NeuroImage 44 (2009) 1022–1031

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Differential patterns of neuronal activation in the brainstem and hypothalamusfollowing peripheral injection of GLP-1, oxyntomodulin and lithium chloride in micedetected by manganese-enhanced magnetic resonance imaging (MEMRI)

James R.C. Parkinson a,1, Owais B. Chaudhri a,1, Yu-Ting Kuo b,c, Benjamin C.T. Field a, Amy H. Herlihy d,Waljit S. Dhillo a, Mohammad A. Ghatei a, Stephen R. Bloom a, Jimmy D. Bell e,⁎a Department of Investigative Medicine, Hammersmith Hospital, Imperial College London, UKb Department of Medical Imaging, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwanc Department of Radiology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwand Biological Imaging Centre, Imaging Sciences Department, MRC Clinical Sciences Centre, Hammersmith Hospital, Imperial College London, UKe Molecular Imaging Group, MRC Clinical Sciences Centre, Hammersmith Hospital, Imperial College London, Du Cane Road, London, W12 0NN UK

⁎ Corresponding author.E-mail address: jimmy.bell@imperial.ac.uk (J.D. Bell)

1 These authors contributed equally to this project.

1053-8119/$ – see front matter © 2008 Elsevier Inc. Alldoi:10.1016/j.neuroimage.2008.09.047

a b s t r a c t

a r t i c l e i n f o

Article history:

We have used manganese- Received 28 May 2008Revised 2 September 2008Accepted 23 September 2008Available online 15 October 2008

Keywords:ManganeseMn2+MRIOxyntomodulinGlucagon-like peptide-1Lithium chlorideHypothalamusObesityGut peptidesAppetiteProglucagon

enhanced magnetic resonance imaging (MEMRI) to show distinct patterns ofneuronal activation within the hypothalamus and brainstem of fasted mice in response to peripheralinjection of the anorexigenic agents glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM) and lithiumchloride. Administration of both GLP-1 and OXM resulted in a significant increase in signal intensity (SI) inthe area postrema of fasted mice, reflecting an increase in neuronal activity within the brainstem. In thehypothalamus, GLP-1 administration induced a significant reduction in SI in the paraventricular nucleus andan increase in the ventromedial hypothalamic nucleus whereas OXM reduced SI in the arcuate andsupraoptic nuclei of the hypothalamus. These data indicate that whilst these related peptides both induce asimilar effect on neuronal activity in the brainstem they generate distinct patterns of activation within thehypothalamus. Furthermore, the hypothalamic pattern of signal intensity generated by GLP-1 closelymatches that generated by peripheral injection of LiCl, suggesting the anorexigenic effects of GLP-1 may be inpart transmitted via nausea circuits. This work provides a framework by which the temporal effects ofappetite modulating agents can be recorded simultaneously within hypothalamic and brainstem feedingcentres.

© 2008 Elsevier Inc. All rights reserved.

Introduction

Appetite regulating hormones are secreted by the gastro-intestinal(GI) tract in response to nutritional stimuli and relay satiety signalsfrom the gut to the central nervous system (CNS) (Stanley et al., 2005).Gut hormones are attractive targets for obesity therapy because,unlike other anti-obesity drugs, they target only the relevant appetitecontrol systems in the brain. Two such peptides, glucagon-like-peptide 1 (GLP-1) and oxyntomodulin (OXM) are the products ofproglucagon cleavage within the GI tract and are released post-prandially in proportion to the amount of calories ingested (Krey-mann et al., 1987; Le et al., 1992; Turton et al., 1996). Central and

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peripheral administration of both GLP-17-36amide, the major bioactiveform of GLP-1 (Orskov et al., 1994), and OXM results in a reduction infood intake in rodents (Turton et al., 1996; Meeran et al., 1999; Dakinet al., 2001, 2002; Tang-Christensen et al., 2001; Baggio et al., 2004;Abbott et al., 2005). In addition, administration of GLP-17-36amide andOXM reduces appetite and caloric intake in lean and obese humans(Gutzwiller et al., 1999; Naslund et al., 1999; Cohen et al., 2003;Wynne et al., 2005).

