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. Author manuscript; available in PMC: 2006 Jan 27.
Published in final edited form as: J Neurochem. 2005 Jun;93(5):1168–1176. doi: 10.1111/j.1471-4159.2005.03105.x

GalR1, but not GalR2 or GalR3, levels are regulated by galanin signaling in the locus coeruleus through a cyclic AMP-dependent mechanism

Jessica J Hawes 1, Darlene H Brunzell 1, David Wynick 2, Venetia Zachariou 3, Marina R Picciotto 1
PMCID: PMC1352153  NIHMSID: NIHMS7536  PMID: 15934937

Abstract

The galanin receptors GalR1, GalR2 and GalR3 are widely expressed throughout the mouse brain and are enriched in catecholaminergic nuclei. Here we show that GalR1 protein levels are regulated by neuronal activity and changes in cyclic AMP levels. GalR1, but not GalR2 or GalR3, is specifically upregulated in the LC-like Cath.a cell line in a cyclic AMP-dependent manner. GalR1 protein and mRNA levels are also upregulated in the LC of galanin knockout mice, whereas GalR2 and GalR3 are not. Lack of galanin-maintained cyclic AMP tone in the galanin knockout mouse appears to result in a loss of negative feedback resulting in increased levels of CREB phosphorylation and increased GalR1 expression. These findings suggest that changes in levels of GalR1 may play an important role in modulating signaling events and neuroplasticity underlying physiological functions of the LC.

** Abbreviations.

Keywords: negative feedback, norepinephrine, tyrosine hydroxylase, knockout mice

Introduction

Galanin and its three known receptors (GalR1, GalR2 and GalR3) are widely expressed throughout the brain with high expression in the noradrenergic neurons of the locus coeruleus (LC) , the predominant noradrenergic nucleus in the brain with ascending fibers that innervate many brain regions (Williams et al. 2001). The influence of galanin on LC neurons may play an important role in physiological processes that correlate with regulation of the LC, such as Alzheimer's disease (Miller et al. 1999), genetic hypertension (Kunkler et al. 1994), stress (Sweerts et al. 1999) and baroreceptor responses (Shih et al. 1996). Galanin has been shown to attenuate the firing rate of LC neurons (Nishibori et al. 1988; Seutin et al. 1989; Sevcik et al. 1993), which correlates with the ability of galanin to attenuate opiate withdrawal symptoms (Zachariou et al. 2003). Galanin has also been shown to have a direct and indirect effect on modulation of the noradrenaline-induced outward current of LC neurons (Xu et al. 2001), but has no effect on LC-stimulated growth hormone release (Mounier et al. 1996). Active wheel running (Van Hoomissen et al. 2004) and estrogen treatment (Tseng et al. 1997) correlate with an induction of galanin gene expression in the LC. Furthermore, galanin binding and gene expression of at least one galanin receptor, GalR1, is increased in the LC during opiate withdrawal (Zachariou et al. 2000). Therefore, galanin receptor regulation may play an important role in galanin-influenced physiological functions of the LC.

Although nothing has been reported regarding the promoter regions of GalR2 and GalR3, the promoter region of GalR1 contains two CRE-response elements, as well as estrogen receptor binding sites (Howard et al. 1997; Zachariou et al. 2001). GalR1 couples to the Gi/Go pathway to decrease adenylyl cyclase activity in neuronal and non-neuronal cell types (Nishibori et al. 1988; Wang et al. 1998). GalR3 is thought to couple to the Gi/Go pathway in a similar manner as GalR1 (Smith et al. 1998), however GalR2 also couples to Gq and Go pathways in some non-neuronal cell types (Wang et al. 1998; Wittau et al. 2000). Nevertheless, galanin-mediated signaling cascades in the brain are still mostly unknown.

Activation of cAMP-dependent protein kinase A (PKA) can lead to phosphorylation of the transcription factor cyclic AMP regulatory element binding protein (CREB) on serine 133 (Herz 1983; Nestler and Greengard 1983; Chao and Nestler 2004; Johannessen et al. 2004; Nestler 2004) which can then regulate the transcription of numerous target genes (Gonzalez and Montminy 1989; Chao and Nestler 2004; Johannessen et al. 2004). Since GalR1 attenuates the cAMP pathway, but its promoter region contains CRE-response elements (Zachariou et al. 2001), it is possible that GalR1 is auto-regulated in the LC through its actions on cyclic AMP signaling cascades. This is consistent with previous reports that GalR1 mRNA is upregulated in the LC during opiate withdrawal (Zachariou et al. 2000), which is associated with increased activity of the cAMP signaling pathway and CRE-mediated transcription (Valentino and Aston-Jones 1983; Nestler and Tallman 1988; Maldonado et al. 1995; Aston-Jones et al. 1997; Punch et al. 1997; Ivanov and Aston-Jones 2001; Nestler 2004). In this report, we provide evidence that GalR1 protein levels are upregulated in a cyclic AMP/CREB-dependent manner as part of a negative feedback mechanism that does not extend to GalR2 and GalR3. In addition to regulation in vitro, there is a significant increase in the number of galanin binding sites in the LC of galanin knockout mice, which is due to a specific upregulation of GalR1 mRNA and protein levels and is accompanied by upregulation of CREB phosphorylation. Furthermore, these observations suggest that GalR1, but not GalR2 or GalR3, can be regulated by events that alter cyclic AMP-dependent signaling pathways in the LC, such as opiate withdrawal.

Materials and Methods

Animals

Galanin wild type and galanin knockout mice were generated as has been described (Wynick and Bacon 2002), reestablished from het x het matings, and maintained on an inbred 129OlaHsd background with homozygous knockout or wild type x wilt type matings. Adult males were used for all experiments. All animal studies were conducted in accordance with guidelines from the National Institutes of Health and approved by the Yale Animal Care and Use Committee.

Galanin Binding

Galanin autoradiography experiments were performed as previously described (Zachariou et al. 2000). Brains from male galanin wild type (n=5) and galanin knockout (n=4) mice were harvested and frozen at −80 °C following rapid decapitation. 14 μm coronal sections were cut on a cryostat at the level of the LC (Bregma −5.34 mm to −5.68 mm) and collected on gelatinized slides. Stereotaxic coordinates for the LC (bregma −5.40 mm) in mouse brain were determined according to (Paxinos and Franklin 2001). Sections were allowed to dry at room temp, then returned to the cryostat and stored at −80°C. Slides were equilibrated to room temp prior to preincubation in a humid chamber with 40 μl/section of buffer A (50 mM Tris pH 7.4, 5 mM MgCl2, 2 mM EGTA) for 30 min. The preincubation solution was removed and sections were incubated for 1h at room temp with buffer A supplemented with 20 μg/ml leupeptin, 1% BSA and [125I]-galanin (Amersham, Piscataway, NJ). Adjacent sections were incubated with 0.01, 0.05, 0.1, 0.25 or 0.5 nM [125I]-galanin to identify differences in Bmax. A duplicate slide was used to determine non-specific binding by competition for binding with 250 nM cold galanin. The slides were then washed twice for 1 min in ice cold 50 mM Tris pH 7.4, dipped in ice cold dH20, dried, and exposed to Hyperfilm (Amersham, Piscataway, NJ) for 5 days. Binding within the LC was quantified using NIH Image software (http://rsb.info.nih.gov/nih-image/about.html).

