanking the BDNF gene (5) and specific mutations in the gene (6) have been associated with the development of addiction to multiple drugs of abuse, including alcohol (ethanol). In addition, much evidence exists suggesting a role for BDNF in regulating neuronal responses to drugs of abuse, at both the molecular and behavioral levels (7 , 8) . BDNF infusion into the ventral tegmental area (VTA) blocks the development of molecular adaptations to chronic morphine and cocaine treatment (9) , and acute administration of (1 , 10 11 12 13) or withdrawal from (14 , 15) multiple drugs of abuse has been shown to increase BDNF levels in various brain regions. Importantly, reduction in BDNF expression results in increased ethanol intake (16 17 18) . For instance, in the central and medial amygdala, reduction of BDNF by antisense oligonucleotides increases both ethanol intake and anxiety-like behaviors, indicating a possible role for amygdalar BDNF in drinking associated with anxiety relief. In addition, the same treatment with BDNF antisense that increased ethanol intake also reduced phosphorylation levels of proteins known to be downstream of BDNF signaling (18) , demonstrating that BDNF may regulate anxiety and ethanol intake via activation of a downstream signaling cascade. Recently, cocaine has also been shown to increase BDNF levels and signaling in the nucleus accumbens although, unlike ethanol, blockade of BDNF decreased cocaine self-administration (19) .

Previously, we found that BDNF in the dorsal striatum participates in a homeostatic pathway triggered in response to ethanol which, in turn, regulates behavioral responding to ethanol (1 , 2) . Specifically, we found that ethanol exposure increased BDNF expression in the dorsal striatum and that elevation of BDNF expression resulted in decreased ethanol intake (1) . In addition, mice expressing half as much BDNF (BDNF+/–) displayed increased sensitivity to ethanol in multiple paradigms, showing increased preference for an ethanol-paired environment, increased locomotor sensitization to ethanol and higher ethanol intake following a period of deprivation, as compared to wild-type (WT) mice (1) . Subsequently, we found that inhibition of BDNF signaling using the Trk inhibitor K252a increased ethanol intake, and this effect was abolished in BDNF+/– mice, in which BDNF signaling is reduced compared to WT mice due to expression of half as much BDNF protein (2) . Thus, we hypothesized that ethanol treatment activates a BDNF-mediated signaling cascade, leading to the up-regulation of downstream proteins whose activities are responsible for BDNF’s ability to control ethanol consumption.

BDNF initiates signaling by binding to its receptor, TrkB, resulting in receptor autophosphorylation and subsequent activation of several signaling cascades, including the MAP kinase cascade (20) . As BDNF-dependent activation of MAP kinase in neurons results in increased gene transcription (21) and acute administration of cocaine, morphine, or nicotine has been shown to increase striatal MAP kinase activity (22) , we hypothesized that ethanol treatment would result in increased activation of the MAP kinase pathway and subsequent up-regulation of genes encoding downstream effectors of BDNF signaling. In addition, we hypothesized that the protein products of these downstream effector genes would be responsible for decreasing ethanol intake.

Because ethanol intake selectively increases BDNF in the dorsal striatum (1) , we sought a downstream effector whose expression in the dorsal striatum could be increased in a BDNF-dependent fashion. Extended administration of BDNF into the striatum has been shown to increase the expression of Pdyn, the mRNA precursor of the dynorphin peptide (23) , and mice heterozygous for BDNF also show reduced striatal Pdyn expression (24) . Additionally, striatal MAP kinase activation is required for induction of Pdyn expression by amphetamine (25) . Interestingly, multiple drugs of abuse, including ethanol, have been reported to increase dynorphin expression in the striatum (26 27 28) . Increasing Pdyn expression in the nucleus accumbens by infusion of HSV-CREB resulted in place aversion to subthreshold doses of cocaine that was dependent on dynorphin signaling (29) . In addition, dynorphin expression is higher in the striatum of DBA/2 mice, who drink lower quantities of alcohol, than in the high-drinking C57BL/6 strain (30) . Additional evidence has implicated dynorphin and its receptor, the kappa opioid receptor (KOR) (31) , in modulation of ethanol intake, as systemic administration of U50,488H, a KOR agonist, decreased two-bottle choice ethanol intake in rats (32) , whereas systemic administration of nor-binaltorphimine (nor-BNI), a KOR antagonist, increased two-bottle-choice ethanol intake in rats (33) . Importantly, polymorphisms in dynorphin and the KOR have been associated with increased risk of alcoholism in humans (34) , indicating that dynorphin may be directly involved in protection against the development of alcohol addiction. In light of these data, we hypothesized that ethanol treatment activates BDNF signaling via the MAP kinase pathway, increasing the expression of dynorphin, whose activity would be responsible for BDNF’s ability to control ethanol consumption.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Recombinant human BDNF, TrkB-Fc, and the Fc fragment of human IgG were purchased from R&D Systems (Minneapolis, MN, USA). U0126 was purchased from Cell Signaling (Danvers, MA, USA). Primary antibodies were obtained as follows: anti-TrkB and anti-phosphotyrosine antibodies from Upstate (Temecula, CA, USA), anti-phospho-Trk and anti-phospho-ERK1/2 antibodies from Cell Signaling, anti-GAD-67, anti-BDNF, and anti-MAP2 antibodies from Chemicon (Temecula, CA, USA), and normal rabbit IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibodies for Western blot detection were purchased from Santa Cruz Biotechnology. AlexaFluor-conjugated secondary antibodies and 4',6-diamidino-2-phenylindole, dilactate (DAPI), as well as all culture media, B-27 and GlutaMAX supplements, protein G agarose beads, and Nu-PAGE gels, were supplied by Invitrogen (Carlsbad, CA, USA). VectaShield mounting medium was obtained from Vector Laboratories (Burlingame, CA, USA). Serum extender was obtained from BD Biosciences (San Jose, CA, USA). Protease and phosphatase inhibitors were obtained from Roche (Basel, Switzerland). TRIzol and the Reverse Transcription System were purchased from Promega Corp. (Madison, WI, USA). All real-time polymerase chain reaction (PCR) reagents, including TaqMan Gene Expression Assays, were obtained from Applied Biosystems, Inc. (Foster City, CA, USA). DNase and nor-binaltorphimine were obtained from Sigma Aldrich (St. Louis, MO, USA). Tat-RACK1 fusion protein was expressed in Escherichia coli and purified as described previously (35) .

