Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Neurochem. 2011 Jan 19;116(6):984–995. doi: 10.1111/j.1471-4159.2010.07137.x

β1-adrenergic receptors activate two distinct signaling pathways in striatal neurons

John Meitzen 1, Jessie I Luoma 1, Christopher M Stern 1, Paul G Mermelstein 1
PMCID: PMC3078043  NIHMSID: NIHMS257938  PMID: 21143600

Abstract

Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and β1-adrenergic receptors may affect striatal physiology.

Keywords: norepinephrine, CREB, nucleus accumbens, striatum, noradrenaline, adrenergic receptor

INTRODUCTION

Monoamine action on striatal neurons plays essential roles in striatal physiology. While dopamine (DA) and its receptors are the focus of most research in this area, norepinephrine (NE) and adrenergic receptors are also crucial in regulating striatal function. There is abundant expression of α- and β-adrenergic receptors in the striatum (Nicholas et al., 1993; Pisani et al., 2003; Paschalis et al., 2009; Rommelfanger et al., 2009) (Hara et al., 2010), and dysregulation of striatal NE signaling plays important roles in both drug addiction and Parkinson's Disease (Fornai et al., 2007; Rommelfanger and Weinshenker, 2007; Weinshenker and Schroeder, 2007; Aston-Jones and Kalivas, 2008; Sofuoglu and Sewell, 2009). Despite this recognition, the intracellular signaling mechanisms by which NE modulates striatal neurons are not well understood. While over thirty years ago activation of striatal β-adrenergic receptors was demonstrated to increase cAMP concentrations (Forn et al., 1974; Harris, 1976), since that time there have been few follow-up studies examining NE-mediated signaling in striatal neurons. This is particularly true regarding β-adrenergic receptors (Hara et al., 2010). Thus, many questions remain regarding striatal NE signaling. Our work has focused on answering three of those questions. Specifically, whether cAMP accumulation is the singular action of β-adrenergic receptors in this brain region, whether activation of β-adrenergic receptors affects transcription factors and activity-dependent gene expression, and which of the β-adrenergic receptors and associated signaling pathways mediate these changes in cellular physiology.

We find that NE activation of β1-adrenergic receptors stimulates two distinct signaling pathways: one novel and one canonical. The novel pathway leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor that functions as a molecular switch underlying neural plasticity (Lonze and Ginty, 2002; Carlezon et al., 2005). β1-adrenergic receptor-mediated CREB phosphorylation is initiated by stimulation of a receptor tyrosine kinase (RTK). Transactivation of the RTK by β1-adrenegic receptors leads to stimulation of a signaling cascade that includes Ras, Raf, MEK, MAPK and MSK. In addition to CREB phosphorylation, we also observed an increase in CRE-dependent transcription and c-fos gene expression. The second signaling pathway is the previously defined canonical pathway in which stimulation of β1-adrenergic receptors leads to an increase in cAMP production. Interestingly, increases in cAMP were not functionally linked to rapid CREB phosphorylation. These findings indicate that NE can act on striatal neurons via different signaling pathways to stimulate both long-term changes in gene expression, as well as rapidly modulate cellular physiology. These data provide a new framework in which to understand monoamine signaling in striatal neurons, whereby NE and adrenergic receptors can modulate striatal physiology.

MATERIALS and METHODS

Cell culture

Striatal neurons were cultured from 1 to 2 day old Sprague-Dawley male rat pups as previously described (Mermelstein et al., 2000; Groth et al., 2008). All protocols were approved by the Animal Care and Use Committee at the University of Minnesota. Chemicals were purchased from Sigma (St. Louis, MO) unless stated otherwise. Following decapitation, the dorsal striatum and nucleus accumbens (striatum) were isolated in ice-cold modified Hank's Balanced Salt Solution (HBSS) containing 20% fetal bovine serum (FBS; HyClone, Logan, UT) and (in mM) 4.2 NaHCO3 and 1 HEPES, pH 7.35, 300 mOsm. The tissue was then washed and digested for 5 min in a trypsin solution (type XI; 10 mg/ml) containing 137 mM NaCl, 5 mM KCl, 7 mM Na2HPO4, 25 mM HEPES, and 1500 U of DNase, pH 7.2, 300 mOsm. After additional washes, tissue was dissociated and pelleted twice by centrifugation (180g for 10 min) to remove contaminants. Cells were then plated onto 10 mm coverslips (treated with Matrigel to promote adherence; BD Biosciences, San Jose, CA) and incubated for 20 min at room temperature. Two milliliters of minimum essential medium (MEM; Invitrogen, Grand Island, NY) containing 28 mM glucose, 2.4 mM NaHCO3, 0.0013 mM transferrin (Calbiochem, La Jolla, CA), 2 mM glutamine, and 0.0042 mM insulin with 1% B-27 supplement (Invitrogen) and 10% FBS, pH 7.35, 300 mOsm, were added to each coverslip. To inhibit glial growth, 1 ml of medium was replaced with a solution containing 4 μM cytosine 1-ß-D-arabinofuranoside and 5% FBS 24 h after plating. Seventy-two hours later, 1 ml of medium was replaced with modified MEM solution containing 5% FBS. Gentamicin (2 μg/ml; Invitrogen) was added to all media solutions to eliminate bacterial growth.

Drugs

The drugs used from Tocris (Ellisville, MO) were: tetrodotoxin (TTX; 1 μM); D(–)-2-amino-5-phosphonopentanoic acid (AP-5; 25 μM); propanolol (30 μM); betaxolol (10 μM); melittin (1 μM); gallein (75 μM); SQ22536 (90 μM); H89 (5 μM); KT5720 (3 μM); PKI 14-22 amide (1 μM); GW 5074 (10 μM); SL0101-1 (10 μM); U0126 (10 μM); PD98059 (25 μM); K252a (100 nM); 8CPT-2Me-cAMP (50 μM); PP1 (5 μM); thapsigargin (1 μM); pertussis toxin (500 ng/mL). The drugs used from Sigma were: norepinephrine (25 μM, unless otherwise stated); isoproterenol (10 μM); RP-cAMPs (10 μM); 6-Chloro-PB hydrobromide (500 nM). The drug used from Molecular Probes was: BAPTA AM (10 μM). The drugs used from Alomone Labs (Jerusalem, Israel) were: recombinant human neurotrophin 3 and neurotrophin-4/5 (NT-3 and NT-4/5, 100 ng/ml), and recombinant human BDNF (100 ng/ml). The drug used from Cayman Chemical (Ann Arbor, Michigan) was: Farnesyl Thiosalicylic Acid (FTA; 25 μM). The drugs used from Ascent Scientific (Princeton, NJ) were: yohimbine (10 μM) and prazosin (5 μM). M119 (5 μM) was a gift of Dr. Kirill Martemyanov (University of Minnesota).