Cell Culture

The LC-like murine cell line, Cath.a, was purchased from American Tissue Culture Collection (ATCC, Manassas, VA) and grown according to ATCC guidelines. Briefly, Cath.a cells were cultured in RPMI 1640 medium with L-glutamine, supplemented with 8% horse serum, 4 % fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, and 1% penicillin-streptomycin (1:100). All tissue culture media reagents were purchased from Gibco (Carlsbad, CA).

Western Blot Analysis

Cath.a cells were grown in 6-well plates for 5 days and then starved overnight in media lacking serum. Cells were treated with 5 μM forskolin for 5 min, 7h, or left untreated, and harvested in cell lysis buffer (20 mM Hepes, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5% Glycerol, 10mM pyrophosphate, 1 mM NaVO3, 1 mM PMSF, 5 μg/ml aprotinin, and 5 μg/ml leupeptin, pH 7.5). Bilateral LC tissue punches from a minimum of 4 animals per genotype were sonicated for 5 s in cell lysis buffer. Cell and tissue punch lysates were allowed to incubate on ice for 30 min, then cleared by centrifugation in a table top centrifuge at 14,000 rpm at 4°C for 15 min. Biorad (Hercules, CA) Lowry reagents were used to determine protein concentrations. 5μg protein of each sample was separated on 8.5% SDS-PAGE gels and transferred to nitrocellulose membranes. Blots were blocked in 5% BSA/TBS (pH 7.4) for 1h, then incubated overnight at 4° C in primary antibody. The antibody raised against phospho-CREB (Ser133) in rabbit was used at a dilution of 1:1000 in 5% BSA/TBS (Cell Signaling, Beverly, MA). Affinity purified anti-GalR1 and anti-GalR3 (Alpha Diagnostic International, ADI, San Antonio, TX) polyclonal antibodies were used at a dilution of 1:1000 in 5% BSA/TBS. Antibody #9883 raised against GalR2, in rabbit, was provided by CURE / Digestive Diseases Research Center, Antibody / RIA Core (UCLA, Los Angeles, CA), NIH Grant # DK41301 and was used at a dilution of 1:3000. Antisera specific for each galanin receptor has been characterized by peptide competition for western analysis and described elsewhere (Hawes and Picciotto 2004). Blots were washed 3 times for 5 min each, then incubated for 30 min at room temp in anti-GAPDH monoclonal primary antibody (Advanced ImmunoChemical Inc., Long Beach, CA) diluted to 1:2000 in 5% BSA/TBS. After washing with TBS (pH 7.4)/0.05% Tween, all blots were incubated with secondary antibodies, peroxidase-labeled anti-rabbit IgG 1:2000 and peroxidase-labeled anti-mouse IgG 1:5000 (Vector Laboratories, Inc., Burlingame, CA) for 1h at room temp. Bands were visualized by applying enhanced chemiluminescence to each blot for 1 min and exposing Kodak biomax MR or AR film (Kodak, Rochester, NY). All bands were quantified using NIH Image software (http://rsb.info.nih.gov/nih-image/about.html). Protein loading was verified using Ponceau staining and GAPDH was used as an internal standard. Significance was determined using ANOVA and the Least Significant Difference test (p<0.05). For comparison of only two groups (i.e. galanin wild type vs. knockout), significance was determined using the t test, paired two samples for means (p< 0.05).

Immunohistochemistry

Galanin wild type and galanin knockout mice were anesthetized with 15% Chloral Hydrate then transcardially perfused for 10 min with 4% paraformaldehyde (PFA). Harvested brains were placed in 4% PFA overnight at 4°C then transferred to 30% sucrose and rotated for 24h at 4°C. 35 μm coronal sections were cut on a freezing microtome and stored in potassium phosphate buffered saline (KPBS, pH 7.1)/0.1% NaAzide at 4°C. LC sections were washed 3 times in KPBS for 10 min each prior to immunostaining. For whole section visualization, LC sections were singly immunostained for GalR1, GalR2, or GalR3 and visualized by infrared immunohistochemistry, in which the slides were scanned using a LICOR instrument as previously described (Hawes and Picciotto 2004). Briefly, sections were permeabilized for 30 min at room temp in 4% normal goat serum (NGS, Vector Laboratories, Inc., Burlingame, CA)/0.3% Triton X-100/KPBS, then blocked for 30 min at room temp in 4% NGS/KPBS. Sections were subsequently incubated with anti-GalR1 (1:250), anti-GalR2 (1:5000), or anti-GalR3 (1:500) primary antibodies overnight at 4° C. Affinity purified anti-GalR1 polyclonal antibody was purchased from Alpha Diagnostic International (ADI, San Antonio, TX) and affinity purified anti-GalR3 polyclonal antibody was purchase from Chemicon (Temecula, CA). Antibody #9883 raised against GalR2, in rabbit, was provided by CURE / Digestive Diseases Research Center, Antibody / RIA Core (UCLA, Los Angeles, CA), NIH Grant # DK41301. Antisera specific for each galanin receptor have been characterized by peptide competition for immunohistochemistry (Hawes and Picciotto 2004). Sections were washed 3 times for 10 min each in KPBS, followed by a 1h incubation at room temp in anti-rabbit IR 800 secondary antibody (LI-COR Biosciences, Lincoln, NB) that excites and emits in the infrared spectrum. All sections were washed 5 times in KPBS for 10 min each, mounted on microscope slides and coverslipped using Permount (Fisher Scientific, Suwanee, GA). No immunoreactivity above baseline was detected in control sections incubated in secondary antibody alone. All slides were scanned simultaneously on a LICOR Odyssey Infrared Imager (LI-COR Biosciences, Lincoln, NB). Images were exported to JPEG files and equally adjusted for contrast using Adobe Photoshop 7.0.