Animals
C57BL/6J mice (7 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Sprague-Dawley pups for primary neuronal culture and Long-Evans rats for BDNF infusion were obtained from Harlan (Indianapolis, IN, USA). Animals were housed under a 12:12-h light-dark cycle, with lights on at 7:00 AM and lights off at 7:00 PM, and they were provided with continuous ad libitum access to food and water. All animal procedures were approved by the Gallo Center Institutional Animal Care and Use Committee and were conducted in agreement with the Guide for the Care and Use of Laboratory Animals, National Research Council (1996).

Primary striatal neuronal culture
A litter of Sprague-Dawley rats was obtained on either the day of birth or the first postnatal day (P0-P1) and were euthanized by rapid decapitation. The striata were dissected out, pooled, and digested in a papain solution for 35 min. Following inhibition of digestion, cells were mechanically dissociated using flamed glass pipettes in Minimum Essential Medium containing 22 mM D-glucose, 5% fetal bovine serum, and serum extender. Neurons were plated on poly-D-lysine-coated plates or CC2-coated chamber slides in Neurobasal-A media (NB-A) containing B-27 and GlutaMax supplements, as well as penicillin and streptomycin. Cultures were maintained for 11 days in vitro (DIV), with 50% of the media changed at 1 DIV and 7 DIV. For phosphorylation studies, media were changed to Basal Medium Eagle (BME) containing penicillin and streptomycin on DIV 10; otherwise, cells remained in NB-A and 50% of the media was changed 3 h before treatment on DIV 11. In addition, 10 µM cytosine arabinoside (AraC) was added on DIV 1 to inhibit multiplication of glial cells.

Immunoprecipitation
Following treatment, media were removed and cells were harvested in RIPA buffer containing 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, and 2 mM EDTA, as well as protease and phosphatase inhibitor cocktails. Cell lysates were precleared for 1 h with protein G agarose, and precleared lysates were then incubated overnight with either TrkB antibodies or normal rabbit IgG. The following day, protein G agarose beads were added for 2 h incubation, then beads were washed thoroughly prior to addition of sample loading buffer. Immunoprecipitates were resolved on a 4–12% gradient reducing Nu-PAGE gel and transferred onto a nitrocellulose membrane. Western blot analysis was performed with the appropriate antibodies; immunoreactivity was detected using ECL Plus (GE Bio-Sciences Healthcare, Piscataway, NJ, USA) and processed using the STORM System (Molecular Dynamics, Sunnyvale, CA, USA).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Primary striatal neurons or brain sections were homogenized in TRIzol and mRNA was isolated according to standard protocol. Samples were treated with DNase prior to reverse transcription using the Reverse Transcription System. The resulting cDNA samples were amplified by TaqMan quantitative PCR using commercially available primer/probe kits.

Acute striatal slice treatment
Sprague-Dawley rats, aged 21–28 days at the time of experimentation, were anesthetized and euthanized by rapid decapitation. The brains were rapidly removed and placed immediately in ice-cold aCSF containing (in mM): 126 NaCl, 1.2 KCl, 1.2 NaH2PO4, 0.01 MgCl2, 2.4 CaCl2, 18 NaHCO3, and 11 glucose, saturated with 95% O2-5% CO2. Coronal slices (175 µm) were cut on a vibratome (Leica, Nussloch, Germany); the dorsal striata were dissected out and transferred to rest in saturated aCSF (as above) at 25°C for 90 min before treatment. At the conclusion of treatment, slices were collected and immediately homogenized in TRIzol, followed by RNA extraction and analysis by qRT-PCR, as detailed above.

Acute infusion of BDNF for Pdyn expression
Adult Long-Evans rats (weighing 350–550 g at the time of surgery) were anesthetized using isoflurane and placed in a standard stereotaxic frame. A 10-µl Hamilton syringe was used to infuse 1 µl BDNF (0.25 µg/µl) unilaterally into the dorsolateral striatum and vehicle (PBS) into the contralateral dorsal striatum (coordinates: anterioposterior +1.20 mm, mediolateral ±3.40 mm, dorsoventral –4.20 mm, relative to bregma). The hemisphere receiving BDNF (left or right) and the order of infusion (BDNF or vehicle first) were counterbalanced across subjects. The infusion was performed over 2 min, and the syringe was left in place an additional 2 min to allow for diffusion from the infusion site. Rats were maintained under anesthesia for 3 h after the BDNF infusion and were then sacrificed. A striatal punch of the region immediately surrounding the tip of the infusion track was collected and processed for mRNA expression by qRT-PCR as above.

