The 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 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 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 (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 non (2 mg͞kg i.p.) significantly attenuated several opiate with- cut at the level of the LC and periacqueductal gray (PAG) by drawal signs in C57BL͞6 mice (Fig. 1A). Backward walking, using a Leica microtome (approximately at Bregma level Ϫ5.52). chewing, diarrhea, jumping, paw tremor, and weight loss were all Based on anatomical markers, including ventricle shape and significantly reduced after galnon administration. Consistent presence of the VII cranial nerve tract, two to four sections per with these changes, the overall withdrawal score was significantly brain were selected for c-fos immunocytochemistry. Sections reduced (P Ͻ 0.01) in galnon-treated vs. vehicle-treated mice were washed in 0.1 M PBS, blocked with 5% normal goat serum, (Fig. 1B). Peripherally administered galnon had no effect on and incubated overnight with anti-c-fos antiserum (1:5,000, pain threshold (Fig. 1C) or morphine-induced analgesia and Santa Cruz Biotechnology) at 4°C for 48 h. After three washes tolerance (Fig. 1D) at the dose used. in 0.1 M PBS, sections were incubated with biotinylated goat Galnon experiments suggested that pharmacological activa- anti-rabbit Ig (1:200), washed three times in 0.1 M PBS, and tion of galanin receptors can decrease opiate withdrawal signs, incubated with biotin-streptavidin-peroxidase complex (1:200), but could not test whether activation of galanin receptors by and the peroxidase reaction product was visualized by 3,3Ј- endogenous galanin normally plays a role in protection against diaminobenzidine (Vector Laboratories). Sections were opiate dependence and withdrawal. Knockout mice lacking the mounted on electrostatic Superfrost glass slides, dehydrated, galanin gene on an inbred 129OlaHsd genetic background were and coverslipped with prolonged antifade medium (Molecular tested for severity of morphine withdrawal. GalϪ͞Ϫ mice Probes). Using NIH IMAGE software (http:͞͞rsb.info.nih.gov͞ showed significantly more withdrawal signs than Galϩ͞ϩ mice nih-image), a defined box was placed over the center of the LC (Fig. 2A). Backward walking, diarrhea, rearing, weight loss, and on each side of the brain (see Fig. 4) for each section, and all wet-dog shakes were all significantly increased in GalϪ͞Ϫ mice. c-fos-positive nuclei were counted within the box. The number In addition, overall withdrawal scores were significantly different of positive nuclei reported represents the average of the right and (P Ͻ 0.01) between Galϩ͞ϩ and GalϪ͞Ϫ mice (Fig. 2B). It is left side from two sections for each mouse. All slides were important to note that the 129OlaHsd strain shows much less NEUROSCIENCE quantitated at the same time under the same conditions. jumping behavior than the C57BL͞6 strain during opiate with- For TH and phosphorylated (P) TH Western blotting studies, drawal [the 129OlaHsd mice showed Ͻ3 jumps over the course mice were killed by rapid decapitation 15 min after naloxone of the experiment (Fig. 2A), whereas C57BL͞6 mice showed administration, and 1-mm punches of the LC [close to Bregma Ͼ120 jumps (Fig. 1A)]. A higher dose of naloxone (4 mg͞kg) Ϫ5.52 mm (23)] were taken and immediately frozen in liquid results in more severe withdrawal signs in 129OlaHsd mice, but nitrogen (24). Groups were identical to those described above for does not result in increased jumping behavior (data not shown). c-fos studies [Sal–Veh (n ϭ 5), Sal–Gal (n ϭ 5), Mor–Veh Thus, the lack of jumping response in this experiment is likely to (n ϭ 5), and Mor–Gal (n ϭ 7)]. LC punches were homogenized be a strain-related phenomenon. in lysis 1 reagent (RPA538, Amersham Biosciences), and protein To determine whether the altered morphine withdrawal seen concentrations were determined (Bio-Rad Lowry reagents). in GalϪ͞Ϫ mice is due, at least in part, to acute effects during Five micrograms of protein for each sample were separated on the expression of withdrawal signs, rather than involvement of SDS͞8.5% PAGE gels and transferred to nitrocellulose mem- galanin during chronic morphine administration or adaptation branes. For quantitation, 10 ␮g of liver extract and 0.0125–0.1 ng to effects of galanin during development, GalϪ͞Ϫ mice were of Ser-40P-TH PC12 standard (generously provided by J. Hay- treated acutely with galnon (2 mg͞kg) or vehicle 15 min before cock, Louisiana State University, Baton Rouge) were also naloxone injection (1 mg͞kg). Galnon administration reversed loaded. Each blot was cut at the 77-kDa protein marker and several signs of opiate withdrawal in GalϪ͞Ϫ mice to wild-type blocked in 5% BSA. The bottom portion of each blot was levels, including backward walking, diarrhea, rearing, tremor, incubated overnight at 4°C in 1:200 Ser-40P-TH antibody (25), weight loss, and wet-dog shakes (Fig. 2A), and reduced the and the top portion was incubated in 1:250 PYK2 antibody overall withdrawal score (Fig. 2B). Paw tremor was reduced (Upstate Biotechnology, Lake Placid, NY). After washing with below the level seen in Galϩ͞ϩ mice, suggesting that this sign Tris-buffered saline͞0.1% Triton X-100, blots were incubated might be particularly sensitive to exogenous galnon administra- with peroxidase-labeled 1:2,000 anti-rabbit IgG (Vector Labo- tion. Withdrawal signs not affected by galnon treatment in ratories) for1hatroom temperature. Bands were visualized by galanin knockout mice were also not strongly expressed in using enhanced chemiluminescence and exposure to Kodak Galϩ͞ϩ mice.