For double-labeling experiments, LC brain sections were blocked in 4% NGS/0.3% Triton X-100/KPBS for 1h at room temp. Sections were then incubated with a mixture of anti-TH (1:3500; Sigma, Saint Louis, MI) monoclonal and anti-GalR1 (1:100), anti-GalR2 (1:1000) or anti-GalR3 (1:100) polyclonal primary antibodies overnight at 4° C. Sections were washed 3 times in KPBS/0.3% Triton X-100 for 10 min, then incubated for 1h at room temp in secondary antibodies, AlexaFluor anti-rabbit antibody 568 (1:2000) and AlexaFluor anti-mouse antibody 488 (1:4000). All Sections were washed 3 times in KPBS/0.3% Triton X-100 for 10 min followed by two washes in KPBS for 5 min. Sections were then stained with 0.7% Sudan Black/70% methanol, briefly dipped in 70% ethanol, rinsed in KPBS, mounted on microscope slides and coverslipped using Gel/mount (Biomeda corp., Foster City, CA). Immunofluorescent images were captured using a confocal microscope. Signal Intensity of galanin receptor staining and TH staining of the LC were quantified using NIH Image software (http://rsb.info.nih.gov/nih-image/about.html). GalR immunoreactivity was measured in a minimum of 5 KO animals and from 3-5 WT animals, with a minimum of 2 sections per animal. Noradrenergic neurons were identified by co-labeling for TH. The GalR immunoreactivities of all of the noradrenergic neurons in each section (12 to 95 neurons per section depending on the level of the LC) were quantitated using an area of similar size throughout each section and averaged per section. The intensity of GalR immunoreactivity per section was then averaged to obtain a value for each animal. To determine the relative intensity of staining in KO animals relative to WT mice, the staining intensity for each animal was divided by the average of the control group (WT mice) and multiplied by 100. These values were then averaged for each genotype to obtain a normalized mean and standard error values. Significance was determined using ANOVA with significance set at p<0.05. Representative images were adjusted for optimal contrast using Adobe Photoshop 7.0.

Real-time PCR

Brains from galanin wild type and knockout mice were harvested by rapid decapitation. An 18 gauge punch was used to isolate LC and cerebellum tissue that was placed in lysis buffer (RNAqueous-micro kit, Ambion) less than 5 min after decapitation and immediately sonicated. Total messenger RNA was isolated using the Ambion RNAqueous-micro kit according to instructions. One hundred ng of total mRNA for LC punches and 325 ng for cerebellum punches was reverse transcribed into cDNA using oligo dT primers and the SS (Superscript) III First-strand synthesis system (Invitrogen). One μl of cDNA from each reverse transcriptase reaction was added to 1 pM forward and reverse primers, 11 μl nuclease-free water, and 12 μl Sybergreen (QuantiTect SYBR Green PCR kit, Qiagen) and analyzed for the number of cycles required to reach threshold using a quantitative real-time PCR Smart Cycler (Cepheid). The theoretical melting temperature for each product was calculated online and compared to experimental melting temperatures. Primers for GalR1, GalR2 and GalR3 were BLAST-searched to verify specificity. Forward and reverse primers for GalR1 were 5′-GCCGCGATGTCTGTGGATCG-3′ and 5′-CGATGGACAGCGCCCAGATG-3′, respectively. Forward and reverse primers for GalR2 were 5′-GTGTGCCACCCAGCGTGGAG-3′ and 5′-TGGTGCGCGCATAGGTCAGG-3′, respectively. Forward and reverse primers for GalR3 were 5′-CCTGCCTCAACCCGCTCGTC-3′ and 5′-TGAAGGCGGTGGTGGTGGTG-3′, respectively. TH real-time PCR reactions were used as a marker of noradrenergic neurons to verify placement of LC tissue punches. Forward and reverse primers for TH were 5′-CCTCCTCGCCACAGCCCAAG-3′ and 5′-CTGCCGCCGTCCAATGAACC-3′, respectively. PCR reactions using β-tubulin primers were used as an internal control. Five μl of each PCR reaction was separated on a 1.2 % DNA gel to determine the PCR product size and number of product bands. PCR products were purified using the Qiaquick PCR purification kit (Qiagen) and sent for sequencing by the Yale Keck facility. Sequences were compared to the nucleotide databases using Mega BLAST to verify PCR product identity. Each PCR product was positively identified as being the correct galanin receptor.

Results

The LC-like cell line, Cath.a, was used to determine potential galanin receptor regulatory mechanisms in the LC. Galanin binding to GalR1 couples to Gi/Go proteins and attenuates adenylyl cyclase activity thereby decreasing cyclic AMP production (Wang et al. 1998) and PKA activity, which normally activates CREB signaling. Furthermore, the GalR1 promoter has a CRE-like binding site which is responsive to forskolin-induced CREB activation (Zachariou et al. 2001). Therefore, we examined the effect of forskolin-stimulated cyclic AMP production on downstream levels of phosphorylated/total CREB as well as on potential regulation of galanin receptor expression in Cath.a cells. The Cath.a cell line was originally isolated from a brainstem tumor of a transgenic mouse expressing the simian virus 40 T antigen under the control of the tyrosine hydroxylase promoter (Suri et al. 1993). Cath.a cells exhibit a neural, noradrenergic phenotype and resemble LC neurons in their signal transduction profile and electrophysiology (Widnell et al. 1994; Baraban et al. 1995; Boundy et al. 1998; Zachariou et al. 2001). Treatment of Cath.a cells with 5μM forskolin for 5 min resulted in an increase in CREB phosphorylation. The effect of forskolin on galanin receptor protein expression was examined 7h after stimulation with 5μM forskolin. Antisera specific for GalR1, GalR2 and GalR3 have recently been characterized (Hawes and Picciotto 2004). These antisera appear to be specific according to band size, peptide competition and internal negative control experiments. Nevertheless, the specificity of these antisera using galanin receptor knockout mice still remains to be determined. GalR2 and GalR3 immunoreactivity levels were unaffected by forskolin treatment, however, GalR1 protein immunoreactivity was increased 3-fold following forskolin treatment (Fig. 1). These data suggest that activation of the cyclic AMP pathway specifically regulates GalR1, but not GalR2 and GalR3 protein levels.

Figure 1.

Figure 1

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GalR1 is upregulated in a cyclic AMP-dependent manner in the LC-like cell line, Cath.a. A) Cath.a cells were stimulated for 5 min with 5 μM forskolin to induce cyclic AMP production and PKA activation. Activation of the cyclic AMP/PKA signaling pathway was analyzed by western analysis. Representative western blots are shown for CREB (Ser133) phosphorylation, total CREB and GAPDH (loading control). An increase in CREB (Ser133) phosphorylation indicates forskolin-induced activation of the cyclic AMP/PKA pathway. B) Cath.a cells were stimulated with 5 μM forskolin for 7h to determine cyclic AMP-dependent changes in galanin receptor protein expression. Representative western blots are shown for GalR1, GalR2, GalR3 and GAPDH. C) Galanin receptor protein levels were quantified using NIH Image software and significance was determined by ANOVA. * p < 0.001.