Immunocytochemistry

Immunocytochemistry protocols followed those described previously (Mermelstein et al., 2001; Boulware et al., 2007). Briefly, cultured striatal neurons (9-10 d.i.v.) were incubated in a Tyrode's solution containing TTX (1 μM) and D-AP-5 (25 μM) at room temperature for 1.5-2.0 h. Unless stated otherwise, cell stimulations (and drug exposure durations before fixation) were performed as follows: vehicle (10 min); all agonists/activators such as norepinephrine and isoproterenol (10 min); 20 mM K+ (5 min). All inhibitors/antagonists were applied 30 min prior to stimulation and then concurrently with stimulation, except for melittin which was applied 15 min prior to stimulation, and pertussis toxin, which was applied 24 hours prior to stimulation (Glass and Felder, 1997; Boulware et al., 2005). Cells were fixed for 20 min after stimulation using ice-cold 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) in PBS containing 4 mM EGTA. After three PBS washes, permeabilization of cells was achieved by a 5 min incubation in a 0.1% Triton X-100 (VWR Scientific, West Chester, PA) solution. After three more washes, cells were blocked at 37°C for 30 min in PBS containing 1% BSA and 2% goat serum (Jackson ImmunoResearch, West Grove, PA). The cells were then incubated at 37°C for 1 h in block solution containing a monoclonal antibody directed against serine 133 phosphorylated CREB (pCREB; 1:500; Upstate Biotechnology, Lake Placid, NY), and to identify individual cell morphology, a polyclonal antibody targeting microtubule-associated protein 2 (MAP2; 1:500; Calbiochem). Cells were then washed three times and incubated for 1 h at 37°C in block solution containing FITC- and CY5-conjugated secondary antibodies for visualization of MAP2 and pCREB, respectively (Jackson ImmunoResearch). After washing off excess secondary antibody, cells were mounted using the antiquenching and mounting medium Citifluor (Ted Pella, Redding, CA). Nuclear fluorescent intensities for pCREB (n ≥ 30 cells per group) were acquired using a Leica DM5500Q confocal system. Data acquired from the Yokogawa system were quantified using MetaMorph software (version 6.0; Universal Imaging, Downington, PA). Data acquired from the Leica system were quantified with Leica LAS AF (version 1.9.0; Leica).

Following established protocols, the confocal excitation and detection settings (i.e., laser intensity, image acquisition time, etc.) for each experiment were determined using coverslips stimulated with 20 mM K+. Inter-coverslip variability was accounted for by subjecting two coverslips to each treatment. For image acquisition, at least 30 neurons were selected randomly across both coverslips using MAP2 fluorescence, allowing the experimenter to remain blind to pCREB intensities. Data were acquired from coverslips in a random order. Images were captured through the approximate midline of each cell. During data analysis, the MAP2 staining was used to draw a region of interest (ROI) outlining the nucleus of each neuron, allowing the experimenter to remain blind to pCREB intensity. The ROI was then transferred to the pCREB image, and average fluorescence intensities within the nucleus were recorded. For all images, background from a region of the image that did not contain pCREB fluorescence was subtracted from the average pCREB fluorescence intensity. Each experiment was performed at least three times to verify results.

cAMP Assay

We measured cAMP concentrations in cultured striatal neurons (9-10 d.i.v.) using a Parameter cAMP kit (R&D Systems, Minneapolis, MN). Cell stimulations were as described above.

Luciferase-based gene reporter assays

Cultured neurons were transfected on 8 d.i.v. with a luciferase-based reporter (1 μg of DNA per coverslip) of CRE-dependent transcription using a calcium phosphate-based method (Deisseroth et al., 1998) or Optifect (Invitrogen). Once transfected, cells were incubated in serum-free DMEM (Invitrogen) with 1% B-27. On 10 d.i.v., cells were stimulated for 4 hours, lysed, and then assayed for luciferase expression using standard protocols and a luminometer (Monolight 3010; PharMingen, San Diego, CA, USA). Each treatment group within a single experiment was comprised of at least 8 coverslips, and all experiments were replicated at least three times.

PCR

qPCR was performed using standard protocols (Mermelstein et al., 2000; Boulware et al., 2007). mRNA was extracted and reverse transcribed from cultured striatal neurons or the striatum of adult rats using a standard kit (RNAeasy Mini kit; QauntiTect kit; Qiagen). In select experiments cultures were exposed to NE, ISO, or vehicle in MEM (Invitrogen) for 1 hour prior to mRNA extraction. Striatal tissue was stored in RNAlater (Qiagen). qPCR amplification was performed using QuantiFast SYBR Green PCR master mix (Qiagen). All qPCR was performed and analyzed using an Opticon 2 (Bio-Rad, Hercules, CA) and standardized to the ribosome-related genes s15 and rpl13a. The critical cycle threshold was set at 25 SDs above baseline. PCR for individual cDNA samples was performed in triplicate, and overall experiments were repeated at least three times. The thermal cycling program used with QuantiFast SYBR was: an initial denaturing step at 95°C for 6 minutes, followed by at least 30 cycles consisting of a 10 sec denaturing step at 95°C, annealing/extension step for 30 seconds at 60°C, and a measurement of fluorescent intensity. At the end of each cycling program a melting curve was run. PCR products were sequenced for verification of product identity.

The primer sequences used were as follows. The upper and lower sequences for adrb1 (GenBank accession number NM_012701) were: 5’- ACC CCA AGT GCT GCG ATT TCG T-3’ and 5’- GCT CGC AGC TGT CGA TCT TCT T-3’. The primer sequences for adrb2 (GenBank accession number NM_012492.2) were 5’- TTC TGT GCC TTC GCC GGT CTT CTT -3’ and 5’- ATG CCA GGG GCT TCC TCA CAA A -3’. The primer sequences for adrb3 (GenBank accession number NM_013108.1) were 5’- AAC TCT GCC TTC AAC CCG CTC A -3’ and 5’- TGA GTT CTG CCT GGA CGC AAC A -3’. The primer sequences for c-fos (GenBank accession number NM_022197.2) were 5’- TGC CAG ATG TGG ACC TGT CTG GTT-3’ and 5’- TAT AGG TAG TGC AGC TGG GAG TGC-3’. The primer sequences for rpl13a (GenBank accession number NM_173340) were 5’- TGC TGC CGC ACA AGA CCA AA-3’ and 5’- AAC TTT CTG GTA GGC TTC AGC CGC-3’. The primer sequences for s15 (GenBank accession number BC094409) were 5’-CCG AAG TGG AGC AGA AGA AG-3’ and 5’-CTC CAC CTG GTT GAA GGT C-3’ (Groth et al., 2007).

Statistics

Experiments were analyzed using ANOVAs and Tukey's multiple comparison post hoc tests, Student's t test, or nonlinear curve fits using Prism 4.03 (GraphPad Software, La Jolla, CA). Statistical differences between all treatment groups are depicted within each figure as different alphabetical characters. Probability values <0.05 were considered a priori as significant. Data are presented as mean ± SEM.

RESULTS

NE induces CREB phosphorylation in striatal neurons

Our initial experiments were designed to test the hypothesis that NE would rapidly induce CREB phosphorylation in cultured striatal neurons. A ten minute exposure of striatal neurons to 25 μM NE increased CREB phosphorylation (Figure 1A, B). NE stimulation triggered CREB phosphorylation in a dose-dependent manner, at concentrations consistent with activation of adrenergic receptors (Figure 1C). Since 25 μM NE was maximally effective in eliciting CREB phosphorylation, this concentration was used for the remainder of the studies. NE-induced CREB phosphorylation in striatal neurons occurred rapidly, within 5 minutes of NE administration, with maximal responses observed after a 10 minute stimulus (Figure 1D). In addition, CREB remained phosphorylated within striatal neurons for at least 120 minutes after a 10 minute exposure to NE (Figure 1E), providing ample time in which CREB-dependent changes in gene expression could occur (see below).

Figure 1.

Figure 1

Open in a new tab

NE induces CREB phosphorylation in striatal neurons. A. Example confocal images of cultured striatal neurons immunolabeled with MAP2 (green) and pCREB (red). Striatal neurons exhibited heightened nuclear staining for pCREB after a 10 min application of norepinephrine (NE; 25 μM). No Stim (i.e. no stimulation) neurons were treated with vehicle only. Scale bar, 20 μm. B. Quantification of the immunolabeling demonstrated that NE significantly increased CREB phosphorylation (t=5.489). Letters within each bar indicate statistically different groups. C. NE increased CREB phosphorylation in a concentration-dependent manner, with an EC50 = 0.43μM. D. Time course of the onset of NE-induced CREB phosphorylation. E. Time course of the recovery of CREB phosphorylation following a 10 minute exposure to 25 μM NE.

