Neuropeptides

Neuropeptides 38 (2004) 55–61

www.elsevier.com/locate/npep

Regulation of feeding by galnon

Urho Abramov a, Anders Flor�en b, David J. Echevarria c, Ariel Brewer c,Honeyleen Manuzon c, John K. Robinson c, Tamas Bartfai d, Eero Vasar a, €Ulo Langel b,*

a Department of Physiology, Tartu University, Ravila 19, Tartu 50 411, Estoniab Department of Neurochemistry and Neurotoxicology, Stockholm University, S. Arrheniusv€agen 21A, Stockholm SE-106 91, Sweden

c Department of Psychology, SUNY at Stony Brook, Stony Brook, NY 11733-2500, USAd Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037, USA

Received 9 June 2003; accepted 6 January 2004

Abstract

Galanin is a neuropeptide that has been implicated in multiple bioactivities, inter alia eating disorders. In this study, we have

examined the effects of galnon, a novel low molecular weight galanin receptor ligand. Previous studies have shown that galnon acts

as a systemically active, blood–brain barrier crossing agonist on galanin signaling both in vitro and in vivo, inhibiting pentylen-

etetrazole-induced seizures. Here, intracerebroventricular (10–20 lg) and intraperitoneal (1.5–5 mg/kg) administration of galnon

induced a strong, dose-dependent reduction of food intake in rats and mice. This reduction in feeding occurred without reducing

general activity and was shown to be attenuated by an intracerebroventricular administration of M35, a peptide galanin antagonist.

These data demonstrate that galnon is a promising tool for studies of the involvement of galanin in feeding disorders and other

behavioral processes.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Galanin; GALR; Galnon; M35; Feeding behavior; Satiety

1. Introduction

The involvement of the neuropeptide galanin in ap-petite and feeding behavior has been well established,

for review see Crawley (1999). However, until today, no

non-peptide ligands to the three known galanin recep-

tors (GALR1–3) have been available that would act in

the brain upon systemic administration. The available

peptide-type receptor antagonists (M40, C7 and M35)

reduce food intake or reverse the orexigenic effect of

galanin, but they have to be administered intracerebro-ventricularly (i.c.v.), and are prone to proteolytic deg-

radation (Crawley et al., 1993). These peptides all

contain the conserved N-terminal sequence of galanin,

where Trp2, Asn5 and Tyr9 are the most important

pharmacophores (Land et al., 1991). Based on this in-

formation, we previously synthesized a library of tri-

peptide analogues to Trp-Asn-Tyr, using molecules with

* Corresponding author. Tel.: +46-8-161-793; fax: +46-8-161-371.

E-mail address: ulo@neurochem.su.se ( €U. Langel).

0143-4179/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.npep.2004.01.001

functional groups that mimic these side chains (Saar

et al., 2002; Wu et al., 2003; Zachariou et al., 2003).

By screening the combinatorial library based on theability to displace [125I]porcine galanin, the most active

compound in this library was selected, 7-((9-fluorenyl-

methoxy-carbonyl)cyclohexylalanyllysyl)amino-4-meth-

ylcoumarin, and named galnon, having an affinity of

approximately 5 lM towards galanin receptor type 1

(GALR1). Galnon was found to behave as an agonist at

galanin receptor GALR1, inhibiting adenylyl cyclase

activity in the rat hippocampal membranes, which arerich in GALR1 (Saar et al., 2002). Galnon also reduced

the intensity and increased the latency of pentylenetet-

razole (PTZ)-induced seizures in rats when given

systemically, acting as a galanin signaling agonist. Co-

administered galnon (i.p.) with M35 (i.c.v.), a peptide

galanin receptor antagonist that is known to facilitate

PTZ-induced seizures, completely abolished the effect of

galnon, confirming that a galanin receptor mediatedantiepileptic action. Pre-treatment of rats with antisense

peptide nucleic acid (PNA) targeted to GALR1 mRNA

56 U. Abramov et al. / Neuropeptides 38 (2004) 55–61

to reduce the expression of the GALR1 abolished theeffect of galnon, suggesting mediation of its anticon-

vulsant properties through this receptor subtype. Re-

cently, it has been demonstrated that galnon is efficient

in diminishing the physical signs of morphine with-

drawal in rats (Zachariou et al., 2003) and in reduction

of heat hyperalgesia in rats with nerve injury (Wu et al.,

2003).