Zachariou et al. PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 ͉ 9029 Downloaded by guest on September 30, 2021 Fig. 1. Galnon attenuates morphine withdrawal. (A) Mice were administered increasing doses of morphine every 8 h for 3 days (20, 40, 60, 80, 100, 100, and 100 mg͞kg s.c.), and withdrawal was precipitated 2 h after the last morphine dose by using naloxone (1 mg͞kg s.c.). One group received naloxone alone (n ϭ 8), and a second group received 2 mg͞kg galnon 15 min before naloxone injection (n ϭ 7). After naloxone administration, withdrawal signs were scored for 30 min by an observer blind to treatment. For each withdrawal sign, comparisons were made between galnon- and vehicle-treated mice by using Student’s t test. (B) The overall withdrawal score was significantly decreased by galnon administration (P Ͻ 0.01). (C) Galnon (2 mg͞kg) alone did not alter pain threshold as measured on the 52°C hotplate. (D) No differences in initial analgesic response to morphine or the development of morphine tolerance was seen between galnon-treated and vehicle-treated control mice. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001.

GalϪ͞Ϫ mice have previously been shown to have alterations crease in nociceptive threshold was seen in GalϪ͞Ϫ mice (Fig. in neuropathic pain, survival of cholinergic neurons, and overall 2C), morphine analgesia and tolerance to morphine were not excitability (22, 26–28). Although a small, but significant, de- significantly different between GalϪ͞Ϫ and Galϩ͞ϩ mice (Fig.

Fig. 2. Knockout of the galanin gene exacerbates morphine withdrawal, and this is reversed by treatment with galnon. (A) GalϪ͞Ϫ mice on the 129OlaHsd background (n ϭ 8) and Galϩ͞ϩ mice (n ϭ 8) were administered increasing doses of morphine every 8 h for 3 days (20, 40, 60, 80, 100, 100, and 100 mg͞kg s.c.), and withdrawal was precipitated 2 h after the last morphine dose by using naloxone (1 mg͞kg s.c.). After naloxone administration, withdrawal signs were scored for 30 min by an observer blind to treatment. An independent group of GalϪ͞Ϫ mice was treated with 2 mg͞kg galnon 15 min before naloxone injection (n ϭ 6). One-way ANOVA was used to compare differences in withdrawal signs between Galϩ͞ϩ mice, GalϪ͞Ϫ mice, and GalϪ͞Ϫ mice treated with galnon. (B)A significant effect of genotype on overall withdrawal scores was identified by one-way ANOVA. Using the least significant difference post hoc test, overall withdrawal scores were significantly different between Galϩ͞ϩ and GalϪ͞Ϫ mice (P Ͻ 0.01), and between GalϪ͞Ϫ mice treated with vehicle or galnon (P Ͻ 0.01). (C) Pain thresholds as measured on the 52°C hotplate were slightly decreased in GalϪ͞Ϫ mice as compared with Galϩ͞ϩ mice. (D) No differences in initial analgesic response to morphine or the development of morphine tolerance was seen between Galϩ͞ϩ and GalϪ͞Ϫ mice. *, P Ͻ 0.05; *, P Ͻ 0.01; ***, P Ͻ 0.001.