Immunofluorescence
Subsequent to treatment on DIV11, primary striatal neuronal cultures were washed briefly in PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. The cells were then washed twice with PBS and once for 10 min with PBS containing 0.1% Triton X-100 (PBS-Triton). Cells were blocked for a minimum of 4 h with 3% normal donkey serum in PBS-Triton. Cells were incubated in primary antibodies overnight at 4°C; primary antibodies were diluted in PBS-Triton containing 1% normal donkey serum at a dilution of 1:200 for phospho-ERK1/2 or 1:2000 for MAP2. The slides were then washed 3x in PBS-Triton, followed by incubation with AlexaFluor-conjugated secondary antibodies for 2 h in the dark. The slides were then washed in PBS twice, incubated in 300 nM DAPI for 2 min, and washed twice more in PBS. Slides were mounted in VectaShield and coverslipped prior to visualization with a laser-scanning confocal microscope (LSM 510 Meta; Carl Zeiss Microimaging, Thornwood, NY, USA).

Image analysis
Neurons were located using MAP2 staining and selected at random for imaging. Quantified images represent the 1-µm-thick plane parallel to the coverslip and midway from the top and bottom of the nucleus. Care was taken to determine the exact center of the nuclear span based on the extent of DAPI staining prior to imaging. While the experimenter was not blind to treatment at the time of imaging, the phospho-ERK1/2 channel was never observed so that selection of neurons for imaging was not biased toward the expected response. Thus, the extent of phospho-ERK1/2 fluorescence was unknown prior to capturing each neuron’s final image, and no images were discarded from quantification. In addition, all images were quantified after removal of treatment labels, and quantification was confirmed by two independent observers who were blind to treatment conditions. Images were processed using a Zeiss LSM Image Browser (Carl Zeiss Microimaging), and average fluorescence intensity over the entire span of the nucleus, as selected based on colocalization with DAPI staining, was analyzed using Adobe Photoshop (Adobe Systems, San Jose, CA, USA).

Two-bottle choice ethanol consumption
C57BL/6J mice were individually housed in double-grommet cages and given ad libitum access to food and water. At least 1 wk after arrival, mice were given 24-h access to two bottles on the home cage: one containing 10% ethanol and the other containing water. Bottles were weighed and their positions were switched every other day to prevent side bias. After a stable level of ethanol intake was obtained (minimally 6 wk from the onset of ethanol exposure), drug treatments began. On the day prior to drug treatment, all mice were injected with vehicle (50% saline/50% PBS containing 10% glycerol). The following day, mice were treated with 4 mg/kg Tat-RACK1 or vehicle in combination with either saline or 30 mg/kg nor-binaltorphimine (nor-BNI). Both treatments were administered 3.5 h prior to lights out in a single intraperitoneal (i.p.) injection. The time point was chosen to allow time for transcription and translation prior to lights out. The dose of Tat-RACK1 has previously been shown to decrease ethanol consumption (1) and the nor-BNI dose is within the range of doses selective for the KOR with minimal mu opioid receptor antagonism (36 , 37) . Bottles were returned to the cages 30 min after injection, and ethanol and water intake was measured 18 h later.

Statistical analysis
Data were analyzed using one- or two-way ANOVAs, as appropriate, followed by post hoc Bonferroni (vs. control) and Student-Neuman-Keuls (differences among treatment groups) tests. Gene expression data calculated as percent control were analyzed by one-sample t test (GraphPad Software, Inc., http://www.graphpad.com/quickcalcs/OneSampleT1.cfm), which infers variation for the control population based on variability in the sample set.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ethanol treatment of primary striatal neurons increases BDNF protein levels and TrkB receptor phosphorylation
Previously, we found that ethanol treatment of dorsal striatal slices increased BDNF mRNA levels, and that in vivo ethanol increased BDNF expression in the dorsal striatum (1) . We hypothesized that this ethanol-mediated increase in BDNF mRNA expression would lead to an increase in the BDNF protein and the consequent activation of the BDNF signaling pathway. To test the hypothesis, we established primary striatal neurons as a model system. As shown in Fig. 1 , most primary striatal neurons in culture display characteristics of medium spiny neurons, with medium-sized cell bodies and multiply branching dendrites (Fig. 1A ), and express the 67-kDa isoform of glutamic acid decarboxylase (GAD-67) (Fig. 1B ), an enzyme required for GABA synthesis that therefore identifies GABAergic neurons (38) . In addition, all cells show high expression of the TrkB receptor (Fig. 1C ), as seen in striatal neurons in vivo (39) , thus making the culture system a suitable model for studying the role of BDNF signaling in the striatal response to ethanol.


Figure 1
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  		Figure 1. Establishment of a primary striatal culture model. Primary striatal neurons were obtained from pooled striata of P0 Sprague-Dawley rats and maintained in Neurobasal-A media for 11 days in vitro. A) Neurons display a medium spiny neuron-like morphology. Neuronal morphology visualized by staining with MAP2. B) Primary striatal neurons are GABAergic. Neurons were costained with MAP2 (red) to show processes and GAD-67 (green), demonstrating that the majority of the cultured neurons are GABAergic. GAD-67 was coexpressed in 88% of MAP2-positive neurons (n=119 of 139 neurons counted from 8 fields selected at random from 4 separate cultures). C) Striatal primary neurons express TrkB receptors. Primary neurons were costained with TrkB (green) and MAP2 (red) antibodies. TrkB receptors were expressed in 100% of MAP2-positive neurons (n=78). Scale bars = 20 µm.