As galanin has been suggested to be involved in theregulation of appetite, we evaluated galnon in experi-

ments exploring the feeding behavior of rodents. Exog-

enously administered galanin has been shown to elicit

feeding in satiated rats (Crawley, 1999).

2. Methods

2.1. Synthesis of peptides and galnon

Synthesis of the peptides was carried out on a model

431A peptide synthesizer (Applied Biosystems, USA)

using t-Boc or Fmoc strategies of solid-phase peptide

synthesis as described earlier (Langel et al., 1992). Gal-

non was synthesized following the scheme described by

Saar et al. (2002). The purity of the products was ana-lyzed by HPLC on an analytical Nucleosil 120-3 C18

RP-HPLC column (0.4� 10 cm). The molecular masses

of the peptides were confirmed by MALDI-TOF mass

spectrometer analysis (Applied Biosystems).

2.2. Behavioral testing in rats

Sprague–Dawley male rats, approximately 120 daysold, were housed individually in plastic tub cages in

controlled humidity and temperature animal house, and

were maintained on/off at 07.00/19.00.

Stereotaxic surgery in the rats was conducted under

ketamine (50 mg/kg i.p.) and xylazine (10 mg/kg i.p.)

anesthesia. Each subject was unilaterally implanted into

the lateral ventricle with a guide cannula made of

stainless-steel hypodermic tubing (24 gauge, 1.7 cm).The coordinates were AP (from bregma) )1.0, DV )3.5,LAT +1.0; from (Paxinos and Watson, 1982). All can-

nula placements were verified histologically to be in the

lateral ventricle.

For the experiments with rats, galnon was dissolved

in a 10% DMSO/10% Emulphor (GAF, Rochester NY)/

80% saline solution. Each rat received 0.5 ll of galnonor 0.5 ll of vehicle i.c.v. 20 min before the start of thefeeding testing. The rats were then placed in an empty

plastic tub cage in the presence of two Nabisco Nilla

WafersTM (Nabisco Brands, Inc., East Hanover, NJ; 5

wt:fat¼ 14%, carbohydrate¼ 79% and protein¼ 7%)

cookies soaked in 10 ml water, in a plastic weighing

boat. The sessions were 10 min in duration. All animals

had been pre-exposed to one cookie in the home-cage

24 h prior to the first feeding testing session to overcomeneophobia. All spillage was collected and included in the

calculations. Activity was also scored during the feeding

testing session using a Digiscan (BRS/LVE, Columbus,

OH). The procedures were conducted in accordance

with the NIH Guide for the Care and Use of Laboratory

Animals (1985) and with the approval of the State

University of New York at Stony Brook Institutional

Animal Care and Use Committee.

2.3. Behavioral testing in mice

Female mice of C57BL6 genetic background (3

months old) were used. The mice were adapted to pri-

vate metabolic cages for 7 days and fed daily at 16:00

with a standard diet (R70, Lactamin, Sweden) that was

ground down for more precise weighing. Tap water wasavailable ad libitum. The temperature and humidity

were controlled, and light–dark cycles were kept on/off

at 07.00/19.00.

The subjects who participated in the experiments in

which drug was only administered i.p. (shown in Figs.

3–5) were allowed nine days of adaptation. Food was

then removed for 24 h prior to the day of experiment to

produce enough food intake at the time of testing to beable to detect a drug-induced reduction in food intake.

On day 9, the subjects were given an i.p. injection of

galnon. The drug was dissolved in saline with a help of

DMSO. The concentration of DMSO in the final solu-

tion was not more than 1%. A 1% solution of DMSO

was also used to test any effect caused by the injection of

vehicle alone. Twenty five minutes after the adminis-

tration of galnon, the animals were allowed access to aweighed amount of food (standard diet R70). All spill-

age was collected and included in the calculations.