β1-adrenergic receptor activation mediates NE-induced CREB phosphorylation

Both α- and β-adrenergic receptors are present in the striatum and nucleus accumbens. α-adrenergic receptors are primarily found on presynaptic terminals (Rommelfanger et al., 2009), whereas β-adrenergic receptors are primarily found on post-synaptic membranes and cell bodies (Pisani et al., 2003; Paschalis et al., 2009; Hara et al., 2010). Because β-adrenergic receptors are found on the post-synaptic membrane and cell bodies, we first tested whether these receptors mediate NE-induced CREB phosphorylation. Using RT-PCR, we found that β1, β2, and β3-adrenergic receptor mRNA is expressed in both cultured striatal neurons (Figure 2A) and adult striatal tissue (data not shown). We found that exposure to 30 μM propanolol, a panspecific β-adrenergic receptor antagonist, blocked NE-induced CREB phosphorylation (Figure 2B). Because striatal neurons express all three β-adrenergic receptor subtypes, we then exposed neurons to 10 μM betaxolol, a β1-adrenergic receptor antagonist, and found that this also blocked NE-induced CREB phosphorylation (Figure 2B). Exposure to 10 μM isoproterenol (ISO), a β1 and β2-adrenergic receptor agonist, mimicked the effect of NE (Figure 2C). The effects of ISO and NE were not additive (Figure 2C). ISO stimulated CREB phosphorylation in a dose-dependent manner, with 10 μM ISO being maximally effective in eliciting CREB phosphorylation. This concentration was used for the remainder of the study. The magnitude of ISO-induced CREB phosphorylation was comparable to that induced by the dopamine D1 receptor agonist 6-Chloro-PB (6-C; Figure 2D), and the effects of ISO and 6-C were not additive (Figure 2D). The effects of ISO were also blocked by betaxolol (Figure 2E) and propanolol (Figure 2F), and did not differ between dorsal striatum and nucleus accumbens enriched cultures (data not shown). To test the specificity of ISO stimulation of β-adrenergic receptors, we exposed striatal neurons to yohimbine, an α2-adrenergic receptor antagonist, and prazosin, an α1 and α2B-adrenegic receptor and melatonin MT3 receptor antagonist. Neither yohimbine (Table 1) nor prazosin (Table 1) blocked the effects of ISO, indicating that the effect of ISO is not mediated via α-adrenergic receptors. Of note, the remainder of the studies used ISO because of its specificity in activating β-adrenergic receptors.

Figure 2.

Figure 2

Open in a new tab

β1-adrenergic receptors mediate NE-induced CREB phosphorylation. A. Reverse transcription (RT)-PCR detection of β1-, β2-, and β3-adrenergic receptor mRNA in striatal cultures. Products were verified by sequencing. B. The effects of NE were blocked after inhibition of β-adrenergic receptors with the panspecific β-adrenergic receptor antagonist propanolol or the β1-adrenergic receptor specific antagonist betaxolol (F=16.50). C. The β1 and β2-adrenergic receptor agonist isoproterenol (ISO) mimics NE action, and the effects of ISO and NE are not additive (F=4.337). D. The effects of ISO and the D1 dopamine receptor agonist 6-chloro-PB (6-C) were also comparable and not additive (F=18.19). E. Betaxolol blocks the effects of ISO (F=6.87). F. Propanolol blocks the effects of ISO (F=13.69).

Table 1.

Drugs that did not block ISO-induced CREB phosphorylation.

Drug Action
Yohimbine α2-adrenergic receptor antagonist
Prazosin α1 and α2B-adrenegic receptor antagonist, melatonin MT3 receptor antagonist
Pertussis Toxin Gi/o G-protein inhibitor
Gallein Gβγ G-protein subunit inhibitor
M119 Gβγ G-protein subunit inhibitor
KT5720 PKA inhibitor
Rp-cAMPs PKA inhibitor
PKI 14-22 amide PKA inhibitor
BAPTA AM Cell-permeable calcium chelator
Thapsigargin Depletes intracellular calcium stores
Calcium-free media Removes external calcium
PP1 Src kinase family inhibitor
SL0101-1 RSK inhibitor
Open in a new tab

None of the drugs listed in this table blocked ISO-induced CREB phosphorylation.

Gαs/olf is implicated in β1-adrenergic receptor signaling

The next experiments were designed to elucidate the G-protein responsible for β1-adrenergic receptor-induced CREB phosphorylation. Similar to the D1 dopamine receptor and adenosine A2A receptor (Herve et al., 1993; Kull et al., 2000; Herve et al., 2001), striatal β1-adrenergic receptors are usually described as activating the Gs family of G-proteins, including Golf and Gs (Hara et al., 2010). To test whether β1-adrenergic receptor-induced CREB phosphorylation is mediated by Gs/olf, we first exposed neurons to melittin (1 μM). Melittin inhibits the Gs family of G proteins, although it has other actions, including activation of the Gi G-protein family, PLA2, and possibly PLC (Fukushima et al., 1998; Raghuraman and Chattopadhyay, 2007). We also note that the only study to date that has examined the effects of melittin used synaptic membranes, not intact neurons (Fukushima et al., 1998). Melittin was found to block ISO-induced CREB phosphorylation (Figure 3A). As a positive control, we also exposed neurons to melittin and the D1 dopamine receptor agonist 6-Chloro-PB. As predicted, melittin blocked 6-Chloro-PB-induced CREB phosphorylation (Figure 3B). To eliminate the possibility that ISO-induced CREB phosphorylation was mediated by stimulating a Gi G-protein, we pretreated neurons with the Gi/o inhibiter pertussis toxin. Pertussis toxin did not block ISO-induced CREB phosphorylation (Table 1). Given that G protein-coupled receptors can stimulate intracellular signaling pathways via both Gα and Gβγ G-protein subunits, we next tested whether β1-adrenergic receptor-induced CREB phosphorylation was mediated via Gβγ (possibly following dissociation from Gαs/olf). We found that the Gβγ inhibitors gallein (Table 1; 75 μM) and M119 (Table 1; 5 μM) did not affect ISO-induced CREB phosphorylation. These same compounds were effective in blocking the actions of corticotropin releasing factor (CRF) on CREB phosphorylation (Stern and Mermelstein, unpublished data), although we note that M119 and gallein individually do not interfere with all Gβγ pathways. Future studies will need to combine both genomic and biochemical experiments to further identity the exact G-proteins involved.

Figure 3.

Figure 3

Open in a new tab

ISO-mediated CREB phosphorylation via Gαs/olf, MEK, TRK, RAS, RAF, MSK signaling. A. Melittin, a Gαs family antagonist and Gαi/o agonist, blocked ISO-induced CREB phosphorylation (F=8.096). The Gαi/o inhibitor pertussis toxin did not block ISO-induced CREB phosphorylaion. B. As a positive control, we exposed neurons to melittin and the D1 receptor agonist 6-Chloro-PB. Melittin also blocked CREB phosphorylation induced by 6-Chloro-PB (F=14.28). C. The effect of ISO was eliminated after inhibition of MEK with U0126 (F=18.34). D. The effect of ISO was eliminated after inhibition of MEK with PD98059 (F=16.11). ISO-induced CREB phosphorylation was not blocked by the PKA inhibitors KT5720, Rp-cAMPs, or PKI 14-22 amide. E. The receptor tyrosine kinase inhibitor K252A also blocked ISO-mediated CREB phosphorylation (F=15.09). F. The Ras inhibitor Farnesylthiosalicylic Acid (FTA) blocked CREB phosphorylation (F=17.57). G. The effect of ISO was also blocked after inhibition of Raf with GW5074 (F=9.671). H. The MSK inhibitor H89 blocked CREB phosphorylation (F=11.73), while the RSK inhibitor SL0101-1 did not.