GalR1 couples to Gi/Go inhibitory signaling proteins, which attenuate adenylyl cyclase activity and cyclic AMP production. Therefore, we examined levels of CREB phosphorylation in the LC of galanin knockout mice and wild type controls. CREB (Fig. 2a) phosphorylation levels are significantly increased in galanin knockout mice. This suggests that a loss of galanin-mediated inhibitory signaling in the LC leads to increased CREB phosphorylation.

Figure 2.

Figure 2

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CREB phosphorylation levels are increased in the LC of galanin knockout mice. CREB phosphorylation levels were determined in LC tissue punches from galanin wild type and knockout mice by western blot analysis. CREB phosphorylation was significantly increased in galanin knockout mice as compared to wild type, whereas total CREB protein levels were unchanged. All protein levels were quantified using NIH Image software and significance was determined by t test, paired two samples for means. * p < 0.05.

Since the cyclic AMP pathway regulates GalR1 expression in the LC-like Cath.a cells and galanin knockout mice have increased activation of signaling components downstream of the cyclic AMP pathway, we examined the effect of a loss of galanin signaling on galanin receptor regulation in galanin knockout mice. Galanin binding sites were examined by equilibrium binding at the level of the LC in galanin wild type and knockout mice. Adjacent sections were incubated with multiple concentrations of iodinated galanin in the absence and presence of cold galanin. The Bmax for Galanin binding was significantly increased in the LC of galanin knockout mice compared to galanin wild-type mice (Fig. 3). There may also be increases in galanin binding in nuclei adjacent to the LC, such as the lateral parabrachial and tegmental nuclei, in the galanin knockout mice, however galanin binding in these areas was not quantified.

Figure 3.

Figure 3

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Upregulation of [125I]-galanin binding sites in galanin knockout mice. A) Galanin binding sites within the LC (arrows) of wild type (WT; n=5) and galanin knockout (KO; n=4) mice were labeled with 0.25 nM [125I]-Galanin and visualized by autoradiography. B) Incubation with 0.01, 0.05, 0.1, 0.25 or 0.5 nM [125I]-galanin results in saturation curves of binding in the LC that plateau at a level significantly higher in the LC of galanin knockout mice as compared to wild type mice.

Real-time PCR was used to examine galanin receptor mRNA levels to determine whether the increase in the number of galanin binding sites in the LC of galanin knockout mice was attributable to differences in the expression of specific galanin receptors. The real-time PCR reaction resulted in a single PCR product of the expected size for each galanin receptor transcript (Fig. 4D). The calculated melting temperatures for the predicted GalR1, GalR2 and GalR3 PCR products are 87 °C, 84 °C and 89 °C and the experimental melting temperatures of the PCR reactions were 87.06 °C, 85.57 °C and 90.35 °C, respectively. The PCR products from each reaction were sequenced and all were identical to the predicted amplified product for each galanin receptor. Significantly fewer cycles were required to reach threshold for amplification of GalR1 mRNA isolated from LC tissue punches taken from galanin knockout mice as compared to wild type mice, suggesting that there is an increase in GalR1 mRNA in the LC of galanin knockout mice (Fig. 4A). In contrast, the number of cycles required to reach threshold for GalR2 and GalR3 PCR reactions were not signicantly different across genotypes (Fig. 4B-C). Although GalR3 expression in the brain has been controversial (Wang et al. 1997; Kolakowski et al. 1998; Smith et al. 1998; Waters and Krause 2000; Mennicken et al. 2002; Hawes and Picciotto 2004), these data support previous reports that GalR3 is expressed widely in the brain (Kolakowski et al. 1998; Hawes and Picciotto 2004). There were no significant differences in the number of cycles to reach threshold for any of the three galanin receptors in the cerebellum of galanin knockout mice compared to wild type mice (data not shown), suggesting that regulation of GalR1 mRNA levels in galanin knockout mice is specific to particular brain regions.

Figure 4.

Figure 4

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GalR1 mRNA levels are upregulated in the LC of galanin knockout mice. Total mRNA was isolated from LC tissue punches of galanin knockout (n=4) and wild type (n=4) mice. The number of cycles to reach threshold was determined by real-time PCR using primers specific for GalR1 (A), GalR2 (B), GalR3 (C) or β-tubulin (internal control; data not shown). The number of cycles for each galanin receptor reaction to reach threshold was averaged according to genotype (D) and significance was determined by ANOVA (* p < 0.05). The real-time PCR reaction for each galanin receptor results in a single product of the expected size of approximately 150 base pairs (D insert).

To determine whether the regulation of GalR1 mRNA in the LC of galanin knockout mice correlates with changes at the protein level, immunoreactivity for GalR1, GalR2 and GalR3 was examined in the LC of wild type and galanin knockout mice using infrared immunohistochemistry. LICOR images of sections through the LC from galanin wild type and galanin knockout mice revealed that immunoreactivity for all three receptors is distinctly present within the LC region and that the patterns of immunoreactivity in the surrounding brain regions are consistent with previously reported galanin receptor expression patterns (Kolakowski et al. 1998; Hawes and Picciotto 2004). In accord with the real-time PCR findings, GalR1 was increased in the LC region of galanin knockout mice, whereas GalR2 and GalR3 protein immunoreactivity remained unchanged (Fig. 5). Although the infrared immunohistochemistry technique using the LICOR instrument is ideal for screening numerous samples for differences in relative immunoreactivity intensity at a macroscopic level, the resolution compared to that obtained from fluorescence microscopy is relatively poor. Confocal microscopy was therefore used to examine the levels of galanin receptor immunoreactivity specifically in the noradrenergic neurons of the LC. TH immunoreactivity was used as a marker for noradrenergic cell bodies. Immunoreactivity for all three receptors colocalized with TH in noradrenergic neurons of the LC, in addition to being present in surrounding cell bodies. However, GalR1 and GalR3 immunoreactivities were higher in TH-positive neurons than in TH-negative cell bodies. Furthermore, GalR1 immunoreactivity was significantly upregulated in the LC of galanin knockout mice, whereas GalR2 and GalR3 were not (Fig. 6). Thus, the increase in galanin binding within the LC of galanin knockout mice is likely due to a specific upregulation of GalR1 protein levels.