Studies measuring the expression of the immediate early genemarker c-Fos indicate that both GLP-17-36amide and OXM act via thevagus nerve and the brainstem (Baggio et al., 2004; Dakin et al., 2004;Abbott et al., 2005). However, other evidence, both in vitro and in vivo,supports the view that OXM acts principally through the hypothala-mus (Dakin et al., 2004). Both GLP-1 and OXM are thought to mediatetheir actions via the GLP-1 receptor (GLP-1R) as the anorexigenicactions of both peptides are absent in GLP-1R null mice (Baggio et al.,2004). The presence of the GLP-1R has been confirmed in a number of

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regions important in appetite regulation in both humans and rodents,including the arcuate nucleus (ARC), paraventricular nucleus (PVN),supra-optic nucleus (SON) and area postrema (AP) (Wei and Mojsov,1995; Shughrue et al., 1996). However, OXM has an in vitro bindingaffinity for the GLP-1R two orders of magnitude less than that of GLP-1(Fehmann et al., 1994) and further differences in action between thetwo peptides have led some researchers to suggest that OXM may beacting via as an yet undiscovered receptor (Cohen et al., 2003; Wynneet al., 2005; Poon et al., 2005).

Nausea and vomiting are more prevalent side-effects after GLP-1analogue administration compared to OXM administration in humans(Fineman et al., 2004; Amori et al., 2007; Cohen et al., 2003;Wynne etal., 2005). Furthermore, GLP-1 has been shown to induce aconditioned taste aversion (CTA) response in both rats (Thiele et al.,1997) and mice (Lachey et al., 2005) and in this respect it acts in asimilar fashion to the toxin lithium chloride (LiCl) (Karniol et al., 1978;McCann et al., 1989; Wolgin and Wade, 1990). Indeed, it has beenshown that the GLP-1R is required to mediate the reduction in foodintake and induction of CTA after peripheral delivery of LiCl in rats(Seeley et al., 2000; Rinaman, 1999). However this effect appears to bespecies specific, as LiCl retains its anorectic and CTA-inducing effectsin GLP-1R−/−mice (Lachey et al., 2005). In combination, these datasuggest that systemic GLP-1 stimulates similar neuronal pathways tothose activated by LiCl.

Increasingly, functional neuroimaging techniques have beenemployed to study aspects of metabolic physiology in both rodents(Stark et al., 2008) and humans (Liu et al., 2000; Tataranni andDelParigi, 2003; Batterham et al., 2007). The ability of paramagneticmanganese (Mn2+) ions to shorten the proton T1 signal, yielding anincreased signal intensity (SI) in specifically weighted magneticresonance imaging (MRI) scans, combined with their capacity toenter cells via voltage-gated calcium channels, has promoted their useas a marker or neuronal activation in magnetic resonance imaging(MRI) (Silva et al., 2004; Lin and Koretsky, 1997). Importantly,manganese-enhanced MRI (MEMRI) overcomes some of the limita-tions associated with utilising the expression of the proto-oncogene c-Fos as an indirect marker of neuronal activation (Harris, 1998;Hoffman and Lyo, 2002). c-fos encodes a nuclear protein, Fos, that israpidly expressed upon depolarising stimuli in neuronal tissues(Herrera and Robertson, 1996; Curran and Morgan, 1995). Immuno-histochemical staining for Fos-like immunoreactivity (FLI) has becomea standard technique for determining changes in neuronal activity.However, c-Fos expression is not coupled to neuronal depolarisationper se, but rather to changes in signal transduction pathways(Luckman et al., 1994) and the absence of c-Fos expression does notnecessarily imply the absence of neuronal activation (Harris, 1998).Furthermore, expression of c-Fos may be induced by stimuli otherthan neuronal activation, including the effects of growth factors,neuronal differentiation and apoptosis (Yu et al., 1994; Yamamori,1991; Roca et al., 2004).