MEK and TRK inhibitors block CREB phosphorylation

At this point in our research, we expected β1-adrenergic receptors to stimulate CREB phosphorylation through the canonical AC/cAMP/PKA pathway (Lands et al., 1967; Ursino et al., 2009). We were therefore surprised to find that specific PKA blockers did not block ISO-induced CREB phosphorylation. We used three separate PKA inhibitors: KT5720 (Table 1; 3 μM), RP-cAMPs (Table 1; 10 μM), and PKI 14-22 amide (Table 1; 1 μM). Instead, CREB phosphorylation was blocked by inhibitors of MEK, including U0126 (Figure 3C; 10 μM) and PD98059 (Figure 3D; 25 μM). Evidently, a signaling pathway distinct from the canonical pathway was responsible for β1-adrenergic receptor-mediated CREB phosphorylation.

β1-adrenergic receptors could activate MEK signaling via several different routes, including transactivation of a receptor tyrosine kinase (Lowes et al., 2002). Indeed, we found that the receptor tyrosine kinase inhibitor K252A (100 nM) blocked ISO-mediated CREB phosphorylation (Figure 3E). To determine whether activation of the receptor tyrosine kinase by ISO was mediated through the release of neurotrophins, we examined the time course of neurotrophin-induced CREB phosphorylation. We reasoned that if neurotrophin release was downstream of β1-adrenergic receptor activation, the time course of neurotrophin-mediated CREB phosphorylation would be at least as fast as ISO-induced CREB phosphorylation. To test this hypothesis, we utilized BDNF, known to elicit CREB phosphorylation via TrkB activation (Finkbeiner et al., 1997; Arthur et al., 2004). We found that ISO-induced CREB phosphorylation occurs more rapidly than BDNF-induced CREB phosphorylation. As mentioned previously, ISO-mediated CREB phosphorylation occurs within 5 minutes of drug administration. In comparison, BDNF did not produce a significant increase in CREB phosphorylation under these conditions (pCREB fluorescence intensity: vehicle: 857 ± 107; ISO: 1908 ± 166, p<0.05 vs vehicle and BDNF; BDNF: 1134 ± 140; F = 14.87; BDNF concentration: 100 ng/ml). BDNF-mediated CREB phosphorylation was first observed 15 minutes following neurotrophin administration. As an additional test, we found the effects of NT3 (100 ng/ml) and NT4 (100 ng/ml) to parallel those of BDNF (data not shown). While there are several possibilities to account for the slower time course of neurotrophin mediated CREB phosphorylation, the data suggests to us that at least several receptor tyrosine kinases responsible for signaling to CREB (including those activated by β1-adrenergic receptors) are not on the extracellular surface (Rajagopal et al., 2004). However, the definitive testing of this hypothesis requires further study (see discussion).

Calcium or src kinase family inhibitors do not block CREB phosphorylation

Both dopamine (Iwakura et al., 2008) and adenosine (Lee and Chao, 2001; Assaife-Lopes et al., 2010) receptors can transactivate receptor tyrosine kinases in neurons via a calcium and/or src kinase dependent mechanism that is independent of neurotrophin binding. We next tested whether this same mechanism also underlies adrenergic receptor-mediated signaling to CREB. We find that blocking calcium action with the cell-permeable calcium chelator BAPTA-AM (Table 1; 10 μM), by depleting intracellular stores with thapsigargin (Table 1; 1 μM), or with incubation in calcium free media (Table 1) did not affect ISO-mediated CREB phosphorylation. We also find that the src kinase family inhibitor PP1 (Table 1; 5 μM) did not block CREB phosphorylation.

RAS, RAF and MSK are necessary for CREB phosphorylation

In our next series of experiments, we determined what signaling molecules lie between the receptor tyrosine kinase and MEK. Receptor tyrosine kinases typically activate the MEK pathway via the Ras/Raf signal transduction cascade. Consistent with this hypothesis, the Ras inhibitor FTA blocked ISO-mediated CREB phosphorylation (Figure 3F; 25 μM). The same was true for the Raf inhibitor, GW5074 (Figure 3G; 10 μM).

CREB phosphorylation due to activation of MEK/MAPK signaling has been intensely studied (Carlezon et al., 2005). MAPK signaling ultimately leads to activation of either RSK and/or MSK, two kinases believed to directly phosphorylate CREB. To determine whether either of these kinases mediated β1-adrenergic receptor-induced CREB phosphorylation, cultures were treated with inhibitors of either RSK (SL0101-1; 10 μM) or MSK (H89; 5 μM). SL0101-1 did not affect ISO-mediated CREB phosphorylation (Table 1), whereas H89 did block the actions of ISO (Figure 3H). At the 5 μM concentration used, H89 also inhibits PKA, creating a potential confound. However, since three more specific PKA inhibitors failed to block CREB phosphorylation (Table 1), along with the knowledge that MAPK signaling often leads to activation of MSK, we attribute the effect of H89 to inhibition of MSK, and not PKA.

β1-adrenergic receptor activation induces cAMP production

While the preceding experiments indicate that NE induces rapid CREB phosphorylation via a novel signaling pathway, the question remained whether we could also observe activation of the canonical β-adrenergic receptor/Gαs/AC/cAMP pathway. We tested this by measuring cAMP concentrations following β1-adrenergic receptor activation. ISO was found to increase cAMP concentrations in our cultured striatal neurons, an effect that was blocked by betaxolol and propanolol (Figure 4A, and data not shown). ISO-mediated increases in cAMP concentrations were also blocked by the adenylyl cyclase inhibitor SQ22536 (Figure 4A; 90 μM). However, SQ22536 did not affect CREB phosphorylation (Figure 4B), indicating that cAMP accumulation was not functionally linked to rapid CREB signaling. Previous reports have indicated that global activation of adenylyl cyclase in striatal neurons using forskolin will in fact produce CREB phosphorylation (Liu and Graybiel, 1996). Consistent with these studies, we found that a 10 minute exposure of our striatal neurons to 25 μM forskolin also induced CREB phosphorylation (data not shown), indicating consistent findings across laboratories. These data suggest that while β1-adrenergic receptors activate AC, either the pool of enzymes activated by these receptors is not functionally linked to rapid CREB phosphorylation, or that a cAMP threshold is not achieved through direct stimulation with ISO.

Figure 4.

Figure 4

Open in a new tab

β1-adrenergic receptor activation induces cAMP formation, although cAMP signaling is not functionally linked to rapid CREB phosphorylation. A. ISO-induced cAMP formation is blocked by the β1-adrenergic receptor antagonist betaxolol and the adenylyl cyclase inhibitor SQ22536 (F=117.7). B. ISO-induced CREB phosphorylation is not affected by pretreatment with SQ22536 (F=14.40). C and D: Activation of β1-adrenergic receptors stimulate CRE-dependent transcription and c-fos gene transcription. C. Application of ISO increased CRE-dependent transcription, as did 6-C. The effects of ISO and 6-C were not additive (F=12.72). D. Exposure to ISO produced an approximate 4-fold increase in c-fos expression (t=3.33).

As an additional test that cAMP accumulation following ISO administration is not functionally linked to rapid CREB signaling, we activated Epac, a cAMP-sensitive guanine nucleotide-exchange factor that is known to link the cAMP and MEK signaling pathways. Exposure to the Epac activator 8CPT-2Me-cAMP (50 μM) did not affect CREB phosphorylation (data not shown).

β1-adrenergic receptors stimulate CRE-dependent transcription and c-fos expression

Because phosphorylation of CREB on serine 133 is necessary but not sufficient for activation of CRE-dependent transcription, we applied isoproterenol to cultured striatal neurons transfected with a luciferase-based CRE reporter construct in order to monitor changes in CRE-dependent transcription. We found that isoproterenol increased CRE-dependent transcription (Figure 4C), as did the D1 dopamine agonist 6-Chloro-PB. The effects of isoproterenol and 6-Chloro-PB were not additive. Similar results were also observed with NE stimulation (data not shown).