9030 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1533224100 Zachariou et al. Downloaded by guest on September 30, 2021 Fig. 3. Galanin overexpression under control of the D␤H promoter can attenuate some signs of morphine withdrawal. (A) Transgenic mice overexpressing galanin under control of the D␤H promoter (n ϭ 10) and their wild-type siblings (n ϭ 9) were administered increasing doses of morphine every 8 h for 3 days (20, 40, 60, 80, 100, 100, and 100 mg͞kg s.c.), and withdrawal was precipitated 2 h after the last morphine dose by using naloxone (1 mg͞kg s.c.). After naloxone administration, withdrawal signs were scored for 30 min by an observer blind to treatment. One-way ANOVA was used to compare differences in withdrawal signs between genotype. (B) The overall withdrawal score was significantly reduced in Gal-tg mice as compared with their wild-type siblings (P Ͻ 0.01). (C)No difference in pain threshold was seen between Gal-tg and their wild-type siblings as measured on the 52°C hotplate. (D) No differences in initial analgesic response to morphine or the development of morphine tolerance was seen between Gal-tg and wild-type mice. tg, Gal-tg mice; wt, wild-type siblings; *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001.

2D). Whereas C57BL͞6 mice showed complete tolerance to A likely mechanism underlying the decreased firing rate of LC morphine analgesia by 3 days of treatment (Fig. 1D), 129OlaHsd neurons reflected in c-fos measurements is a decrease in cAMP mice required treatment for 5 days to achieve full morphine levels as a result of activation of GalRs. It has previously been tolerance (Fig. 2D). shown that Ser-40 in TH is specifically phosphorylated by One method of identifying the site of action for galnon or cAMP-dependent protein kinase (PKA) (25). Thus, TH Ser-40 endogenous galanin in attenuating opiate withdrawal is to phosphorylation can be used as a measure of cellular cAMP determine whether localized overexpression of galanin can levels in noradrenergic neurons. Further, because only norad- mimic the effect of peripherally administered galnon. Trans- renergic neurons in this brain region express TH, the examina- genic mice expressing the galanin cDNA under the control of a tion of the phosphorylation state of TH is a sensitive assay for D␤H promoter that targets expression to noradrenergic neurons changes in cAMP in this subclass of neurons (24). Morphine (20) were examined for opiate withdrawal signs. Significant withdrawal significantly increased the stoichiometry of Ser-40 Ͻ decreases were seen in backward walking, chewing, diarrhea, phosphorylation (P 0.001; Fig. 4C). In contrast, galnon jumping, tremor, and wet-dog shakes in Gal-tg mice as compared treatment decreased the phosphorylation of Ser-40 back to NEUROSCIENCE Ͻ with their wild-type siblings (Fig. 3A), as well as in overall baseline levels (P 0.05, galnon vs. vehicle). Total TH levels withdrawal scores (Fig. 3B). These data suggest that galanin appeared to increase as a result of morphine withdrawal, but it effects on opiate withdrawal signs may be mediated through was not possible to determine whether this was a true increase or reflected residual IgG bound to P-Ser-40 (blots for total TH actions on the noradrenergic (or potentially the adrenergic) were performed after stripping after P-Ser-40 blotting to allow system and͞or targets of noradrenergic innervation in the brain measurement of total TH and P-TH from the same LC samples). and periphery. Pain thresholds of galanin transgenic mice were Because ratios of P-Ser-40 to total TH were used for all similar to those of their wild-type littermates in the hotplate test measurements, the increase in stoichiometry after morphine (Fig. 3C), and no differences were observed for responses to withdrawal may represent an underestimate. morphine or morphine tolerance (Fig. 3D) between Gal-tg mice and their wild-type siblings. Discussion Based on the transgenic mouse studies and previous physio- The circuitry underlying the development of addiction and logical studies (6), a possible mechanism underlying galnon tolerance has been widely explored (for review, see refs. 29 and antagonism of opiate withdrawal signs could be decreased firing 30). In contrast, endogenous pathways that oppose addictive of noradrenergic neurons. We used c-fos as a marker of neuronal processes have been more difficult to study but may be critical activation and compared c-fos immunoreactivity in the LC of in developing pharmacological interventions to combat opiate wild-type mice treated with galnon or vehicle before naloxone- dependence and withdrawal. Using pharmacological and genetic precipitated withdrawal (Fig. 4 A and B). A decrease in c-fos- models, we have shown that the galanin system modulates labeled LC neurons was observed in mice treated with galnon morphine withdrawal. When galanin is not expressed, as in alone in the absence of opiate withdrawal, but this did not reach GalϪ͞Ϫ mice, opiate withdrawal signs are more severe. In significance. There was a significant increase in c-fos immuno- contrast, decreased opiate withdrawal is seen when galanin is reactivity in the LC after morphine withdrawal (P Ͻ 0.0004), and overexpressed under the control of the D␤H promoter in this increase was significantly attenuated in galnon-treated mice transgenic mice. Systemic administration of a as compared with vehicle-treated mice (P Ͻ 0.05). agonist also alleviates opiate withdrawal symptoms, suggesting