We first set out to determine whether BDNF protein levels increased following ethanol treatment in striatal primary neurons, since the expression profiles of BDNF mRNA and protein are not always identical (40) . Because ethanol treatment increased BDNF mRNA expression after 30 min (1) , we hypothesized that BDNF protein expression would increase as well, but at a later time point due to the temporal constraints of protein synthesis. As illustrated in Fig. 2 , we observed that 2 h ethanol exposure resulted in an increase in BDNF protein expression in striatal neurons.


Figure 2
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  		Figure 2. Ethanol increases BDNF protein levels in primary striatal neuronal cell cultures. Primary striatal neurons were treated without or with 100 mM ethanol as indicated. Fixed cells were stained with anti-BDNF (red, left panels) and anti-MAP2 (green, middle panels). Right panels show merged images. Visible anti-BDNF fluorescence was observed in 16% of control neurons (n=9 of 58 over 2 fields) and 85% of ethanol-treated neurons (n=64 of 75 over 2 fields). Scale bars = 10 µm.

Next, we determined whether ethanol treatment would also result in the activation of the BDNF receptor, TrkB. Binding of BDNF to the TrkB receptor results in receptor dimerization and autophosphorylation of tyrosine residues on the intracellular tail of the receptor (20) . To determine the level of TrkB activation following ethanol treatment, TrkB was immunoprecipitated (IPed) and Western blot membranes were probed with antiphosphotyrosine antibodies. As shown in Fig. 3 A, B, treatment of primary striatal neurons with ethanol for 4 h resulted in a significant activation of the TrkB receptor within the 4-h ethanol incubation period [main effect of treatment, F(1, 17)=5.566; P<0.05]. This was confirmed by immunofluorescent analysis of Trk phosphorylation, as ethanol treatment increased pTrk signal (Fig. 3C ). These results suggest that exposure of striatal neurons to ethanol leads to the activation of the BDNF signaling cascade.


Figure 3
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  		Figure 3. Ethanol activates TrkB receptors in primary striatal neuronal cell cultures. A) Primary striatal neurons were treated with 100 mM ethanol as indicated or 500 pg/ml BDNF as a positive control. TrkB was immunoprecipitated from total cell lysate, and membranes were probed for tyrosine phosphorylation with anti-p-tyrosine antibodies (pY, top row). Membranes were stripped and reprobed for TrkB (bottom row). Representative images show increased TrkB phosphorylation following BDNF or 4 h ethanol treatment. B) Quantitation of change in percent tyrosine phosphorylation of TrkB receptors as shown in A. Data are presented as mean ± SE; *P < 0.05, one-sample t test; n = 9. C) Representative images demonstrating Trk phosphorylation in control as compared to 4-h ethanol-treated neurons. Fixed cells were stained with anti-pTrk(Y490) (green, left panels) and anti-MAP2 (red, middle panels). Right panels show merged images. pTrk fluorescence was visualized in 90% of ethanol-treated neurons counted (n=36 of 40 in a single x10 field), as compared to 26% of control neurons (n=7 of 34 in a single x10 field). Scale bars = 10 µm.

Ethanol treatment of striatal neurons leads to the activation of the MAP kinase pathway via BDNF
BDNF-mediated activation and thus phosphorylation of the TrkB receptor activates MAP kinase signaling by recruiting and phosphorylating the adaptor protein Shc, which interacts with the adaptor protein Grb-2. Grb-2 then recruits a guanine nucleotide exchange factor for Ras, resulting in the activation of this small G protein (41) . Activated Ras triggers a cascade of phosphorylation by activating the MAP kinase kinase Raf, which, in turn, phosphorylates MEK1/2. MEK1/2 then phosphorylates the MAP kinase extracellular signal-regulated kinase 1/2 (ERK1/2) (42) . Activated ERK1/2 can then translocate to the nucleus, where it can activate transcription factors to alter gene expression (43) . BDNF signaling via the MAP kinase cascade has been shown to play a role in downstream gene expression after BDNF treatment of primary neurons (44 45 46) . As ethanol treatment resulted in TrkB receptor phosphorylation, we hypothesized that the activation of the TrkB receptor would lead to the activation of the MAP kinase signaling cascade and thus to an increase in ERK1/2 phosphorylation and nuclear localization. To test this possibility, we treated primary striatal neurons with ethanol for 4 h and visualized changes in the amount and nuclear localization of phospho-ERK1/2 using confocal microscopy. We quantified the level of phospho-ERK1/2 in the nucleus, which we isolated using DAPI as a nuclear marker. As shown in Fig. 4 , ethanol treatment increased the level of phospho-ERK1/2 in primary striatal neurons (Fig. 4A left, top left panel (control) vs. bottom left panel (ethanol), with phospho-ERK1/2 shown in green). Importantly, we found that this activation of ERK1/2 by ethanol was BDNF-dependent, as addition of TrkB-Fc, a fusion protein that competes with endogenous TrkB receptors for BDNF binding (47) , blocked the ethanol-induced ERK phosphorylation. [Fig. 4A , right bottom panel (ethanol+TrkB-Fc) vs. left bottom panel (ethanol alone)]. Quantitation of nuclear phospho-ERK1/2 levels revealed a main effect of treatment [F(3, 309)=4.000, P<0.01], which was accounted for by increased ERK1/2 phosphorylation in ethanol-treated cells as compared to all other groups (Fig. 4B ). ERK phosphorylation was also observed after 2 h treatment with ethanol (data not shown, 50 mM ethanol, 35% over control, n=17 cells for control, 14 cells for 2 h ethanol treatment, P<0.005). Thus, at the time point at which ethanol increases BDNF expression, ERK phosphorylation also begins to increase. Taken together, these results show that ethanol exposure of striatal neurons leads to the activation of the MAP kinase signaling cascade via BDNF, yielding an increase in nuclear phospho-ERK1/2, suggesting that ethanol via BDNF may alter transcription of downstream genes.