We then tested whether activation of β1-adrenergic receptors induces changes in mRNA expression by exposing cultured striatal neurons to isoproterenol and then measuring changes in c-fos cDNA using qPCR. The c-fos gene was chosen because it is a known target of CREB that plays a significant role in striatal plasticity (Konradi et al., 1994). Administration of ISO produced an approximate four-fold increase in the abundance of c-fos (Figure 4D), indicating that activation of β1-adrenergic receptors drives changes in gene expression in striatal neurons.

DISCUSSION

This study found that NE acts on striatal neurons to stimulate both novel and canonical signaling pathways that induce rapid CREB phosphorylation, affect gene expression, and initiate cAMP production. While both rapid CREB phosphorylation and cAMP accumulation are induced by activating β1-adrenergic receptors, the signaling pathways diverge to induce cAMP accumulation through canonical signaling, and rapid CREB phosphorylation via a receptor tyrosine kinase/Ras/Raf/MEK/ MAPK/MSK pathway (Figure 5). These findings establish that β1-adrenergic receptors can activate multiple signaling pathways in striatal neurons, including those that affect striatal plasticity and function through changes in gene expression. These data provide a potential new mechanism underlying the influence of NE and adrenergic receptors on striatal function.

Figure 5.

Figure 5

Open in a new tab

β1-adrenergic receptors activate multiple pathways in striatal neurons. β1-adrenergic receptor stimulation induces canonical cAMP production and the activation of a receptor tyrosine kinase. Receptor tyrosine kinase activation leads to the stimulation of the Ras/Raf/MEK/MAPK/MSK signaling pathway, ultimately leading to rapid CREB phosphorylation. We note that receptor tyrosine kinases can be present on both the cellular and internal membranes.

A novel signaling pathway for NE action in the striatum

β-adrenergic receptors are classically described as stimulating the AC/cAMP/PKA pathway (Lands et al., 1967; Ursino et al., 2009), with β-adrenergic activation of cAMP in the striatum having been described since the 1970s (Forn et al., 1974; Harris, 1976; Harris, 1978; Daly et al., 1981). Here we describe a new action of NE in the striatum: β1-adrenergic receptors can rapidly induce CREB phosphorylation and downstream gene expression via activation of a receptor tyrosine kinase. This finding adds to recent research in other systems, which has found that a number of different pathways can be activated by β-adrenergic receptors depending on the specific receptor and cell type (Giembycz and Newton, 2006; Hein, 2006; Chen et al., 2007; Grimm and Brown, 2009; Ursino et al., 2009).

One interesting aspect of this novel pathway is the link between the β1-adrenergic receptor and receptor tyrosine kinase (RTK) signaling. Our working hypothesis is that the β1-adrenergic receptor transactivates RTKs via an intracellular pathway that is independent of neurotrophin binding (Lee et al., 2002; Lowes et al., 2002), perhaps similar to that linking the D1 dopamine receptor to TrkB receptors in cultured striatal neurons (Iwakura et al., 2008) or that linking adenosine receptors to Trk receptors located on Golgi membranes in cultured basal forebrain or hippocampal neurons (Lee and Chao, 2001; Rajagopal et al., 2004). Indeed, in another system the β2-adrenergic receptor transactivates Trk via intracellular mechanisms (Maudsley et al., 2000). While we favor the intracellular pathway hypothesis, we acknowledge that other possible mechanisms could link the β1-adrenergic receptor to Trk. One such mechanism is a direct physical coupling of β1-adrenergic receptors to Trk. Another possibility is a β-arrestin depdendent pathway (Reiner et al., 2010). Alternatively, β1-adrenergic receptor activation could lead to BDNF or other neurotrophin release which then activates a Trk receptor (Chen et al., 2007). We do not favor this hypothesized mechanism given the rapidity of ISO-induced CREB phosphorylation compared to that induced by neurotrophins, including BDNF (Finkbeiner et al., 1997). Future experiments will more explicitly test these various hypotheses, and attempt to identify the specific Trk. That said, it is difficult to directly test this hypothesis due to a lack of specific inhibitors of RTKs, and knockdown or inhibition of these receptors have pronounced effects on cell viability (Ghosh et al., 1994).

NE and DA signaling in the nucleus accumbens and striatum

Though DA is the principal neuromodulator studied in the context of striatal physiology, NE and adrenergic receptors are also present in the nucleus accumbens and striatum. The brainstem noradrenergic cell groups A1 and A2 project to the nucleus accumbens (Berridge et al., 1997; Delfs et al., 1998; Tong et al., 2006), while noradrenergic cell bodies in the locus ceruleus (LC) project to many brain regions, including the striatum (Moore and Bloom, 1979). These projections are sparser than those associated with dopamine release, but NE signaling within the striatum is highly relevant. Baseline striatal NE concentrations are approximately half of that of DA, and as with DA, striatal NE concentrations are significantly elevated following injection of psychostimulants, as measured using in vivo microdialysis techniques (Li et al., 1998; McKittrick and Abercrombie, 2007). In parallel, NE neurotransmission is disrupted in PD (Rommelfanger and Weinshenker, 2007). Dopamine has also been shown to activate α- and β- adrenergic receptors (Malenka and Nicoll, 1986; Cornil and Ball, 2008). As such, it should not be surprising that α- and β- adrenergic receptors are abundantly present in the striatum, on both medium spiny projection neurons and cholinergic interneurons (Nicholas et al., 1993; Pisani et al., 2003; Paschalis et al., 2009; Rommelfanger et al., 2009). The extent to which β1-adrenergic receptors co-localize with D1 and D2 DA receptor expressing neurons is unknown, although the non-additive effects of ISO and 6-Chloro=PB (Figure 2D) suggest that β1-adrenergic receptors at least co-localize with D1 receptors.

NE and striatal pathologies

NE has long been studied in the context of drug addiction and PD. NE was in fact the first candidate for the essential “reward transmitter,” but then fell out of favor in the late 1970s. The importance of NE has since re-emerged following the development of more sophisticated models of drug addiction (Weinshenker and Schroeder, 2007; Aston-Jones and Kalivas, 2008; Sofuoglu and Sewell, 2009). Exposure to many drugs of abuse enhance NE neurotransmission throughout the nervous system, including within the striatum (Li et al., 1998; McKittrick and Abercrombie, 2007). For instance, NE signaling is required for the full amphetamine-induced increase in locomotor activity, as well as maximal behavioral sensitization following repeated drug exposure (Kostowski et al., 1982; Archer et al., 1986; Mohammed et al., 1986; Harris et al., 1996; Weinshenker et al., 2002; Vanderschuren et al., 2003). Furthermore, experiments in dopamine transporter knockout mice find substantial DA-independent amphetamine-induced locomotion (Sotnikova et al., 2005). Later in the addiction cycle, NE signaling is known to affect drug relapse (Davis et al., 1975; Weinshenker and Schroeder, 2007; Smith and Aston-Jones, 2008; Sofuoglu and Sewell, 2009). These and other studies in animal models have lead to clinical studies involving drugs that manipulate various aspects of noradrenergic neurotransmission (Szerman et al., 2005; Sofuoglu and Sewell, 2009).

NE has similarly been implicated in PD, as NE-producing neurons in the LC die alongside the dopaminergic neurons of the substantia nigra pars compacta (Mann and Yates, 1983; Mann et al., 1983; Rommelfanger and Weinshenker, 2007). In nonhuman primates and other animal models, the symptoms of PD following dopamine depletion are exacerbated by lesions of the LC (Mavridis et al., 1991; Marien et al., 1993; Fornai et al., 1997; Srinivasan and Schmidt, 2003; Rommelfanger et al., 2007). It has been suggested that NE plays a neuroprotective role essential for the maintenance of striatal control of motor function, with the activity of surviving LC neurons perhaps compensating for depleted DA concentrations (Marien et al., 2004; Rommelfanger and Weinshenker, 2007).