Previous work carried out by our laboratory indicates that, evenin the presence of an intact blood brain barrier (BBB), Mn2+ions arecapable of entering the hypothalamus, enabling detection of asignificant difference in activity-dependent Mn2+uptake into neu-rons in response to gut hormone administration (Kuo et al., 2005,2007). We have previously used MEMRI to demonstrate that OXMand GLP-17-36amide generate distinct patterns of activation within themurine hypothalamus (Chaudhri et al., 2006). Here, we presentevidence that MEMRI without BBB disruption is capable ofsimultaneously measuring neuronal activity in both the hypothala-mus and brainstem and demonstrate significant differences in fastedcompared to ad libitum fed mice. We have further investigated theactions of GLP-17-.36amide and OXM in specific nuclei of thehypothalamus and brainstem and compared this data with thepattern of c-Fos expression. We have also used MEMRI and c-Fosexpression to determine the regions of the CNS activated by LiCl

administration and carried out phenotypic analysis in metaboliccages following peripheral injection of GLP-17-36amide, OXM and LiCl.

Methods and materials

Materials

Synthetic human GLP-17-36amide and OXM were purchased fromBachem (Merseyside, UK). All other chemicals were purchased fromSigma-Aldrich Company Ltd. (Poole, Dorset, UK) unless otherwisestated.

Animal preparation

All studies were performed in accordance with the Animals(Scientific Procedures) Act 1986 (UK) (license number 70/5632).Animals were allowed ad libitum access to standard chow (RM1,Special Diets Services Ltd, Essex, UK) and drinking water unless statedotherwise. Male C57BL/6 mice (16–24 weeks old, Harlan, UK) wereacclimatised in a holding room adjacent to the MRI laboratory for 24 hbefore scanning.

MRI parameters

Spin-echomulti-slice T1-weighted sequence imagingwas performedas previously described (Kuo et al., 2005) with the following scanningparameters: repetition time (TR)=600 ms, echo time (TE)=10 ms, 1average and 10 contiguous transverse slices of 1mm thickness. The scantime for each acquisition was 1 min 57 s. The data matrix was 256×192and the field of view 25 mm×25 mm, giving an in-plane spatialresolution of approximately 100 μm per voxel. The slice offset for eachmousewas aligned to identifiable anatomical features with reference toa standardmouse brain atlas (Franklin and Paxinos,1997) to ensure thatthe region of brain scanned was in register for all mice. Thehypothalamus was placed at the foremost point of the 10 transverseslices in order to enable measurement of Mn2+accumulation in bothbrainstem and hypothalamic regions concurrently.

Experiment 1: comparison of Mn2+uptake in the hypothalamus andbrainstem of fasted and ad libitum fed mice

The degree of manganese accumulation within the hypothala-mus and brainstem of fasted and ad libitum fed mice was measuredto provide control signal intensity (SI) profiles for subsequentcomparison with those generated by GLP-17-36amide, OXM and LiCl.Fasted animals had their chow removed 18 h prior to scanning andwere allowed ad libitum access to water. All scans were performedin the early light phase as previously described (Chaudhri et al.,2006; Kuo et al., 2006, 2007). Mice (n=5 fasted; n=6 ad libitumfed) were anaesthetised with isoflurane via a facemask (1.5%isoflurane-oxygen mixture for induction, reduced to 1% for main-tenance). The tail vein was cannulated for intravenous (i.v.) infusionof MnCl2 and an intraperitoneal (i.p.) catheter sited. The head wascentrally located inside a quadrature mouse head coil with aninternal diameter of 25 mm (Magnetic Resonance Laboratories,Oxford, UK) and a phantom consisting of a glass tube containing0.9% saline was scanned simultaneously with each animal. Therectal temperature of each mouse was monitored and maintained at35.5±0.5 °C for the duration of each scan by a heating system (SAInstruments, NY, USA). Infusion of MnCl2 was commenced after 3initial baseline acquisitions. Each mouse received a bolus i.p.injection of 100 μl 0.9% saline and an infusion 5 μl/g bodyweightof 100 mM MnCl2 at a rate of 0.2 ml/h via the tail vein. A further 63acquisitions were obtained, each acquisition commencing immedi-ately after completion of the one before, lasting approximately120 min in total.

