The Neuropeptide Galanin Modulates Behavioral and Neurochemical Signs of Opiate Withdrawal

The neuropeptide galanin modulates behavioral and neurochemical signs of opiate withdrawal Venetia Zachariou*†, Darlene H. Brunzell*, Jessica Hawes*, Diann R. Stedman*, Tamas Bartfai‡, Robert A. Steiner§, David Wynick¶,U¨ lo Langelʈ, and Marina R. Picciotto*,** *Department of Psychiatry, Yale University School of Medicine, New Haven, CT 06508; †Faculty of Medicine, Department of Pharmacology, University of Crete, 711 10 Crete, Greece; ‡Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037; §Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195; ¶Department of Medicine, Bristol University, Bristol BS2 8HW, United Kingdom; and ʈDepartment of Neurochemistry and Neurotoxicology, Stockholm University, 106 91 Stockholm, Sweden Edited by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, and approved June 2, 2003 (received for review May 23, 2003) Much research has focused on pathways leading to opiate addic- galanin-mediated effects on morphine withdrawal were exam- tion. Pathways opposing addiction are more difficult to study but ined in transgenic mice that overexpress the peptide under the may be critical in developing interventions to combat drug depen- control of the dopamine-␤-hydroxylase (D␤H) promoter, which dence and withdrawal. Galanin decreases firing of locus coeruleus normally drives gene expression in noradrenergic neurons (20), neurons, an effect hypothesized to decrease signs of opiate with- and in studies of c-fos and tyrosine hydroxylase (TH) phosphor- drawal. The current study addresses whether galanin affects mor- ylation in the LC of wild-type mice administered galnon. phine withdrawal signs by using a galanin agonist, galnon, that crosses the blood–brain barrier, and mice genetically engineered Methods to under- or overexpress galanin peptide. Galnon significantly Animals. Galanin knockout mice. Experimental animals were mice ͞ decreased morphine withdrawal signs in C57BL 6 mice. Further, homozygous for a targeted mutation of the galanin gene on an knockout mice lacking galanin showed exacerbated morphine inbred 129OlaHsd background (F18; GalϪ͞Ϫ; ref. 19), and withdrawal signs, suggesting that endogenous galanin normally controls were 129OlaHsd wild-type mice (Galϩ͞ϩ). counteracts opiate withdrawal. Transgenic mice overexpressing Galanin-overexpressing mice. Mice overexpressing galanin under the galanin in noradrenergic neurons also showed decreased mor- control of D␤H promoter (20) were backcrossed onto the phine withdrawal signs, suggesting a possible neuroanatomical ͞ locus for these effects of galanin. Both c-fos immunoreactivity, a C57BL 6 background for seven generations. Experimental an- marker of neuronal activity, and phosphorylation of tyrosine imals were generated by crossing mice heterozygous for the ͞ hydroxylase at Ser-40, a marker of cAMP levels, are decreased in transgene with C57BL 6 wild-type mice to generate littermate the locus coeruleus by galnon treatment after morphine with- controls of which 50% were positive for the transgene (Gal-tg) drawal, suggesting a possible molecular mechanism for the be- and 50% were negative for the transgene (wild type). havioral effects of galanin. These studies suggest that galanin Pharmacological studies. Galnon was synthesized as has been de- normally acts to counteract opiate withdrawal and that small scribed (18). C57BL͞6 mice (The Jackson Laboratory) or molecule galanin agonists could be effective in diminishing the 129OlaHsd mice were used for pharmacological studies. physical signs of withdrawal. For all studies, mice were used at 8–12 weeks of age and were age- and sex-matched. he neuropeptide galanin is widely expressed in brain (1) and Tdecreases neurochemical and behavioral effects related to Opiate Withdrawal. Mice were injected i.p. with escalating mor- opiate reinforcement. Galanin is neither rewarding nor aversive, phine doses every 8 h (20, 40, 60, 80, 100, 100, and 100 mg͞kg). but the peptide attenuates morphine place preference (2). Two hours after the last morphine injection, mice were injected Galanin also inhibits mesolimbic dopamine neurotransmission with naloxone (1 mg͞kg s.c.), and withdrawal symptoms (jump- (3, 4), an effect critical for rewarding properties of drugs of abuse ing, wet-dog shakes, backward walking, paw tremor, tremor, (5). In addition, galanin inhibits the firing of locus coeruleus diarrhea, ptosis, and weight loss) were monitored for 30 min (LC) neurons (6), which may affect expression of opiate with- after naloxone administration. In addition to measuring indi- drawal signs (7). Further, stress increases galanin mRNA in the vidual withdrawal signs, an overall opiate withdrawal score was LC (8), and at least one galanin receptor