Conclusion

Over thirty years ago, NE exposure was found to induce cAMP production in striatal tissue. Since that time, few studies have examined NE and β-adrenergic receptor mediated signaling in this brain region. Juxtaposed to the paucity of research on this topic, many studies have demonstrated the overall importance of NE neurotransmission in both normal striatal function and the phenotypes of striatal-mediated pathologies. Given this, it is important to understand how NE and adrenergic receptors signals in striatal neurons. In a broader context, that β1-adrenergic receptors trigger multiple signaling pathways may be relevant to other brain regions, given that NE is found across the brain and has been implicated in a host of basic processes and pathologies, including but not limited to sleep (Mitchell and Weinshenker, 2009), PTSD (Krystal and Neumeister, 2009) and Alzheimer's disease (Weinshenker, 2008).

Acknowledgements

We thank Drs. Robert Meisel, William Engeland, Virginia S. Seybold and their laboratory members for their support. We also thank Kyla Britson, Krista Tuomela and Mikaela Hofer for technical assistance. This work was supported by National Institutes of Health Grant NS41302 (P.G.M.) and T32 DA07234 (J.M. and C.M.S.). The authors declare no competing interests.

Abbreviations used

CREB

cAMP Response Element Binding Protein

DA

dopamine

NE

norepinephrine

RTK

receptor tyrosine kinase

ISO

isoproterenol

PD

Parkinson's Disease

6-C

6-Chloro-PB

REFERENCES

  1. Archer T, Fredriksson A, Jonsson G, Lewander T, Mohammed AK, Ross SB, Soderberg U. Central noradrenaline depletion antagonizes aspects of d-amphetamine-induced hyperactivity in the rat. Psychopharmacology (Berl) 1986;88:141–6. doi: 10.1007/BF00652230. [DOI] [PubMed] [Google Scholar]
  2. Arthur JS, Fong AL, Dwyer JM, Davare M, Reese E, Obrietan K, Impey S. Mitogen- and stress-activated protein kinase 1 mediates cAMP response element-binding protein phosphorylation and activation by neurotrophins. J Neurosci. 2004;24:4324–32. doi: 10.1523/JNEUROSCI.5227-03.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Assaife-Lopes N, Sousa VC, Pereira DB, Ribeiro JA, Chao MV, Sebastiao AM. Activation of adenosine A2A receptors induces TrkB translocation and increases BDNF-mediated phospho-TrkB localization in lipid rafts: implications for neuromodulation. Journal of Neuroscience. 2010;30:8468–80. doi: 10.1523/JNEUROSCI.5695-09.2010. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  4. Aston-Jones G, Kalivas PW. Brain norepinephrine rediscovered in addiction research. Biol Psychiatry. 2008;63:1005–6. doi: 10.1016/j.biopsych.2008.03.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berridge CW, Stratford TL, Foote SL, Kelley AE. Distribution of dopamine beta-hydroxylase-like immunoreactive fibers within the shell subregion of the nucleus accumbens. Synapse. 1997;27:230–41. doi: 10.1002/(SICI)1098-2396(199711)27:3<230::AID-SYN8>3.0.CO;2-E. [DOI] [PubMed] [Google Scholar]
  6. Boulware MI, Kordasiewicz H, Mermelstein PG. Caveolin proteins are essential for distinct effects of membrane estrogen receptors in neurons. J Neurosci. 2007;27:9941–50. doi: 10.1523/JNEUROSCI.1647-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boulware MI, Weick JP, Becklund BR, Kuo SP, Groth RD, Mermelstein PG. Estradiol activates group I and II metabotropic glutamate receptor signaling, leading to opposing influences on cAMP response element-binding protein. J Neurosci. 2005;25:5066–78. doi: 10.1523/JNEUROSCI.1427-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carlezon WA, Jr., Duman RS, Nestler EJ. The many faces of CREB. Trends Neurosci. 2005;28:436–45. doi: 10.1016/j.tins.2005.06.005. [DOI] [PubMed] [Google Scholar]
  9. Chen MJ, Nguyen TV, Pike CJ, Russo-Neustadt AA. Norepinephrine induces BDNF and activates the PI-3K and MAPK cascades in embryonic hippocampal neurons. Cell Signal. 2007;19:114–28. doi: 10.1016/j.cellsig.2006.05.028. [DOI] [PubMed] [Google Scholar]
  10. Cornil CA, Ball GF. Interplay among catecholamine systems: dopamine binds to alpha2-adrenergic receptors in birds and mammals. J Comp Neurol. 2008;511:610–27. doi: 10.1002/cne.21861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Daly JW, Padgett W, Creveling CR, Cantacuzene D, Kirk KL. Cyclic AMP-generating systems: regional differences in activation by adrenergic receptors in rat brain. J Neurosci. 1981;1:49–59. doi: 10.1523/JNEUROSCI.01-01-00049.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davis WM, Smith SG, Khalsa JH. Noradrenergic role in the self-administration of morphine or amphetamine. Pharmacol Biochem Behav. 1975;3:477–84. doi: 10.1016/0091-3057(75)90059-3. [DOI] [PubMed] [Google Scholar]
  13. Deisseroth K, Heist EK, Tsien RW. Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature. 1998;392:198–202. doi: 10.1038/32448. [DOI] [PubMed] [Google Scholar]
  14. Delfs JM, Zhu Y, Druhan JP, Aston-Jones GS. Origin of noradrenergic afferents to the shell subregion of the nucleus accumbens: anterograde and retrograde tract-tracing studies in the rat. Brain Res. 1998;806:127–40. doi: 10.1016/s0006-8993(98)00672-6. [DOI] [PubMed] [Google Scholar]
  15. Finkbeiner S, Tavazoie SF, Maloratsky A, Jacobs KM, Harris KM, Greenberg ME. CREB: a major mediator of neuronal neurotrophin responses. Neuron. 1997;19:1031–47. doi: 10.1016/s0896-6273(00)80395-5. [DOI] [PubMed] [Google Scholar]
  16. Forn J, Krueger BK, Greengard P. Adenosine 3',5'-monophosphate content in rat caudate nucleus: demonstration of dopaminergic and adrenergic receptors. Science. 1974;186:1118–20. doi: 10.1126/science.186.4169.1118. [DOI] [PubMed] [Google Scholar]
  17. Fornai F, Bassi L, Bonaccorsi I, Giorgi F, Corsini GU. Noradrenaline loss selectivity exacerbates nigrostriatal toxicity in different species of rodents. Funct Neurol. 1997;12:193–8. [PubMed] [Google Scholar]
  18. Fornai F, di Poggio AB, Pellegrini A, Ruggieri S, Paparelli A. Noradrenaline in Parkinson's disease: from disease progression to current therapeutics. Curr Med Chem. 2007;14:2330–4. doi: 10.2174/092986707781745550. [DOI] [PubMed] [Google Scholar]
  19. Fukushima N, Kohno M, Kato T, Kawamoto S, Okuda K, Misu Y, Ueda H. Melittin, a metabostatic peptide inhibiting Gs activity. Peptides. 1998;19:811–9. doi: 10.1016/s0196-9781(98)00027-8. [DOI] [PubMed] [Google Scholar]
  20. Ghosh A, Carnahan J, Greenberg ME. Requirement for BDNF in activity-dependent survival of cortical neurons. Science. 1994;263:1618–23. doi: 10.1126/science.7907431. [DOI] [PubMed] [Google Scholar]
  21. Giembycz MA, Newton R. Beyond the dogma: novel beta2-adrenoceptor signalling in the airways. Eur Respir J. 2006;27:1286–306. doi: 10.1183/09031936.06.00112605. [DOI] [PubMed] [Google Scholar]