For the experiments involving i.c.v. injections into the

lateral ventricle of M35, all other procedures were the

same except that after day 7 of adaptation, the animals

were anesthetized with halothane and the scalp over the

region of i.c.v. injection area removed. On the 9th day,

the mice were anesthetized with halothane again and ani.c.v. injection of M35 was performed according to the

atlas of Franklin and Paxinos (1997), coordinates from

the bregma: caudal, 0.22 mm; lateral, 1 mm and depth,

1.5 mm.

M35 was dissolved in physiological saline (S. NaCl

0.9%) 0.1 lg/1 ll and injected i.c.v. via a syringe per-

fusor at the rate 1 ll/min for 20 s, thus each animal was

injected with 33 ng/0.33 ll. The injection of physiolog-ical saline was used as a control. All cannula placements

were verified histologically to be in the lateral ventricle.

The food intake was measured to the nearest 0.01 g at

30- and 60-min intervals after access to food. All ex-

periments started at 16:00� 30 min. All procedures were

in accordance with the European Communities directive

U. Abramov et al. / Neuropeptides 38 (2004) 55–61 57

86/609/EEC and approved by University of Tartu Ani-mal Care Committee.

For the study of locomotor activity in mice, an ad-

ditional experiment was conducted separate from the

feeding test in which the animals were placed individu-

ally into the photoelectric motility boxes (448�448� 450 mm) connected to a computer (TSE – Tech-

nical and Scientific Equipment, GMBH, Germany). The

illumination level of the transparent test boxes was �250lux. After removing a mouse from the box the floor was

cleaned by using 5% alcohol solution. Duration in

movement (s), the total distance of movement (m), the

number of rearing and corner entries were registered

during the 30-min observation period. The locomotor

effects of galnon (2, 5 and 10 mg/kg) were studied in

mice. Galnon or vehicle was injected intraperitoneally

15 min before the experiment. The animals that were notadapted to the experimental environment were not used

in this experiment.

Fig. 1. The effects of galnon in rats (10.0 or 20.0 lg i.c.v) on: (a) the

consumption of cookie mash and (b) activity (photobeam interruption)

per session. Means� SEM are presented. ��p < 0:01 by Fisher�s PLSDtest. The 10% DMSO/10% Emulphor/80% saline vehicle group N ¼ 4,

10.0 lg group N ¼ 4 and the 20.0 lg group N ¼ 3.

Fig. 2. The effects of galnon in rats (20.0 lg i.c.v), galanin (20.0 lgi.c.v) and three control treatments on the consumption of cookie mash.

Means� SEM are presented. Galanin stimulated cookie mash intake

and galnon reduced cookie mash intake compared to the controls.�p < 0:05 by Fisher�s PLSD test compared to saline; ��p < 0:01 com-

pared to vehicle. The no-injection group N ¼ 12, the saline group

N ¼ 9, vehicle group N ¼ 8, the galanin group N ¼ 7 and the galnon

group N ¼ 9.

2.4. Statistical analysis

The data on food consumption and activity following

i.c.v. administration in rats were analyzed using one-way

analysis of variance (ANOVA), following the removal of

any observation that was more than 4 SD from the

group mean to increase statistical power. This excluded

one observation from the 20-lg group of the first test(Fig. 1), which produced a power of 0.89 for the food

consumption and 0.35 for the activity analyses. No

subjects were removed following this screen in the sec-

ond test (Fig. 2). In female mice, the behavioral studies

were also analyzed using one-way ANOVA. Post hoc

comparisons between individual groups were performed

by means of Tukey HSD test using the program Stat-

istica for Windows software.

2.5. Displacement of galanin by galnon

A binding assay was used to quantify ability of gal-

non to displace [125I]porcine–galanin in membrane

preparations from rat hypothalamus. The assay is de-

scribed in detail in a previous study (Saar et al., 2002).

3. Results

As shown in Fig. 1, food consumption was reduced

significantly by i.c.v. galnon administration (F2;8 ¼ 9:1,

Fig. 4. The effect of i.p. galnon (5 mg/kg) and i.c.v. M35 (33 ng) (alone

and co-administered on food consumption in mice. Means�SEM are

presented. The striped bars indicate consumption at a 30-min time-

point and the solid bars indicate consumption in the same animals at a

60-min timepoint. Galnon (5 mg/kg) reduced the food intake roughly

by half and the effect attenuated by M35. The effect of M35 did not

differ significantly from the control animals. The effect was more

pronounced in the first half hour of the experiment. �p < 0:05 (com-

pared to i.p. vehicle + i.c.v. saline-treated mice, Tukey HSD test after

the significant one-way ANOVA). The saline+ 1%DMSO vehicle

group N ¼ 8, the saline+ galnon group N ¼ 9, the M35+1% DMSO

group N ¼ 10 and the M35+galnon group N ¼ 11.