subtype, GalR1, is calculated as (no. of backward walking steps ϫ 0.1) ϩ (diar- up-regulated in the LC during opiate withdrawal (9). Thus, rhea ϫ 2) ϩ (no. of jumps ϫ 0.1) ϩ (paw tremor ϫ 0.1) ϩ (ptosis) galanin may counteract opiate withdrawal through effects on the ϩ (tremor) ϩ (% weight loss ϫ 5) ϩ (no. of wet-dog shakes) noradrenergic system. (modified from ref. 21). Galanin mediates its effects through activation of at least three receptor subtypes coupled to Gi, Gq, or Go (10, 11). These Morphine Analgesia and Tolerance Tests. For morphine tolerance receptors show distinct patterns of distribution and activate studies, mice were placed on a 52°C hot plate apparatus, and different second messenger pathways, depending on cell type latency to paw lick was measured. A cutoff time of 30 s was set (11–14). The galanin receptors and peptide are expressed in to avoid tissue damage. Mice were tested once daily for 3–5 days. many brain regions, and all are expressed in the LC (8, 15–17). Data were calculated as percent maximal possible effect [MPE ϭ Galnon, a galanin agonist, can cross the blood–brain barrier, ␮ (test latency Ϫ baseline latency)͞(cutoff Ϫ baseline latency)]. has moderate affinity for GalRs (4.8 M), does not distinguish ͞ among GalR subtypes, and has significantly lower affinity when Morphine (10 mg kg) was administered s.c. 30 min before screened against a broad panel of nongalanin receptors (includ- ing opiate receptors; ref. 18). Thus, this compound can be This paper was submitted directly (Track II) to the PNAS office. considered a relatively selective galanin agonist. The current Abbreviations: D␤H, dopamine-␤-hydroxylase; LC, locus coeruleus; P, phosphorylated; TH, study was designed to determine whether galnon could affect tyrosine hydroxylase. morphine withdrawal signs. In addition, the role of endogenous **To whom correspondence should be addressed at: Department of Psychiatry, Yale galanin in modulating opiate withdrawal was studied in galanin University School of Medicine, 34 Park Street, New Haven, CT 06508. E-mail: marina. knockout mice (19). Further, potential site(s) of action for [email protected]. 9028–9033 ͉ PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1533224100 Downloaded by guest on September 30, 2021 testing. Galnon (or vehicle) was administered i.p. (2 mg͞kg) 15 Biomax MR film. The bottom portion of each blot was subse- min before morphine administration. quently stripped with 0.2 M NaOH and reprobed with 1:2,000 TH antibody (Chemicon), followed by secondary 1:2,000 anti- c-fos Immunohistochemistry and TH Western Blotting. Four groups of rabbit IgG antibody. All bands were quantified by using NIH mice were used for c-fos experiments (n ϭ 6 per group) and TH IMAGE. Protein loading was verified by using Ponceau staining. Western blotting experiments (n ϭ 5–7 per group). The Sal–Veh Band intensities were corrected for background and normalized group received chronic saline treatment as described for mor- to PYK2 levels, which had previously been shown to remain phine withdrawal studies and an acute vehicle injection, and 15 constant across treatment groups. Band intensities from the liver min later receiveda1mg͞kg injection of naloxone. The Sal–Gal sample were used as an internal control for comparison between group received chronic saline treatment and an acute galnon individual blots. PC12 cell extracts at known concentrations were injection (2 mg͞kg), and 15 min later receiveda1mg͞kg used to generate a concentration curve and calculate the total injection of naloxone. The Mor–Veh group received chronic nanograms of TH, as well as nanograms of TH phosphorylated morphine treatment and a vehicle injection, and 15 min later on Ser-40 (P-Ser-40), for each LC sample. Using these standard receiveda1mg͞kg injection of naloxone. The Mor–Gal group curves, the percentage of TH phosphorylated on Ser-40 was received chronic morphine treatment and a galnon injection (2 determined ([P-Ser-40]͞[total TH] ϫ 100). Significance was mg͞kg), and 15 min later receiveda1mg͞kg injection of determined by using repeated-measures ANOVA and the least naloxone. significant difference post hoc test (P Ͻ 0.05). c-fos immunohistochemistry was performed essentially as has been described (22). Briefly,2hafternaloxone injection, mice Results were injected with chloral hydrate and perfused with 0.1 M PBS The small molecule galanin agonist galnon has been shown to act for 5 min, followed by a 15-min perfusion with 4% paraformal- as an anticonvulsant (18), but its effects on opiate-related dehyde. Brains were removed, postfixed for 24 h, and cryopro- behaviors have not been tested. Systemically administered gal- tected in 20% sucrose. Forty-micrometer coronal sections were

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