Zachariou et al. PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 ͉ 9031 Downloaded by guest on September 30, 2021 Fig. 4. c-fos immunoreactivity and TH phosphorylation in the LC after galnon administration after naloxone-stimulated withdrawal. Mice treated with morphine and naloxone showed significantly more c-fos immunoreactivity and phosphorylation of TH Ser-40 in the LC than mice treated with naloxone alone. Galnon significantly attenuated the induction of c-fos immunoreactivity, as well as TH phosphorylation, in morphine- and naloxone-treated mice. A decrease in c-fos immunoreactivity was seen in the LC of naloxone-treated mice after galnon (2 mg͞kg) administration, but this did not reach significance. (A) Representative photomicrographs of c-fos staining at the level of the LC. A section through the LC after morphine withdrawal is shown at left to identify the areas of c-fos immunoreactivity quantified for all treatment groups. The boxed region shown at higher magnification for each treatment group represents the area counted for each section. Counts were averaged from left and right LC across two sections per mouse matched for anatomical markers (23). (B) Quantitation of c-fos immunoreactivity. There was a significant effect of naloxone-precipitated withdrawal on c-fos immunoreactivity in the LC (ANOVA, P Ͻ 0.0005). *, P Ͻ 0.05 galnon vs. vehicle. (C) Stoichiometry of TH phosphorylation at Ser-40. Representative Western blots are shown for Ser-40-P, total TH, and pyk2 (as a control for protein loading). There was a significant increase in Ser-40-P levels and stoichiometry in mice treated with morphine and naloxone that was significantly attenuated by galnon administration. S-V, saline–vehicle group; S-G, saline–galnon group; M-V, morphine–vehicle group; M-G, morphine–gainon group. *, P Ͻ 0.05 galnon vs. vehicle.