58 U. Abramov et al. / Neuropeptides 38 (2004) 55–61

p < 0:01). Fisher�s PLSD post hoc test revealed thatboth the vehicle ðp < 0:01Þ and 10-lg ðp < 0:01Þ groupswere significantly different from the 20-lg group, but

not different from each other. In contrast, activity was

not significantly altered by galnon administration

ðF2;8 ¼ 2:4; p ¼ 0:15Þ. The striking difference by i.c.v.-

administered galnon was affirmed in additional rats

given galnon (Fig. 2), especially when compared to rats

receiving 20 lg of galanin i.c.v. ðp < 0:01Þ as comparedto the 10% DMSO/10% Emulphor/80% saline vehicle, a

saline vehicle, a cannulated, no-injection group (main

effect: F4;40 ¼ 94:4; p < 0:01Þ. No significant effect of

any treatment on activity was detected (main effect:

F4;40 ¼ 0:65; data not shown).

The anorexigenic effect was also seen in mice (Fig. 3).

In the first experiment, female mice were given i.p. in-

jections containing 2 or 5 mg/kg of galnon and foodintake assessed at 30 and 60 min timepoints. The ap-

plication of one-way ANOVA revealed differences in the

anorexigenic effect in the mice (the effect of galnon:

F 2; 16 ¼ 12:4; p < 0:001 at 30 min; F 2; 16 ¼ 6:01;p < 0:05 at 60 min). Post hoc comparisons (Tukey HSD

test, p < 0:01) established that galnon (5 mg/kg) caused

a significant suppression of 30 and 60 min feeding in

mice compared to both vehicle and 2 mg/kg groups.In the co-administration experiments (Fig. 4), female

mice received i.p. 5 mg/kg of galnon/vehicle and 33 ng of

M35/vehicle i.c.v., and measurements were conducted as

above. Mice receiving galnon consumed roughly half the

amount of food compared to the control group at the

30-min timepoint ðF3;34 ¼ 3:06; p < 0:05Þ. Animals

Fig. 3. The effects of galnon (2.0 or 5.0 mg/kg i.p.) on food con-

sumption in mice. Means�SEM are presented. The control group

received an i.p. injection of vehicle alone. The striped bars indicate

consumption at a 30-min timepoint and the solid bars indicate con-

sumption in the same animals at a 60-min timepoint. �p < 0:05

(compared with vehicle-treated mice by Tukey HSD test). The vehicle

group N ¼ 7, 2 mg/kg group N ¼ 6 and the 5 mg/kg group N ¼ 6.

Fig. 5. The effects of three control treatments (no injection, saline i.p.

and 1% DMSO i.p.) on food consumption in mice. Means�SEM are

presented. The striped bars indicate consumption at a 30-min time-

point and the solid bars indicate consumption in the same animals at a

60-min timepoint. The no-injection group N ¼ 5, the saline group

N ¼ 5, 1% DMSO vehicle group N ¼ 8.

Fig. 6. The effect of galnon (2–10 mg/kg i.p.) on locomotor activity in mice. Means� SEM are presented. The numbers below the bars: 0–1% DMSO

vehicle; 2, 5, 10 – the doses of galnon in mg/kg. �p < 0:05 (compared with vehicle-treated mice, Tukey HSD test after significant one-way ANOVA).

The 1% DMSO vehicle group N ¼ 11, the 2.0 mg/kg group N ¼ 10, the 5.0 mg/kg group N ¼ 10 and the 10.0 mg/kg group N ¼ 10.

U. Abramov et al. / Neuropeptides 38 (2004) 55–61 59

receiving M35 and M35+ galnon consumed almost the

same as the control group receiving only vehicle.