Figure 4
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  		Figure 4. Ethanol increases ERK1/2 phosphorylation via BDNF. A) Representative confocal images showing the central nuclear plane of primary striatal neurons treated without or with 100 mM ethanol for 4 h or 25 ng/ml BDNF for 30 min as a positive control, in the absence or presence of the BDNF inhibitor TrkB-Fc (250 ng/ml). Fixed cells were stained with anti-pERK1/2 (green, left panels), anti-MAP2 (red) and DAPI to mark nuclei (blue); right panel for each treatment shows merged image. Scale bars = 2.5 µm. B) Quantitation of average nuclear pERK intensity. Histogram displays mean ± SE pERK intensity by treatment. *P < 0.05 vs. all other treatments; n = 71 for control; n = 82 for TrkB-Fc; n = 77 for ethanol; n = 80 for ethanol + TrkB-Fc. Nuclear images were obtained from 19–24 fields per condition from 2 independent experiments.

Ethanol increases Pdyn expression in primary striatal neurons via a BDNF-dependent mechanism
Thus far, we have demonstrated that ethanol exposure of striatal neurons increases BDNF protein levels, resulting in an activation of the TrkB receptor, which leads to increased levels of phospho-ERK1/2 in the nucleus. As BDNF can increase expression of downstream genes via activation of the MAP kinase pathway (21 , 44) , and infusion of BDNF into the striatum increases Pdyn expression (23) , we hypothesized that ethanol, via BDNF-mediated activation of ERK1/2, would increase Pdyn expression in primary striatal neurons. To test this, primary striatal neurons were treated with 100 mM ethanol for 2–4 h. Following treatment, total RNA was isolated from cells and mRNA expression was assessed using qRT-PCR. As shown in Fig. 5 A, we found a time-dependent increase in Pdyn, with elevated Pdyn levels observable 2 h after ethanol treatment and with maximal expression following 4 h ethanol treatment [main effect of treatment length, F(3, 25)=12.023, P<0.001]. Importantly, we found 4 h of ethanol treatment to be effective at increasing Pdyn expression even at the lowest dose tested (10 mM) (Fig. 5B ), a physiologically relevant concentration roughly equivalent to the blood alcohol level achieved following consumption of 1 or 2 alcoholic drinks (48) .


Figure 5
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  		Figure 5. Ethanol time- and dose-dependently increases Pdyn expression. A) Striatal primary neurons were treated without or with 100 mM ethanol for the times indicated. Data are expressed as mean ± SE percentage increase over control Pdyn/GAPDH expression. *P < 0.05; **P < 0.005; ***P < 0.001; n = 6. B) Striatal primary neurons were treated without or with ethanol for 4 h at the doses indicated. Data are expressed as mean ± SE percentage increase over control Pdyn/GAPDH expression. *P < 0.05; **P < 0.005; n = 6.

Next, we set out to determine whether these observed increases in Pdyn expression were dependent on BDNF signaling via the MAP kinase pathway. To test this hypothesis, primary striatal neurons were treated with ethanol in the presence or absence of the BDNF inhibitor TrkB-Fc, or in the presence or absence of the MEK inhibitor U0126, to block the activation of ERK1/2. As shown in Fig. 6 , we found a significant increase in Pdyn expression following ethanol treatment that was blocked by the addition of TrkB-Fc (Fig. 6B ), demonstrating that ethanol treatment increases Pdyn via BDNF. In addition, we found that ethanol-induced increases in Pdyn expression were also blocked by the MEK inhibitor U0126 (Fig. 6C ), indicating that ethanol treatment increases Pdyn by activating MAP kinase signaling.


Figure 6
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  		Figure 6. BDNF and ethanol via BDNF increase Pdyn expression both in vivo and in vitro by activating the MAP kinase pathway. A) Infusion of 0.25 µg BDNF into the dorsolateral striatum increases Pdyn mRNA expression 3 h postinfusion. Data are expressed as mean ± SE Pdyn/GAPDH expression. *P < 0.05 vs. vehicle-infused control; n = 8. B) Striatal primary neurons were treated without or with 100 mM ethanol or with 25 ng/ml BDNF in the absence or presence of the BDNF inhibitor TrkB-Fc (250 ng/ml). Data are expressed as mean ± SE percentage increase over control Pdyn/GAPDH expression. *P < 0.05; n = 6. C) Striatal primary neurons were treated without or with 100 mM ethanol or with 25 ng/ml BDNF in the absence or presence of the MEK inhibitor U0126 (20 µM, 30 min pretreatment). Data are expressed as mean ± SE percentage increase over control Pdyn/GAPDH expression. *P < 0.05 vs. control; #P < 0.05 vs. BDNF; n = 3.