  22. Glass M, Felder CC. Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors augments cAMP accumulation in striatal neurons: evidence for a Gs linkage to the CB1 receptor. J Neurosci. 1997;17:5327–33. doi: 10.1523/JNEUROSCI.17-14-05327.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Grimm M, Brown JH. Beta-adrenergic receptor signaling in the heart: role of CaMKII. J Mol Cell Cardiol. 2009;48:322–30. doi: 10.1016/j.yjmcc.2009.10.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Groth RD, Coicou LG, Mermelstein PG, Seybold VS. Neurotrophin activation of NFAT-dependent transcription contributes to the regulation of pro-nociceptive genes. J Neurochem. 2007;102:1162–74. doi: 10.1111/j.1471-4159.2007.04632.x. [DOI] [PubMed] [Google Scholar]
  25. Groth RD, Weick JP, Bradley KC, Luoma JI, Aravamudan B, Klug JR, Thomas MJ, Mermelstein PG. D1 dopamine receptor activation of NFAT-mediated striatal gene expression. Eur J Neurosci. 2008;27:31–42. doi: 10.1111/j.1460-9568.2007.05980.x. [DOI] [PubMed] [Google Scholar]
  26. Hara M, Fukui R, Hieda E, Kuroiwa M, Bateup HS, Kano T, Greengard P, Nishi A. Role of adrenoceptors in the regulation of dopamine/DARPP-32 signaling in neostriatal neurons. J Neurochem. 2010;113:1046–59. doi: 10.1111/j.1471-4159.2010.06668.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Harris GC, Hedaya MA, Pan WJ, Kalivas P. beta-adrenergic antagonism alters the behavioral and neurochemical responses to cocaine. Neuropsychopharmacology. 1996;14:195–204. doi: 10.1016/0893-133X(95)00089-V. [DOI] [PubMed] [Google Scholar]
  28. Harris JE. Beta adrenergic receptor-mediated adenosine cyclic 3',5'-monophosphate accumulation in the rat corpus striatum. Mol Pharmacol. 1976;12:546–58. [PubMed] [Google Scholar]
  29. Harris JE. beta-Adrenergic receptor-sensitive adenosine cyclic 3',5'-monophosphate accumulation in homogenates of the rat corpus striatum. A comparison with the dopamine receptor-coupled adenylate cyclase. Biochem Pharmacol. 1978;27:2919–25. doi: 10.1016/0006-2952(78)90209-5. [DOI] [PubMed] [Google Scholar]
  30. Hein L. Adrenoceptors and signal transduction in neurons. Cell Tissue Res. 2006;326:541–51. doi: 10.1007/s00441-006-0285-2. Epub 2006 Aug 1. [DOI] [PubMed] [Google Scholar]
  31. Herve D, Le Moine C, Corvol JC, Belluscio L, Ledent C, Fienberg AA, Jaber M, Studler JM, Girault JA. Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum. J Neurosci. 2001;21:4390–9. doi: 10.1523/JNEUROSCI.21-12-04390.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Herve D, Levi-Strauss M, Marey-Semper I, Verney C, Tassin JP, Glowinski J, Girault JA. G(olf) and Gs in rat basal ganglia: possible involvement of G(olf) in the coupling of dopamine D1 receptor with adenylyl cyclase. J Neurosci. 1993;13:2237–48. doi: 10.1523/JNEUROSCI.13-05-02237.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Iwakura Y, Nawa H, Sora I, Chao MV. Dopamine D1 receptor-induced signaling through TrkB receptors in striatal neurons. J Biol Chem. 2008;283:15799–806. doi: 10.1074/jbc.M801553200. Epub 2008 Apr 1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Konradi C, Cole RL, Heckers S, Hyman SE. Amphetamine regulates gene expression in rat striatum via transcription factor CREB. J Neurosci. 1994;14:5623–34. doi: 10.1523/JNEUROSCI.14-09-05623.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kostowski W, Plaznik A, Pucilowski O, Malatynska E. Effect of lesions of the brain noradrenergic systems on amphetamine-induced hyperthermia and locomotor stimulation. Acta Physiol Pol. 1982;33:383–7. [PubMed] [Google Scholar]
  36. Krystal JH, Neumeister A. Noradrenergic and serotonergic mechanisms in the neurobiology of posttraumatic stress disorder and resilience. Brain Res. 2009;1293:13–23. doi: 10.1016/j.brainres.2009.03.044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kull B, Svenningsson P, Fredholm BB. Adenosine A(2A) receptors are colocalized with and activate g(olf) in rat striatum. Mol Pharmacol. 2000;58:771–7. doi: 10.1124/mol.58.4.771. [DOI] [PubMed] [Google Scholar]
  38. Lands AM, Arnold A, McAuliff JP, Luduena FP, Brown TG., Jr. Differentiation of receptor systems activated by sympathomimetic amines. Nature. 1967;214:597–8. doi: 10.1038/214597a0. [DOI] [PubMed] [Google Scholar]
  39. Lee FS, Chao MV. Activation of Trk neurotrophin receptors in the absence of neurotrophins. Proc Natl Acad Sci U S A. 2001;98:3555–60. doi: 10.1073/pnas.061020198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lee FS, Rajagopal R, Chao MV. Distinctive features of Trk neurotrophin receptor transactivation by G protein-coupled receptors. Cytokine Growth Factor Rev. 2002;13:11–7. doi: 10.1016/s1359-6101(01)00024-7. [DOI] [PubMed] [Google Scholar]
  41. Li XM, Perry KW, Wong DT, Bymaster FP. Olanzapine increases in vivo dopamine and norepinephrine release in rat prefrontal cortex, nucleus accumbens and striatum. Psychopharmacology (Berl) 1998;136:153–61. doi: 10.1007/s002130050551. [DOI] [PubMed] [Google Scholar]
  42. Liu FC, Graybiel AM. Spatiotemporal dynamics of CREB phosphorylation: transient versus sustained phosphorylation in the developing striatum. Neuron. 1996;17:1133–44. doi: 10.1016/s0896-6273(00)80245-7. [DOI] [PubMed] [Google Scholar]
  43. Lonze BE, Ginty DD. Function and regulation of CREB family transcription factors in the nervous system. Neuron. 2002;35:605–23. doi: 10.1016/s0896-6273(02)00828-0. [DOI] [PubMed] [Google Scholar]
  44. Lowes VL, Ip NY, Wong YH. Integration of signals from receptor tyrosine kinases and g protein-coupled receptors. Neurosignals. 2002;11:5–19. doi: 10.1159/000057317. [DOI] [PubMed] [Google Scholar]
  45. Malenka RC, Nicoll RA. Dopamine decreases the calcium-activated afterhyperpolarization in hippocampal CA1 pyramidal cells. Brain Res. 1986;379:210–5. doi: 10.1016/0006-8993(86)90773-0. [DOI] [PubMed] [Google Scholar]
  46. Mann DM, Yates PO. Pathological basis for neurotransmitter changes in Parkinson's disease. Neuropathol Appl Neurobiol. 1983;9:3–19. doi: 10.1111/j.1365-2990.1983.tb00320.x. [DOI] [PubMed] [Google Scholar]
  47. Mann DM, Yates PO, Hawkes J. The pathology of the human locus ceruleus. Clin Neuropathol. 1983;2:1–7. [PubMed] [Google Scholar]
  48. Marien M, Briley M, Colpaert F. Noradrenaline depletion exacerbates MPTP-induced striatal dopamine loss in mice. Eur J Pharmacol. 1993;236:487–9. doi: 10.1016/0014-2999(93)90489-5. [DOI] [PubMed] [Google Scholar]
  49. Marien MR, Colpaert FC, Rosenquist AC. Noradrenergic mechanisms in neurodegenerative diseases: a theory. Brain Res Brain Res Rev. 2004;45:38–78. doi: 10.1016/j.brainresrev.2004.02.002. [DOI] [PubMed] [Google Scholar]
  50. Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, Lefkowitz RJ, Luttrell LM. The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem. 2000;275:9572–80. doi: 10.1074/jbc.275.13.9572. [DOI] [PubMed] [Google Scholar]