The administration of saline or 1% DMSO vehicle did

not influence food intake in mice at either the 30-min

ðF2;12 ¼ 0:26; p ¼ 0:77Þ or 60-min ðF2;12 ¼ 0:81; p ¼0:47Þ timepoint (Fig. 5).

The administration of galnon at lower doses (2 and 5

mg/kg) did not reduce locomotor activity (Fig. 6). Thehighest dose of galnon (10 mg/kg) reduced all compo-

nents of locomotor activity: time in locomotion (F3;37 ¼4:60; p < 0:01, distance traveled (F3;37 ¼ 4:24; p < 0:05,number of rearings ðF3;37 ¼ 6:89; p < 0:001Þ and num-

ber of corner entries ðF3;37 ¼ 5:21; p < 0:01Þ.Previous work showed that galnon binds to galanin

receptors and displaces [125I]galanin in membranes from

rat ventral hippocampus with a KD value of 4.8 lM(Saar et al., 2002) and from rat spinal cord membranes

with a KD value of 6.0 lM (Wu et al., 2003). Presently,

the hypothalamic galanin receptors displayed a similar

affinity, with a KD of 6.2 lM (data not shown).

4. Discussion

Previous studies of galnon concluded that systemi-

cally administered galnon crosses the blood–brain bar-

rier and acts as a galanin receptor agonist on PTZ-

induced seizure model in rats (Saar et al., 2002), on heat

hyperalgesia model in rats with nerve injury (Wu et al.,

2003) and on attenuation of opiate withdrawal in rats

(Zachariou et al., 2003). The effects of galnon on PTZ

seizures and heat hyperalgesia were reversible by the

galanin receptor antagonist M35 peptide. These results

suggest that galnon acts through low-affinity activationof galanin receptors of yet non-specified type. Galnon

has been suggested to be an analgesic candidate due to

the potentiation of morphine analgesia and decrease of

morphine abuse potential (Zachariou et al., 2003).

However, mechanical and cold allodynia-like behavior

after nerve injury was not affected by i.p. galnon, which

have previously been shown to be affected by i.t. galanin

(Wu et al., 2003). Hence, galnon may activate signalingsystems additional and distant to galanin signaling

systems.

Presently, we extend these studies by investigating the

effects of galnon on feeding behavior. Galnon-applied

i.c.v. in rats and i.p. in mice strongly reduced food intake,

showing an opposite result than predicted, as galanin

injected i.c.v. or intrahypothalamically typically stimu-

lates consumption of food (Corwin et al., 1993; Koegleret al., 1999; Kyrkouli et al., 1990). It is important to stress

that galnon was effective at doses not affecting locomotor

60 U. Abramov et al. / Neuropeptides 38 (2004) 55–61

activity in either case, though the highest i.p. dose, 10 mg/kg, did reduce the activity in mice. This finding suggests

that the anorexigenic action of galnon is not due to the

non-specific suppression of behavior.

Our earlier studies on seizure-control suggest that

galnon passes the blood–brain barrier (Saar et al., 2002).

In order to verify that the effect is asserted via galanin

receptors in the CNS, we repeated the experiment with

an additional i.c.v. administration of M35, the peptidegalanin receptor antagonist previously used in the sei-

zure-model (Saar et al., 2002). Indeed, the feeding in-

hibition effect of galnon was reduced. The effect of M35

alone was comparable to an injection of vehicle and did

not produce any significant change in feeding. The effect

of M35 on feeding behavior has not been thoroughly

evaluated, as is the case for M40 and C7 (Crawley et al.,

1993). The receptor antagonist peptides have beenshown to have different effects depending on the site of

administration. Intracerebroventricular injections of

M40 to the third ventricle have been shown to have no

other effect than inhibiting the effect of co-administrated

galanin, while it can attenuate feeding when adminis-

tered to the nucleus of the solitary tract. These chimeric

galanin receptor antagonist peptides, M15, M35, M40

and C7, were synthesized before the three galanin re-ceptor subtypes had been identified, and they were

classified as galanin antagonists based on their blockade

of the effects of galanin in in vivo studies (Kahl et al.,

2002). However, as the now known three subtypes have

been identified and cloned, the chimeric peptides when

studied on heterologously expressed galanin receptors in

cell culture appear to act as partial agonists on a sig-

naling level, cf. review (Flor�en et al., 2000).The effects of galanin upon feeding behavior and