Regulation of ethanol intake by BDNF requires dynorphin
Because increasing BDNF expression resulted in a decrease in ethanol consumption in vivo (1) and ethanol increased Pdyn expression via BDNF (Fig. 6B ), we hypothesized that reduction of ethanol consumption by BDNF would require the expression of dynorphin and subsequent signaling via its receptor, the KOR (31) . First, we determined whether BDNF would increase Pdyn in the dorsal striatum in vivo. Previous work from this laboratory has demonstrated a 2–3 h delay between increased BDNF expression and elevated expression of another downstream effector, the dopamine D3 receptor (1 , 2) . Therefore we infused BDNF into the dorsolateral striatum of rats and assessed changes in gene expression 3 h later (Fig. 6A ). We found that BDNF infusion significantly increased Pdyn expression as compared to vehicle infusion [F(1, 15)=6.716; P<0.05]. Next, we assessed dynorphin’s involvement in the regulation of ethanol intake by BDNF. To increase BDNF expression, we used Tat-RACK1, a Tat fusion protein that we have shown increases BDNF expression in the striatum when administered systemically (1) . RACK1 is a scaffolding protein that translocates to the nucleus on ethanol exposure (49) , where it triggers increased transcription of genes, including BDNF (35 , 50) . The expression of RACK1 as a Tat fusion protein allows systemically delivered Tat-RACK1 to cross the blood-brain barrier (51) and increase BDNF mRNA expression in the brain (1) . Importantly, we have shown that systemic administration of Tat-RACK1 significantly decreases ethanol intake via BDNF (1 , 2) and that administration of Tat-RACK1 directly into the dorsal striatum decreases ethanol self-administration (2) . Thus, we first determined the ability of Tat-RACK1 to increase striatal Pdyn expression and found that, as predicted, treatment of dorsal striatal slices with Tat-RACK1 for 4 h significantly increased Pdyn expression [Fig. 7 A; F(1, 19)=5.407; P<0.05]. This dose and time point also increase BDNF expression in dorsal striatal slices (1) . To test whether dynorphin is required for the reduction of ethanol intake by BDNF following Tat-RACK1 treatment, C57 mice consuming ethanol under a standard two-bottle choice paradigm were injected with vehicle or Tat-RACK1 concurrently with vehicle or nor-BNI, an antagonist of the KOR. As most ethanol consumption occurs during the dark cycle, treatments were given 3.5 h prior to dark cycle onset to allow sufficient time for expression of downstream genes such as Pdyn, and bottles were returned to the cages 30 min after injection. As previously reported (1 , 2) , mice injected with Tat-RACK1 showed a highly significant 61% reduction in ethanol consumption (Fig. 7B ). However, when mice were given nor-BNI along with Tat-RACK1, they displayed only a 32% reduction in ethanol consumption (Fig. 7B ). Two-way ANOVA found a significant effect of treatment [F(1, 23)=168.8; P<0.0001], and a significant treatment by group interaction [F(2, 23)=15.36, P<0.0001], but not a main effect of treatment [F(2, 23)=0.651, P=0.53]. The interaction is accounted for by differences between each treatment group and its vehicle control (all values of P<0.005), as well as by differences between the treatment groups on the drug injection day (P<0.05 for Tat-RACK1 vs. Tat-RACK1+nor-BNI, P<0.05 for Tat-RACK1 vs. nor-BNI alone). Importantly, the Tat-RACK1 + nor-BNI group does not differ from the nor-BNI alone treatment group (P>0.05). We did observe a decrease in ethanol intake in the nor-BNI alone group as compared to vehicle control (P<0.005 for vehicle vs. nor-BNI); however, the direction of this effect cannot account for the normalizing effect of nor-BNI when given in combination of Tat-RACK1. This may be due to short-lived antagonism of the mu opioid receptor at the onset of ethanol access following nor-BNI treatment (37) . Because neither treatment affected water intake, changes in total fluid consumption and ethanol preference match those described for ethanol consumption (data not shown). Thus, inhibition of the KOR significantly reduced the ability of Tat-RACK1 to alter ethanol intake, indicating that dynorphin is responsible, at least in part, for the ability of BDNF to control ethanol consumption.


Figure 7
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  		Figure 7. Tat-RACK1 reduces ethanol intake by increasing dynorphin production and signaling. A) Tat-RACK1 increases Pdyn expression in striatal slice. Dorsal striatal slices were treated with 1 µM Tat-RACK1 for 4 h. Data are expressed as mean ± SE percentage increase over control Pdyn/GAPDH expression. *P < 0.05; n = 10. B) Tat-RACK1 decreases ethanol intake via increased dynorphin signaling in C57BL/6J male mice. Mice were treated with vehicle or 4 mg/kg Tat-RACK1, with or without 30 mg/kg nor-BNI, as indicated. Ethanol intake was recorded over the 18-h period following injection. Histograms depict mean ± SE ethanol consumption (g/kg body weight) per 18 h. *P < 0.05; **P < 0.005; n = 8/group for Tat-RACK1 and Tat-RACK1+ nor-BNI; n = 10/group for nor-BNI alone. For graphical purposes, only one vehicle bar is shown, containing average vehicle response from all 3 groups.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we show that ethanol treatment of primary striatal neurons results in activation of the BDNF receptor, TrkB, and subsequent activation of the MAP kinase cascade, in a BDNF-dependent fashion. We also identify dynorphin as a downstream effector of BDNF signaling in striatal neurons, as ethanol treatment results in increased production of Pdyn via a BDNF- and MAP kinase-dependent mechanism. Importantly, we show that blockade of dynorphin signaling inhibited the ability of BDNF to reduce ethanol intake, suggesting that dynorphin is required, at least in part, for the reduction of ethanol intake by BDNF. Taken together, these results indicate that the BDNF homeostatic response to ethanol triggers activation of MAP kinase signaling, initiating production of the downstream effector dynorphin, whose expression and signaling is required for the reduction of ethanol intake by BDNF (Fig. 8 ).