  51. Mavridis M, Degryse AD, Lategan AJ, Marien MR, Colpaert FC. Effects of locus coeruleus lesions on parkinsonian signs, striatal dopamine and substantia nigra cell loss after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in monkeys: a possible role for the locus coeruleus in the progression of Parkinson's disease. Neuroscience. 1991;41:507–23. doi: 10.1016/0306-4522(91)90345-o. [DOI] [PubMed] [Google Scholar]
  52. McKittrick CR, Abercrombie ED. Catecholamine mapping within nucleus accumbens: differences in basal and amphetamine-stimulated efflux of norepinephrine and dopamine in shell and core. J Neurochem. 2007;100:1247–56. doi: 10.1111/j.1471-4159.2006.04300.x. [DOI] [PubMed] [Google Scholar]
  53. Mermelstein PG, Bito H, Deisseroth K, Tsien RW. Critical dependence of cAMP response element-binding protein phosphorylation on L-type calcium channels supports a selective response to EPSPs in preference to action potentials. J Neurosci. 2000;20:266–73. doi: 10.1523/JNEUROSCI.20-01-00266.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Mermelstein PG, Deisseroth K, Dasgupta N, Isaksen AL, Tsien RW. Calmodulin priming: nuclear translocation of a calmodulin complex and the memory of prior neuronal activity. Proc Natl Acad Sci U S A. 2001;98:15342–7. doi: 10.1073/pnas.211563998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Mitchell HA, Weinshenker D. Good night and good luck: norepinephrine in sleep pharmacology. Biochem Pharmacol. 2009;79:801–9. doi: 10.1016/j.bcp.2009.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Mohammed AK, Danysz W, Ogren SO, Archer T. Central noradrenaline depletion attenuates amphetamine-induced locomotor behavior. Neurosci Lett. 1986;64:139–44. doi: 10.1016/0304-3940(86)90089-3. [DOI] [PubMed] [Google Scholar]
  57. Moore RY, Bloom FE. Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Annu Rev Neurosci. 1979;2:113–68. doi: 10.1146/annurev.ne.02.030179.000553. [DOI] [PubMed] [Google Scholar]
  58. Nicholas AP, Pieribone V, Hokfelt T. Distributions of mRNAs for alpha-2 adrenergic receptor subtypes in rat brain: an in situ hybridization study. J Comp Neurol. 1993;328:575–94. doi: 10.1002/cne.903280409. [DOI] [PubMed] [Google Scholar]
  59. Paschalis A, Churchill L, Marina N, Kasymov V, Gourine A, Ackland G. beta1-Adrenoceptor distribution in the rat brain: an immunohistochemical study. Neurosci Lett. 2009;458:84–8. doi: 10.1016/j.neulet.2009.04.023. [DOI] [PubMed] [Google Scholar]
  60. Pisani A, Bonsi P, Centonze D, Martorana A, Fusco F, Sancesario G, De Persis C, Bernardi G, Calabresi P. Activation of beta1-adrenoceptors excites striatal cholinergic interneurons through a cAMP-dependent, protein kinase-independent pathway. J Neurosci. 2003;23:5272–82. doi: 10.1523/JNEUROSCI.23-12-05272.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Raghuraman H, Chattopadhyay A. Melittin: a membrane-active peptide with diverse functions. Biosci Rep. 2007;27:189–223. doi: 10.1007/s10540-006-9030-z. [DOI] [PubMed] [Google Scholar]
  62. Rajagopal R, Chen ZY, Lee FS, Chao MV. Transactivation of Trk neurotrophin receptors by G-protein-coupled receptor ligands occurs on intracellular membranes. J Neurosci. 2004;24:6650–8. doi: 10.1523/JNEUROSCI.0010-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Reiner S, Ambrosio M, Hoffmann C, Lohse MJ. Differential signaling of the endogenous agonists at the {beta}2-adrenergic receptor. J Biol Chem. 2010;285:36188–36198. doi: 10.1074/jbc.M110.175604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Rommelfanger KS, Edwards GL, Freeman KG, Liles LC, Miller GW, Weinshenker D. Norepinephrine loss produces more profound motor deficits than MPTP treatment in mice. Proc Natl Acad Sci U S A. 2007;104:13804–9. doi: 10.1073/pnas.0702753104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Rommelfanger KS, Mitrano DA, Smith Y, Weinshenker D. Light and electron microscopic localization of alpha-1 adrenergic receptor immunoreactivity in the rat striatum and ventral midbrain. Neuroscience. 2009;158:1530–40. doi: 10.1016/j.neuroscience.2008.11.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Rommelfanger KS, Weinshenker D. Norepinephrine: The redheaded stepchild of Parkinson's disease. Biochem Pharmacol. 2007;74:177–90. doi: 10.1016/j.bcp.2007.01.036. [DOI] [PubMed] [Google Scholar]
  67. Smith RJ, Aston-Jones G. Noradrenergic transmission in the extended amygdala: role in increased drug-seeking and relapse during protracted drug abstinence. Brain Struct Funct. 2008;213:43–61. doi: 10.1007/s00429-008-0191-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Sofuoglu M, Sewell RA. Norepinephrine and stimulant addiction. Addict Biol. 2009;14:119–29. doi: 10.1111/j.1369-1600.2008.00138.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Sotnikova TD, Beaulieu JM, Barak LS, Wetsel WC, Caron MG, Gainetdinov RR. Dopamine-independent locomotor actions of amphetamines in a novel acute mouse model of Parkinson disease. PLoS Biol. 2005;3:e271. doi: 10.1371/journal.pbio.0030271. Epub 2005 Aug 2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Srinivasan J, Schmidt WJ. Potentiation of parkinsonian symptoms by depletion of locus coeruleus noradrenaline in 6-hydroxydopamine-induced partial degeneration of substantia nigra in rats. Eur J Neurosci. 2003;17:2586–92. doi: 10.1046/j.1460-9568.2003.02684.x. [DOI] [PubMed] [Google Scholar]
  71. Szerman N, Peris L, Mesias B, Colis P, Rosa J, Prieto A. Reboxetine for the treatment of patients with Cocaine Dependence Disorder. Hum Psychopharmacol. 2005;20:189–92. doi: 10.1002/hup.677. [DOI] [PubMed] [Google Scholar]
  72. Tong J, Hornykiewicz O, Kish SJ. Identification of a noradrenaline-rich subdivision of the human nucleus accumbens. J Neurochem. 2006;96:349–54. doi: 10.1111/j.1471-4159.2005.03546.x. [DOI] [PubMed] [Google Scholar]
  73. Ursino MG, Vasina V, Raschi E, Crema F, De Ponti F. The beta3-adrenoceptor as a therapeutic target: current perspectives. Pharmacol Res. 2009;59:221–34. doi: 10.1016/j.phrs.2009.01.002. [DOI] [PubMed] [Google Scholar]
  74. Vanderschuren LJ, Beemster P, Schoffelmeer AN. On the role of noradrenaline in psychostimulant-induced psychomotor activity and sensitization. Psychopharmacology (Berl) 2003;169:176–85. doi: 10.1007/s00213-003-1509-8. [DOI] [PubMed] [Google Scholar]
  75. Weinshenker D. Functional consequences of locus coeruleus degeneration in Alzheimer's disease. Curr Alzheimer Res. 2008;5:342–5. doi: 10.2174/156720508784533286. [DOI] [PubMed] [Google Scholar]
  76. Weinshenker D, Miller NS, Blizinsky K, Laughlin ML, Palmiter RD. Mice with chronic norepinephrine deficiency resemble amphetamine-sensitized animals. Proc Natl Acad Sci U S A. 2002;99:13873–7. doi: 10.1073/pnas.212519999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Weinshenker D, Schroeder JP. There and back again: a tale of norepinephrine and drug addiction. Neuropsychopharmacology. 2007;32:1433–51. doi: 10.1038/sj.npp.1301263. [DOI] [PubMed] [Google Scholar]

RESOURCES