seizure-control are not strictly comparable. In the sei-

zure-model, galanin acts as an anticonvulsant decreasing

the occurrence and the duration of the seizures, whilst

M35 attenuates the seizures, i.e., acting as an inverse

agonist rather than an antagonist compared to galanin

(Kokaia et al., 2001; Mazarati et al., 1998). However, in

feeding studies, galanin typically increases the food in-take, while the peptide antagonists usually act in a more

proper antagonist manner, reducing the effect of gala-

nin, but, as antagonists, they exert no opposite effect to

that of galanin (Crawley, 1999).

Galanin-applied i.c.v. is restricted in its actions to the

CNS galanin receptors, as the peptide does not cross the

blood–brain barrier. In addition, galanin has the same

high affinity for all three galanin receptor subtypes(Flor�en et al., 2000) and it has a short half-life of deg-

radation (Bedecs et al., 1995). In contrast, galnon ad-

ministered i.p. can affect peripheral as well as CNS

galanin receptors. Peripheral signals such as leptin and

ghrelin are known to affect feeding behavior (Bara-

nowska et al., 1999; Ueta et al., 2003). Nevertheless, the

partial reversal of the galnon effects by i.c.v.-applied

galanin antagonist M35 suggests that galnon effects weremostly exerted at CNS galanin receptors. However, it is

unclear if there are large differences in galnon affinity to

the galanin receptor subtypes in the brain (in cell culture

the affinities for GALR1 and GALR2 are low but

comparable), and galnon metabolites may have differ-

ential effects on the different galanin receptor subtypes.

The distribution of GALR1 mRNA is highest in the

supraoptic nucleus of the hypothalamus, amygdala,ventral hippocampus, thalamus, brainstem and dorsal

horn of the spinal cord (Gustafson et al., 1996). The

mRNA for the GALR2 was widely distributed, with

highest levels in the hypothalamus, dorsal hippocampus,

amygdala and pyriform cortex (Depczynski et al., 1998).

GALR3 mRNA was detected at highest levels in the

hypothalamus and not in the hippocampus (Smith et al.,

1998), for review cf. (Branchek et al., 2000; Kahl et al.,2002). It is not known whether one of these subtypes

may function as an autoreceptor inhibitory of galanin

release. Additionally, it is possible that galnon effects in

other brain structures mediate the general reduction of

feeding. Galnon inhibits forskolin-stimulated adenylyl

cyclase activity in ventral hippocampus (IC50 of 1.1 nM

and 8 lM for galanin and galnon, respectively), sug-

gesting that galnon exhibited galanin receptor agonist-like properties (Saar et al., 2002). Galnon�s affinity

towards the galanin-binding sites in the hypothalamus

(6.2 lM) was found to be similar to the affinity in the rat

ventral hippocampus (4.8 lM) (Saar et al., 2002) and rat

spinal cord (6.0 lM) (Wu et al., 2003), but whether the

anorexigenic effect is asserted via GALR1 receptors in

this area is not certain.

The effect of galnon on feeding is transient. Althoughthe effect in 1 h is less pronounced than the effect in 30

min, this does not necessarily implicate that the action of

galnon is reduced over this period, it can just as well be a

consequence of the experimental setup. Since the mice

have been food-deprived for 24 h prior to the experi-

ment they are likely to be hungry, and a hungry animal

is more likely to eat most of their meal in the first half of

a 1-h experiment. It may be that satiation occurs earlierin a galnon-treated mouse, causing it to stop feeding.

In conclusion, the anorexigenic effect of systemically

applied galnon is contrary to the effect of i.c.v. galanin,

but this effect, like that of galanin can be reversed by the

galanin antagonist M35. In the future, galnon might be a

useful tool when investigating the involvement of galanin

in feeding disorders that are connected to appetite.

Acknowledgements

This work was supported by the research grant from

the Swedish Research Council; US National Institute on

Aging (1RO3 AG21295-01) to J.K.R. and Estonian

Science Foundation (Grant No. 5528) to E.V.

U. Abramov et al. / Neuropeptides 38 (2004) 55–61 61

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