Figure 8
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  		Figure 8. BDNF homeostatic regulation of ethanol intake via MAP kinase and dynorphin. We propose the following model: 1) ethanol increases BDNF protein production and release; 2) BDNF binds to the TrkB receptor, resulting in receptor dimerization and autophosphorylation; 3) the phosphorylated TrkB receptor activates downstream signaling via the MAP kinase pathway, resulting in 4) increased production of Pdyn; following translation, dynorphin is released and 5) acts via activation of the KOR, which, in turn, decreases ethanol intake.

Ethanol increases BDNF protein levels and TrkB receptor activation
We have previously demonstrated increased striatal BDNF mRNA expression following acute ethanol treatment (1) and have provided in vivo evidence to suggest the involvement of TrkB signaling in the control of ethanol intake by BDNF (2) . Here, we demonstrate that ethanol exposure increases BDNF protein levels and consequently increases activation of the TrkB receptor via a direct effect on striatal neurons. While others have demonstrated an involvement of striatal BDNF signaling in response to drugs of abuse (2 , 10) , as well as changes in cocaine self-administration in response to increases or decreases in BDNF (19) , to our knowledge, this is the first demonstration that exposure to any drug of abuse triggers activation of striatal TrkB by BDNF produced within the striatum, as striatal primary cultures are devoid of cortical input, which has been shown to be a major source of BDNF for the striatum in vivo (52) .

MAP kinase activation by ethanol
Acute exposure to multiple drugs of abuse activates MAP kinase signaling in the striatum (22) , and exposure to cocaine-related cues in withdrawal increases BDNF and MAP kinase activation (53) . Additionally, evidence demonstrating the involvement of MAP kinase signaling downstream of BDNF in the central and medial nuclei of the amygdala in response to acute ethanol (18) indicates that BDNF may act via MAP kinase signaling in the striatum to control ethanol intake. We provide here direct evidence that ethanol activates MAP kinase signaling in striatal neurons in a BDNF-dependent fashion. Interestingly, we observed an increase in the phosphorylation of the MAP kinase ERK1/2 in a subset of the ethanol-treated neurons analyzed. This raises the possibility that ethanol-induced BDNF might act selectively on a subset of striatal neurons in vivo, in particular, the striatonigral projection neurons that express dynorphin (54) . However, this suggestion should be taken cautiously, as cultured striatal neurons, in the absence of extrastriatal connections, may not necessarily maintain the subtype distinctions observed in vivo (57) , and exogenous application of BDNF to the striatum increases both Pdyn and preproenkephalin (23) , indicating that both subtypes of medium spiny neurons are capable of responding to BDNF. Alternatively, the induction of ERK phosphorylation in a subset of neurons following ethanol treatment may result from the quiescent nature of striatal cultures, as primary striatal neurons devoid of cortical input display limited network activity (58) . Nonetheless, given the data presented here, investigating a subpopulation-selective striatal neuronal activation by BDNF would be an enlightening future study.

Increased expression of Pdyn by ethanol via BDNF and MAPK
Striatal dynorphin expression can be upregulated by cocaine (27 , 28 , 59 60 61) and amphetamine (61 62 63) ; however, responsiveness of striatal dynorphin expression to ethanol treatment has been mixed, with some reports showing increased expression (64) and others showing no change (59 , 65) . This may be due, in part, to differences in ethanol administration protocols and time points of analyses with respect to the final ethanol administration. In addition, some data have shown increases in dynorphin levels only upon ethanol withdrawal (26 , 66 67 68 69) ; however, these changes were all found outside the dorsal striatum and thus may demonstrate intrinsic brain region-specific differences in regulation of dynorphin expression. We found that acute ethanol treatment, even at the pharmacologically relevant dose of 10 mM, increases Pdyn expression in primary striatal neurons via a BDNF- and MAP kinase-dependent mechanism. Importantly, this induction requires time, as ethanol must first increase BDNF mRNA and protein production and release to trigger the signaling cascade, which culminates in increased Pdyn expression. It should be noted that our cultures do not specifically differentiate between dorsal and ventral striatum (nucleus accumbens), and a rapid increase in dynorphin release in the nucleus accumbens shell has been demonstrated following ethanol treatment (70) . Therefore, a second longer-acting mechanism, as described here, may follow a rapid release mechanism.