Figure 5.

Figure 5

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GalR1 is upregulated in the LC of galanin knockout mice. LC (arrows) sections of galanin wild type (WT) and galanin knockout (KO) mice were immunolabeled with anti-GalR1 (A), anti-GalR2 (B), or anti-GalR3 (C) antibodies and scanned on a LICOR scanner to visualize galanin receptor expression levels by infrared immunohistochemistry.

Figure 6.

Figure 6

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GalR1 immunoreactivity is upregulated in the noradrenergic cell bodies of the LC in galanin knockout mice. Galanin wild type (WT) and galanin knockout (KO) LC sections were double-labeled for tyrosine hydroxylase (TH) and GalR1 (A), GalR2 (B) or GalR3 (C) and visualized by confocal microscopy. D) Galanin receptor expression was quantified using NIH Image software. Intensity of galanin receptor immunoreactivity was determined for each noradrenergic neuron per section, averaged per animal, then calculated as percent control compared to wild type. GalR1 WT (n=5); GalR1 KO (n=5); GalR2 WT (n=4); GalR2 KO (n=5); GalR3 WT (n=3); GalR3 KO (n=5). Significance was determined by ANOVA. * p < 0.05.

Discussion

A large body of evidence supports a role for adaptations in the cyclic AMP pathway in mediating many aspects of neural plasticity (Waltereit and Weller 2003). We show here that GalR1, but not GalR2 or GalR3, is specifically upregulated in the noradrenergic Cath.a cell line in a cyclic AMP- and CREB-dependent manner. Furthermore, GalR1 mRNA and immunoreactivity are also upregulated in the LC of galanin knockout mice, whereas GalR2 and GalR3 are not. GalR1 signals through inhibitory Gi/Go proteins to attenuate adenylyl cyclase activity (Nishibori et al. 1988; Zachariou et al. 2003); however, the cyclic AMP pathway positively regulates GalR1 expression (Fig. 7). Consequently, increased signaling through the cyclic AMP/CREB pathway can lead to increased GalR1 expression that would in turn lead to increased attenuation of adenylyl cyclase and the cyclic AMP/CREB pathway. Although other signaling pathways are likely to regulate GalR1 expression and may also be involved in GalR1 upregulation in the LC of galanin knockout mice, these data suggest that GalR1 expression is controlled by a cyclic AMP-dependent negative-feedback pathway. In galanin knockout mice, galanin is not present to stimulate GalR1 inhibition of the cyclic AMP pathway. Thus, loss of galanin/GalR1-mediated attenuation of adenylyl cyclase results in increased activity of the cyclic AMP/CREB pathway and subsequent upregulation of GalR1 (Fig. 6). It is also possible that expression of other signaling proteins is altered in the LC of galanin knockout mice; however, it is clear that the basal level of CREB phosphorylation is increased in the LC of galanin knockout mice, and this appears to be correlated with an increase in GalR1 levels.

Figure 7.

Figure 7

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Model for GalR1 neuroplasticity within the LC. Galanin binds to GalR1 and signals through the Gi/Go pathway to inhibit adenylyl cyclase activity. In contrast, activation of the cyclic AMP/PKA/CREB pathway upregulates GalR1 expression. Thus, GalR1 expression is controlled by a cyclic AMP-dependent, negative-feedback pathway. In galanin knockout (KO) mice, galanin is not present to stimulate GalR1, relieving a tonic inhibition of adenylyl cyclase activity, cyclic AMP production and PKA activity. Loss of galanin-mediated attenuation of adenylyl cyclase activity results in increased cyclic AMP/PKA/CREB activity in the LC and consequent upregulation of GalR1.

Increases in galanin binding in the LC of galanin knockout mice is likely to be the result of increased receptor number rather than a change in galanin receptor affinity because it is accompanied by an increase in Bmax as well as an increase in GalR1 mRNA and immunoreactivity. Galanin binding and galanin receptor immunoreactivity in the lateral parabrachial and tegmental nuclei adjacent to the LC were not quantitated and it is possible that GalR1 is regulated in these brainstem areas as well. The lack of an increase in GalR1 mRNA levels in the cerebellum of galanin knockout mice is consistent with a lack of apparent increase in galanin binding (Fig. 3) and GalR1 immunoreactivity (Fig. 5). This further suggests that GalR1 is differentially regulated in specific regions of the brain. It is possible that an as yet unidentified, fourth GalR is responsible for the increased galanin binding outside the LC in galanin knockout mice. In addition, galanin may bind to a subset of GalRs semi-irreversibly, thus the lack of endogenous galanin peptide may result in greater availability of galanin binding sites, although we believe this is unlikely since we have preincubated brain slices for 30 min at room temperature which is generally thought to eliminate the endogenous ligand from the brain slice. Taken together, these data support the idea that GalR1 upregulation in the LC is likely to underlie the increased galanin binding in this brain area in galanin knockout mice, but other mechanisms may also be recruited outside this brain area.

There is growing evidence for galanin receptor neuroplasticity, which is also subtype- and region-specific. An increase in GalR1 mRNA, immunoreactivity and receptor binding was demonstrated in neurons within layers II and V of neocortex and in piriform cortex after cortical spreading depression, whereas GalR2 mRNA expression was largely unaltered (Shen et al. 2003). In contrast, GalR2 mRNA levels increase after unilateral facial nerve crush, whereas GalR1 mRNA levels remain unchanged (Burazin and Gundlach 1998). A significant change in GalR1 mRNA was not detected in the periventricular, dorsomedial and ventromedial nuclei of galanin knockout mice (Hohmann et al. 2003), which may imply region or cell-type specificity for cyclic AMP-mediated regulation of GalR1. On the other hand, salt-loading leads to an increase in GalR1 mRNA and protein levels specifically in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus (Burazin et al. 2001), which correlates with salt-loading-induced increases in cyclic AMP specific to the SON and PVN (Carter and Murphy 1989). This suggests that GalR1 upregulation also correlates with upregulation of cyclic AMP in the PVN and SON of the hypothalamus. Together, these data suggest that the galanin receptors are differentially regulated by neuronal activity in several neuronal cell types and that the regulatory mechanisms controlling GalR1 transcription in some brain areas other that the LC may also depend on cyclic AMP signaling.

The promoter regions of GalR2 and GalR3 have not yet been characterized and regulation of GalR3 has not yet been examined. However, the lack of GalR2 and GalR3 upregulation following forskolin treatment in Cath.a cells suggests that these genes do not have a CRE site in their promoter or that activation of the CREB pathway alone is not sufficient to induce transcription. Nevertheless, the differential regulation of GalR1 suggests that galanin receptor subtypes are regulated through distinct molecular mechanisms.