that the galanin system could be a target for the development of galanin, because the peptide can be rapidly proteolyzed. In agents that reduce opiate withdrawal. addition, mice that received galnon were not cannulated, The present study provides evidence that modulation of opiate whereas cannulated mice may have experienced stress as a result withdrawal by galanin is mediated, at least in part, through of the surgical procedure that could modulate the effects of effects in noradrenergic neurons. It is also possible that galanin galanin. We cannot rule out that peripheral actions of galnon are and galanin receptors localized beyond the noradrenergic system responsible for its behavioral effects, although this is unlikely given could be involved in modulation of morphine dependence as that many withdrawal signs have been shown to depend on central well. Gal-tg mice overexpress galanin in the noradrenergic activity (32) and that peripherally administered galanin had no nuclei, the piriform and entorhinal cortices, and the subiculum effect on withdrawal signs in the current study (data not shown). (26), and also have increased galanin expression in the pituitary Galanin receptors are modulated during opiate dependence, and adrenal glands (J. G. Hohmann and R.S., unpublished data). because both galanin receptor levels, as measured by radioligand Thus, actions of galnon outside the noradrenergic system may binding, and GalR1 mRNA levels are increased in the LC after contribute to its effects on opiate withdrawal signs. opiate withdrawal (9). In contrast, galanin peptide mRNA levels Data obtained by using galnon to alleviate opiate withdrawal do not change during withdrawal (31). This finding suggests that signs suggest that the galanin system is critical during the levels of galanin receptors, and their regulation, are a critical expression of withdrawal signs. One concern in studies of mice factor in determining the actions of the endogenous galanin with constitutive knockout or overexpression of a gene of system on opiate withdrawal. Galanin (33) and GalR1 (34) interest is that phenotypic differences reflect a role for the expression are also regulated after activation of the cAMP protein during development and not during adulthood. For pathway. Up-regulation of the cAMP pathway results in the example, galanin knockout mice show deficits in development of increased neuronal activity observed in the LC during with- the dorsal root ganglia (27) and basal forebrain (28). The effects drawal (35–37) and in adaptive changes in expression of a variety seen here on opiate withdrawal are less likely to be due to an of genes in several brain regions (38). Because GalR1 is nega- irreversible developmental defect, however. First, galnon can be tively coupled to cAMP generation (12), and this effect appears administered to adult mice, and its effect is similar to the to be responsible for the ability of galanin to decrease firing rate decrease in opiate withdrawal signs seen in Gal-tg mice. Further, in the LC (39), up-regulation of the particular receptors might the increased withdrawal signs seen in GalϪ͞Ϫ mice can be indicate a compensatory mechanism for the increased neuronal reversed in adulthood by administration of galnon. These phar- activity during opiate withdrawal. We have shown that periph- macological data suggest that galanin plays a role acutely in the eral galnon administration attenuates the increase in LC cAMP adult mouse in the expression of opiate withdrawal signs. due to morphine withdrawal. Thus, it is likely that the action of However, the fact that galnon did not reduce withdrawal signs GalRs on adenylyl cyclase activity contributes to the effect of other than paw tremor in GalϪ͞Ϫ mice below those of untreated galnon on behavioral signs of opiate withdrawal. Galϩ͞ϩ mice could suggest that a higher dose of galnon might Although GalR1 is a likely candidate for mediating the effects be necessary to see a baseline effect on opiate withdrawal signs of galanin on LC firing, both GalR1 and GalR2 are found in in mice on the 129OlaHsd background. areas involved in the development of morphine dependence, A previous study infused a single dose of the galanin peptide including the LC, the periaqueductal gray, the nucleus accum- into the cerebral ventricle of rats and did not observe an effect bens, and the spinal cord. Recent studies have also reported that on opiate withdrawal (31). This discrepancy suggests that local the GalR3 subtype is expressed in the LC (16). GalR3 is also administration of galanin might not be sufficient to counteract negatively coupled to adenylyl cyclase and therefore might play the broad spectrum of opiate withdrawal symptoms because of a role in attenuating behaviors related to opiate withdrawal. limited diffusion, rapid proteolysis, or the necessity of galanin Galanin affects behavioral signs of morphine dependence, but action at sites outside the brain. In the current study, galnon was tolerance to morphine analgesia does not appear to be modu- administered at a high concentration and was administered lated by the peptide. Pain thresholds were normal in galanin- peripherally. Galnon also has a longer duration of action than overexpressing mice and in mice treated with galnon. Consistent

9032 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1533224100 Zachariou et al. Downloaded by guest on September 30, 2021 with previous observations (22), galanin knockout mice had nizes morphine place preference (2). We show here that a lower pain thresholds for thermal stimuli. In addition, transgenic systemically administered galanin agonist also attenuates opiate overexpression of galanin in the dorsal root ganglia attenuates withdrawal. In addition, we present evidence that the underlying allodynia (40). However, neither pharmacological nor genetic mechanism for these behavioral effects could be decreased manipulation of the galanin system had an effect on the devel- cAMP signaling in the LC (although effects on other brain areas opment of opiate tolerance in mice in the current study. Al- may also contribute). Together, these findings suggest that though galnon did not have an effect on the development of galanin agonists would be ideal analgesics because they would opiate analgesia or tolerance at the dose used here, we cannot potentiate morphine analgesia and decrease morphine abuse exclude the possibility that this compound might affect nocicep- potential. Further, the current results indicate that galanin tive transmission when applied in higher doses or when used in agonists could also have pharmacological value for the allevia- different pain models. tion of opiate withdrawal in dependent individuals. A number of studies have suggested that galanin agonists could have pharmacological value as analgesics because intra- We thank Dr. John W. Haycock for antiserum against TH Ser-40 and thecal galanin administration potentiates morphine analgesia Christina Lee for technical assistance. This work was supported by (predominantly in models of chronic pain), and thus, lower National Institutes of Health Grants DA15425 and DA00436 and the morphine doses could be coadministered with galanin to pro- Christiane Brooks Johnson Foundation. U¨ .L. is supported by the Swed- duce an analgesic effect (41–45). In addition, galanin antago- ish Medical Research Council.

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