Decreased ethanol intake due to increased BDNF is mediated via dynorphin signaling
While numerous studies have investigated the role of the kappa opioid system in modulating ethanol intake, to date, results have been somewhat divergent (32 , 33 , 71 72 73 74 75 76 77) . However, we show that blockade of the KOR by the antagonist nor-BNI inhibits the ability of Tat-RACK1—which increases striatal BDNF expression in slices and in vivo (1) and Pdyn mRNA expression in striatal slices—to decrease ethanol intake. These results suggest an important role for endogenous dynorphin as a key regulator of the homeostatic control of ethanol intake by BDNF in the dorsal striatum. It is important to note that under our current paradigm, mice had been consuming ethanol voluntarily for more than 6 wk at the point of treatment. While it has been previously shown that mice do not develop ethanol dependence under a continuous access paradigm (78) , and thus we believe this paradigm models social rather than dependent drinking, it would be an informative line of future study to determine the role that dynorphin plays in regulating the response to a single acute exposure to ethanol. Nonetheless, our data are in agreement with studies showing decreased two-bottle choice ethanol intake following administration the KOR agonist U50,488H in rats (32) , as well as data demonstrating lower expression of dynorphin and the KOR in C57BL/6J mice, which drink more ethanol, as compared to low-drinking DBA mice (30) . However, this contrasts with data from both preprodynorphin and KOR knockout mice (72 , 73 , 76) , in which ethanol intake is decreased in male and female (72) or only female (73) knockout mice. It is possible that the effects observed in the knockout mice are due to compensatory changes in the remaining components of the opioid system, as the Pdyn knockout mice appear to have upregulated KOR signaling (79) and the KOR knockout mice may have similar developmental compensations. In addition, as the homeostatic control of ethanol intake by BDNF is dorsal striatum specific, the global deletion of dynorphin or the KOR may mask the effects of a dorsal striatum-specific role for these proteins due to alterations in signaling in other brain regions.

Possible mechanism of action for the dynorphin-mediated reduction in ethanol consumption
According to our model of homeostatic control of ethanol intake by BDNF and its downstream signaling partner dynorphin (Fig. 8) , we hypothesize that dynorphin may modulate ethanol intake by providing postingestive feedback to regulate subsequent ethanol consumption bouts. We theorize that BDNF homeostasis could break down over time, concomitant with the development of an addictive phenotype, such that in a state of ethanol dependence, each drinking bout will cease to result in increased BDNF and dynorphin production. This loss of dynorphin feedback could result in oversensitivity of the KOR system, as suggested by Walker and Koob (74) , who demonstrated that the KOR antagonist nor-BNI decreased ethanol intake in ethanol-dependent rats but not in ethanol-experienced, nondependent rats. The inability of nor-BNI to alter ethanol intake in nondependent animals, as observed by ourselves and others (75 , 80) , even after a period of alcohol deprivation (71) , supports a role for dynorphin in between-bout rather than within-bout regulation of ethanol intake. Research from Mitchell et al. (33) showing an increase in ethanol intake several days after nor-BNI administration may add additional support for this hypothesis, as the blockade of dynorphin feedback may require multiple ethanol intake sessions in nondependent animals to impact consumption.

How dynorphin functions to decrease ethanol intake is as yet unknown; however, the reduction of neurotransmitter release following activation of KOR signaling presents one possible mechanism (81 , 82) , as repeated administration of KOR agonists blocks cocaine-induced elevation of dopamine release (83) . Like all drugs of abuse, acute ethanol induces dopamine release in the nucleus accumbens (84) , which was reported to be increased by nor-BNI administration (75) . As expected, KOR knockout mice show elevated ethanol-evoked dopamine release in the accumbens (85) . Since acute ethanol treatment increases dynorphin release in the nucleus accumbens (70) , and dynorphin, via activation of KOR signaling, decreases neurotransmitter release in the nucleus accumbens and striatum (86 , 87) , one function of the BDNF/dynorphin homeostatic feedback could be to protect against dysregulation of dopamine release following ethanol exposure, as ethanol-dependent rats show increased dopamine release while intoxicated but reduced basal dopamine tone in withdrawal (88) . While acute ethanol induces dopamine release in the dorsal striatum as it does in the nucleus accumbens (89 , 90) , it is as yet unknown whether dopamine release in the dorsal striatum is altered in ethanol-dependent animals. Of particular interest is the possible progression to alteration of basal and/or stimulus-evoked dopamine release following extensive ethanol experience, which would be in line with both our hypothesis and the dysregulation of dynorphin signaling observed by Walker and Koob (74) in dependent rats. Thus, the production of dynorphin and the activation of the KOR may provide feedback regarding the previous ethanol consumption bout and possibly counteract the development of addiction by reducing overactivation of presynaptic neurotransmitter release. Note that an increase in Pdyn expression was observed following BDNF infusions directly into the dorsolateral striatum—a region implicated in habit formation (55) . As addiction can be conceptualized as a persistent habit (56) , this suggests that the proposed reduction in neurotransmitter release via dynorphin signaling may homeostatically regulate ethanol intake by preventing the development of the habitual nature of alcohol intake, which characterizes the addictive phenotype. Further research to address the function of dynorphin in providing homeostatic feedback, as well as determination of the mechanism by which this system fails to control ethanol intake over time, shows promise not only for furthering our understanding of the neuronal changes underlying the development of addiction but also for finding a possible treatment for alcoholism—and possibly addiction to other drugs of abuse.


   ACKNOWLEDGMENTS
 
This work was supported by a National Research Service Award-National Institute on Alcohol Abuse and Alcoholism grant (F31 AA015462) (M.L.L.), and by the State of California for Medical Research on Alcohol and Substance Abuse through the University of California, San Francisco (P.H.J. and D.R.).

Received for publication September 21, 2007. Accepted for publication February 7, 2008.


   REFERENCES
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 RESULTS
 DISCUSSION
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