The current studies provide evidence that GalR1 levels are regulated in the LC by changes in cyclic AMP signaling. These data are in good agreement with previous studies showing that the GalR1 promoter contains a CRE-like binding site (Zachariou et al. 2001) and that opiate withdrawal, which is associated with increased cyclic AMP levels (Duman et al. 1988; Nestler 2004), leads to increased GalR1 mRNA expression in the LC (Zachariou et al. 2000). GalR1 signaling through Gi/Go proteins may represent a new therapeutic target for the treatment of opiate withdrawal, since galanin decreases opiate withdrawal signs by attenuating markers of cyclic AMP signaling in the LC (Zachariou et al. 2003). Together, these findings suggest that GalR1 signaling and neuroplasticity may play an important role in modulating cyclic AMP-mediated signaling pathways, as is seen during opiate dependence and withdrawal.

Acknowledgements

This work was supported by grants DA15425 and DA00436 from the National Institutes of Health.

Footnotes

**

Abbreviations: LC, locus coeruleus; TH, tyrosine hydroxylase; PKA, protein kinase A, MAPK, p44/42 mitogen activated protein kinase; CREB, cre-binding protein.

Citations

  1. Aston-Jones G, Hirata H, Akaoka H. Local opiate withdrawal in locus coeruleus in vivo. Brain Res. 1997;765:331–336. doi: 10.1016/s0006-8993(97)00682-3. [DOI] [PubMed] [Google Scholar]
  2. Baraban SC, Lothman EW, Lee A, Guyenet PG. Kappa opioid receptor-mediated suppression of voltage-activated potassium current in a catecholaminergic neuronal cell line. J Pharmacol Exp Ther. 1995;273:927–933. [PubMed] [Google Scholar]
  3. Bedecs K, Berthold M, Bartfai T. Galanin--10 years with a neuroendocrine peptide. Int J Biochem Cell Biol. 1995;27:337–349. doi: 10.1016/1357-2725(95)00008-d. [DOI] [PubMed] [Google Scholar]
  4. Boundy VA, Chen J, Nestler EJ. Regulation of cAMP-dependent protein kinase subunit expression in CATH.a and SH-SY5Y cells. J Pharmacol Exp Ther. 1998;286:1058–1065. [PubMed] [Google Scholar]
  5. Burazin TC, Gundlach AL. Inducible galanin and GalR2 receptor system in motor neuron injury and regeneration. J Neurochem. 1998;71:879–882. doi: 10.1046/j.1471-4159.1998.71020879.x. [DOI] [PubMed] [Google Scholar]
  6. Burazin TC, Larm JA, Gundlach AL. Regulation by osmotic stimuli of galanin-R1 receptor expression in magnocellular neurones of the paraventricular and supraoptic nuclei of the rat. J Neuroendocrinol. 2001;13:358–370. doi: 10.1046/j.1365-2826.2001.00640.x. [DOI] [PubMed] [Google Scholar]
  7. Carter DA, Murphy D. Cyclic nucleotide dynamics in the rat hypothalamus during osmotic stimulation: in vivo and in vitro studies. Brain Res. 1989;487:350–356. doi: 10.1016/0006-8993(89)90839-1. [DOI] [PubMed] [Google Scholar]
  8. Chao J, Nestler EJ. Molecular neurobiology of drug addiction. Annu Rev Med. 2004;55:113–132. doi: 10.1146/annurev.med.55.091902.103730. [DOI] [PubMed] [Google Scholar]
  9. Chartoff EH, Papadopoulou M, Konradi C, Carlezon WA., Jr. Dopamine-dependent increases in phosphorylation of cAMP response element binding protein (CREB) during precipitated morphine withdrawal in primary cultures of rat striatum. J Neurochem. 2003;87:107–118. doi: 10.1046/j.1471-4159.2003.01992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duman RS, Tallman JF, Nestler EJ. Acute and chronic opiate-regulation of adenylate cyclase in brain: specific effects in locus coeruleus. J Pharmacol Exp Ther. 1988;246:1033–1039. [PubMed] [Google Scholar]
  11. Ericson E, Ahlenius S. Suggestive evidence for inhibitory effects of galanin on mesolimbic dopaminergic neurotransmission. Brain Res. 1999;822:200–209. doi: 10.1016/s0006-8993(99)01144-0. [DOI] [PubMed] [Google Scholar]
  12. Funada M, Suzuki T, Sugano Y, Tsubai M, Misawa M, Ueda H, Misu Y. Role of beta-adrenoceptors in the expression of morphine withdrawal signs. Life Sci. 1994;54:PL113–118. doi: 10.1016/0024-3205(94)90010-8. [DOI] [PubMed] [Google Scholar]
  13. Gonzalez GA, Montminy MR. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell. 1989;59:675–680. doi: 10.1016/0092-8674(89)90013-5. [DOI] [PubMed] [Google Scholar]
  14. Guitart X, Thompson MA, Mirante CK, Greenberg ME, Nestler EJ. Regulation of cyclic AMP response element-binding protein (CREB) phosphorylation by acute and chronic morphine in the rat locus coeruleus. J Neurochem. 1992;58:1168–1171. doi: 10.1111/j.1471-4159.1992.tb09377.x. [DOI] [PubMed] [Google Scholar]
  15. Hawes JJ, Picciotto MR. Characterization of GalR1, GalR2, and GalR3 immunoreactivity in catecholaminergic nuclei of the mouse brain. J Comp Neurol. 2004;479:410–423. doi: 10.1002/cne.20329. [DOI] [PubMed] [Google Scholar]
  16. Herz A. Multiple opiate receptors and their functional significance. J Neural Transm Suppl. 1983;18:227–233. [PubMed] [Google Scholar]
  17. Hohmann JG, Jureus A, Teklemichael DN, Matsumoto AM, Clifton DK, Steiner RA. Distribution and regulation of galanin receptor 1 messenger RNA in the forebrain of wild type and galanin-transgenic mice. Neuroscience. 2003;117:105–117. doi: 10.1016/s0306-4522(02)00798-4. [DOI] [PubMed] [Google Scholar]
  18. Howard AD, Tan C, Shiao LL, Palyha OC, McKee KK, Weinberg DH, Feighner SD, Cascieri MA, Smith RG, Van Der Ploeg LH, Sullivan KA. Molecular cloning and characterization of a new receptor for galanin. FEBS Lett. 1997;405:285–290. doi: 10.1016/s0014-5793(97)00196-8. [DOI] [PubMed] [Google Scholar]
  19. Ivanov A, Aston-Jones G. Local opiate withdrawal in locus coeruleus neurons in vitro. J Neurophysiol. 2001;85:2388–2397. doi: 10.1152/jn.2001.85.6.2388. [DOI] [PubMed] [Google Scholar]
  20. Johannessen M, Delghandi MP, Moens U. What turns CREB on? Cell Signal. 2004;16:1211–1227. doi: 10.1016/j.cellsig.2004.05.001. [DOI] [PubMed] [Google Scholar]
  21. Kolakowski LF, Jr., O'Neill GP, Howard AD, Broussard SR, Sullivan KA, Feighner SD, Sawzdargo M, Nguyen T, Kargman S, Shiao LL, Hreniuk DL, Tan CP, Evans J, Abramovitz M, Chateauneuf A, Coulombe N, Ng G, Johnson MP, Tharian A, Khoshbouei H, George SR, Smith RG, O'Dowd BF. Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3. J Neurochem. 1998;71:2239–2251. doi: 10.1046/j.1471-4159.1998.71062239.x. [DOI] [PubMed] [Google Scholar]
  22. Koob GF, Nestler EJ. The neurobiology of drug addiction. J Neuropsychiatry Clin Neurosci. 1997;9:482–497. doi: 10.1176/jnp.9.3.482. [DOI] [PubMed] [Google Scholar]
  23. Kunkler PE, Wang GM, Hwang BH. Galanin-containing neurons in the solitary nucleus and locus coeruleus of spontaneously hypertensive rats are associated with genetic hypertension. Brain Res. 1994;651:349–352. doi: 10.1016/0006-8993(94)90718-8. [DOI] [PubMed] [Google Scholar]
  24. Maldonado R, Koob GF. Destruction of the locus coeruleus decreases physical signs of opiate withdrawal. Brain Res. 1993;605:128–138. doi: 10.1016/0006-8993(93)91364-x. [DOI] [PubMed] [Google Scholar]
  25. Maldonado R, Valverde O, Garbay C, Roques BP. Protein kinases in the locus coeruleus and periaqueductal gray matter are involved in the expression of opiate withdrawal. Naunyn Schmiedebergs Arch Pharmacol. 1995;352:565–575. doi: 10.1007/BF00169392. [DOI] [PubMed] [Google Scholar]
  26. Mennicken F, Hoffert C, Pelletier M, Ahmad S, O'Donnell D. Restricted distribution of galanin receptor 3 (GalR3) mRNA in the adult rat central nervous system. J Chem Neuroanat. 2002;24:257–268. doi: 10.1016/s0891-0618(02)00068-6. [DOI] [PubMed] [Google Scholar]
  27. Miller MA, Kolb PE, Leverenz JB, Peskind ER, Raskind MA. Preservation of noradrenergic neurons in the locus ceruleus that coexpress galanin mRNA in Alzheimer's disease. J Neurochem. 1999;73:2028–2036. [PubMed] [Google Scholar]
  28. Mounier F, Bluet-Pajot MT, Moinard F, Kordon C, Epelbaum J. Activation of locus coeruleus somatostatin receptors induces an increase of growth hormone release in male rats. J Neuroendocrinol. 1996;8:761–764. doi: 10.1046/j.1365-2826.1996.05115.x. [DOI] [PubMed] [Google Scholar]
  29. Nestler EJ. Historical review: Molecular and cellular mechanisms of opiate and cocaine addiction. TRENDS in Pharmacological Sciences. 2004;25:210–218. doi: 10.1016/j.tips.2004.02.005. [DOI] [PubMed] [Google Scholar]
  30. Nestler EJ, Greengard P. Protein phosphorylation in the brain. Nature. 1983;305:583–588. doi: 10.1038/305583a0. [DOI] [PubMed] [Google Scholar]
  31. Nestler EJ, Tallman JF. Chronic morphine treatment increases cyclic AMP-dependent protein kinase activity in the rat locus coeruleus. Mol Pharmacol. 1988;33:127–132. [PubMed] [Google Scholar]
  32. Nishibori M, Oishi R, Itoh Y, Saeki K. Galanin inhibits noradrenaline-induced accumulation of cyclic AMP in the rat cerebral cortex. J Neurochem. 1988;51:1953–1955. doi: 10.1111/j.1471-4159.1988.tb01185.x. [DOI] [PubMed] [Google Scholar]
  33. O'Donnell D, Ahmad S, Wahlestedt C, Walker P. Expression of the novel galanin receptor subtype GALR2 in the adult rat CNS: distinct distribution from GALR1. J Comp Neurol. 1999;409:469–481. [PubMed] [Google Scholar]
  34. Paxinos G, Franklin KBJ. The mouse brain in stereotaxic coordinates. 2nd Academic; San Diego, Calif. London: 2001. [Google Scholar]
  35. Punch LJ, Self DW, Nestler EJ, Taylor JR. Opposite modulation of opiate withdrawal behaviors on microinfusion of a protein kinase A inhibitor versus activator into the locus coeruleus or periaqueductal gray. J Neurosci. 1997;17:8520–8527. doi: 10.1523/JNEUROSCI.17-21-08520.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Seutin V, Verbanck P, Massotte L, Dresse A. Galanin decreases the activity of locus coeruleus neurons in vitro. Eur J Pharmacol. 1989;164:373–376. doi: 10.1016/0014-2999(89)90481-0. [DOI] [PubMed] [Google Scholar]
  37. Sevcik J, Finta EP, Illes P. Galanin receptors inhibit the spontaneous firing of locus coeruleus neurones and interact with mu-opioid receptors. Eur J Pharmacol. 1993;230:223–230. doi: 10.1016/0014-2999(93)90806-s. [DOI] [PubMed] [Google Scholar]
  38. Shaw-Lutchman TZ, Barrot M, Wallace T, Gilden L, Zachariou V, Impey S, Duman RS, Storm D, Nestler EJ. Regional and cellular mapping of cAMP response element-mediated transcription during naltrexone-precipitated morphine withdrawal. J Neurosci. 2002;22:3663–3672. doi: 10.1523/JNEUROSCI.22-09-03663.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shen PJ, Larm JA, Gundlach AL. Expression and plasticity of galanin systems in cortical neurons, oligodendrocyte progenitors and proliferative zones in normal brain and after spreading depression. Eur J Neurosci. 2003;18:1362–1376. doi: 10.1046/j.1460-9568.2003.02860.x. [DOI] [PubMed] [Google Scholar]
  40. Shih CD, Chan SH, Chan JY. Participation of endogenous galanin in the suppression of baroreceptor reflex response by locus coeruleus in the rat. Brain Res. 1996;721:76–82. doi: 10.1016/0006-8993(96)00163-1. [DOI] [PubMed] [Google Scholar]
  41. Smith KE, Walker MW, Artymyshyn R, Bard J, Borowsky B, Tamm JA, Yao WJ, Vaysse PJ, Branchek TA, Gerald C, Jones KA. Cloned human and rat galanin GALR3 receptors. Pharmacology and activation of G-protein inwardly rectifying K+ channels. J Biol Chem. 1998;273:23321–23326. doi: 10.1074/jbc.273.36.23321. [DOI] [PubMed] [Google Scholar]
  42. Suri C, Fung BP, Tischler AS, Chikaraishi DM. Catecholaminergic cell lines from the brain and adrenal glands of tyrosine hydroxylase-SV40 T antigen transgenic mice. J Neurosci. 1993;13:1280–1291. doi: 10.1523/JNEUROSCI.13-03-01280.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sweerts BW, Jarrott B, Lawrence AJ. Expression of preprogalanin mRNA following acute and chronic restraint stress in brains of normotensive and hypertensive rats. Brain Res Mol Brain Res. 1999;69:113–123. doi: 10.1016/s0169-328x(99)00095-9. [DOI] [PubMed] [Google Scholar]
  44. Tseng JY, Kolb PE, Raskind MA, Miller MA. Estrogen regulates galanin but not tyrosine hydroxylase gene expression in the rat locus ceruleus. Brain Res Mol Brain Res. 1997;50:100–106. doi: 10.1016/s0169-328x(97)00164-2. [DOI] [PubMed] [Google Scholar]
  45. Tsuda K, Tsuda S, Nishio I, Masuyama Y, Goldstein M. Effects of galanin on dopamine release in the central nervous system of normotensive and spontaneously hypertensive rats. Am J Hypertens. 1998;11:1475–1479. doi: 10.1016/s0895-7061(98)00168-x. [DOI] [PubMed] [Google Scholar]
  46. Valentino RJ, Aston-Jones G. Activation of locus coeruleus neurons in the rat by a benzazocine derivative (UM 1046) that mimics opiate withdrawal. Neuropharmacology. 1983;22:1363–1368. doi: 10.1016/0028-3908(83)90225-3. [DOI] [PubMed] [Google Scholar]
  47. Van Hoomissen JD, Holmes PV, Zellner AS, Poudevigne A, Dishman RK. Effects of beta-adrenoreceptor blockade during chronic exercise on contextual fear conditioning and mRNA for galanin and brain-derived neurotrophic factor. Behav Neurosci. 2004;118:1378–1390. doi: 10.1037/0735-7044.118.6.1378. [DOI] [PubMed] [Google Scholar]
  48. Waltereit R, Weller M. Signaling from cAMP/PKA to MAPK and synaptic plasticity. Mol Neurobiol. 2003;27:99–106. doi: 10.1385/MN:27:1:99. [DOI] [PubMed] [Google Scholar]
  49. Wang S, Hashemi T, He C, Strader C, Bayne M. Molecular cloning and pharmacological characterization of a new galanin receptor subtype. Mol Pharmacol. 1997;52:337–343. doi: 10.1124/mol.52.3.337. [DOI] [PubMed] [Google Scholar]
  50. Wang S, Hashemi T, Fried S, Clemmons AL, Hawes BE. Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes. Biochemistry. 1998;37:6711–6717. doi: 10.1021/bi9728405. [DOI] [PubMed] [Google Scholar]
  51. Waters SM, Krause JE. Distribution of galanin-1, -2 and -3 receptor messenger RNAs in central and peripheral rat tissues. Neuroscience. 2000;95:265–271. doi: 10.1016/s0306-4522(99)00407-8. [DOI] [PubMed] [Google Scholar]
  52. Widnell KL, Russell DS, Nestler EJ. Regulation of expression of cAMP response element-binding protein in the locus coeruleus in vivo and in a locus coeruleus-like cell line in vitro. Proc Natl Acad Sci U S A. 1994;91:10947–10951. doi: 10.1073/pnas.91.23.10947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Williams JT, Christie MJ, Manzoni O. Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev. 2001;81:299–343. doi: 10.1152/physrev.2001.81.1.299. [DOI] [PubMed] [Google Scholar]
  54. Wittau N, Grosse R, Kalkbrenner F, Gohla A, Schultz G, Gudermann T. The galanin receptor type 2 initiates multiple signaling pathways in small cell lung cancer cells by coupling to G(q), G(i) and G(12) proteins. Oncogene. 2000;19:4199–4209. doi: 10.1038/sj.onc.1203777. [DOI] [PubMed] [Google Scholar]
  55. Wynick D, Bacon A. Targeted disruption of galanin: new insights from knock-out studies. Neuropeptides. 2002;36:132–144. doi: 10.1054/npep.2002.0888. [DOI] [PubMed] [Google Scholar]
  56. Xu ZQ, Tong YG, Hokfelt T. Galanin enhances noradrenaline-induced outward current on locus coeruleus noradrenergic neurons. Neuroreport. 2001;12:1779–1782. doi: 10.1097/00001756-200106130-00052. [DOI] [PubMed] [Google Scholar]
  57. Zachariou V, Parikh K, Picciotto MR. Centrally administered galanin blocks morphine place preference in the mouse. Brain Res. 1999;831:33–42. doi: 10.1016/s0006-8993(99)01476-6. [DOI] [PubMed] [Google Scholar]
  58. Zachariou V, Thome J, Parikh K, Picciotto MR. Upregulation of galanin binding sites and GalR1 mRNA levels in the mouse locus coeruleus following chronic morphine treatments and precipitated morphine withdrawal. Neuropsychopharmacology. 2000;23:127–137. doi: 10.1016/S0893-133X(00)00094-4. [DOI] [PubMed] [Google Scholar]
  59. Zachariou V, Georgescu D, Kansal L, Merriam P, Picciotto MR. Galanin receptor 1 gene expression is regulated by cyclic AMP through a CREB-dependent mechanism. J Neurochem. 2001;76:191–200. doi: 10.1046/j.1471-4159.2001.00018.x. [DOI] [PubMed] [Google Scholar]
  60. Zachariou V, Brunzell DH, Hawes J, Stedman DR, Bartfai T, Steiner RA, Wynick D, Langel U, Picciotto MR. The neuropeptide galanin modulates behavioral and neurochemical signs of opiate withdrawal. Proc Natl Acad Sci U S A. 2003;100:9028–9033. doi: 10.1073/pnas.1533224100. [DOI] [PMC free article] [PubMed] [Google Scholar]

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