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Experiment 2: the effect of peripheral administration of GLP-17-36amide,

OXM and LiCl on Mn2+uptake in the hypothalamus and brainstem offasted mice

In order to measure the effects of GLP-17-36amide, OXM and LiCl uponneuronal activation within the hypothalamus and brainstem, MEMRIscans were performed on mice fasted overnight (18 h) and injected i.p.with GLP-17-36amide (900 nmol/kg), OXM (1400 nmol/kg), LiCl (0.15 M at2% of bodyweight (bwt)) or vehicle (0.9% saline) (n=4–5/group). Allother parameters were as in experiment 1. In this study mice werefasted since fasting is a state associated with low circulating levels ofGLP-17-36amide and OXM and provides a receptive physiological back-ground onto which these peptides can be effectively tested (Parkinsonet al., 2008). The effects of GLP-17-36amide, OXM and LiCl aremagnified inthe fasted state, significantly reducing food intake following i.p.injection at the doses implemented here (Parkinson et al., 2008; Lacheyet al., 2005; Neary et al., 2005; Dakin et al., 2001). In addition, fastedmice demonstrate a significantly increased SI in a number ofhypothalamic regions compared to ad libitum fed animals (Chaudhriet al., 2006; Kuo et al., 2006, 2007). Region specific increases in SIrecorded in the fasted state are attenuated by anorexigenic agents suchas PYY3-36 , GLP-17-36amide and OXM; in effect turning the SI profile of afasted animal into that of an ad libitum fed animal (Kuo et al., 2007;Chaudhri et al., 2006). Furthermore, MEMRI has previously been used todemonstrate dose-dependent effects on SI following i.p. injection of900 nmol and 5400 nmol of OXM delivered on a fasted background(Chaudhri et al., 2006).

Experiment 3: the effect of intraperitoneal GLP-17-36, OXM and LiCladministration on food intake in fasted mice as measured by CLAMSmetabolic cages

In order to compare the neuronal activation data obtained byMEMRI analysis with the temporal effect of GLP-17-36amide, OXM andLiCl upon food intake, detailed feeding studies were carried out. MaleC57BL/6micewere continuouslymonitored using a 24 chamber open-circuit Oxymax Comprehensive Lab Animal Monitoring System(CLAMS; Columbus instruments, Columbus, OH). Mice were main-tained at 24 °C under a 12:12 h light-dark cycle (light period 7am–

7pm). Animalswere individually housed inplexiglass cages.Waterwasavailable ad libitum. Allmicewere acclimatized to their cages for 2 daysprior to the study. Powdered chow was removed for an overnight fast(18 h) prior to early light phase i.p. injection of either vehicle (0.9%saline), GLP-17-36amide (900 nmol/kg) or OXM (1400 nmol/kg) (n=8/group). Animals were returned to their home cage with ad libitumaccess to food. During CLAMS monitoring, the food intake of eachindividually housed animal was measured simultaneously everyminute for 240 min. After 3 days of recovery mice were fastedovernight (18 h) and injected with either saline or LiCl (0.15M LiCl 2%bodyweight) (n=12/group) and food intake was measured as above.Data are represented as a 5 min mean of cumulative food intake.Cumulative food intake data are presented for the first 140 min of thestudy in order to allow comparison with the MEMRI SI profiles.Cumulative activity along the x axis (XAMB) as measured byconsecutive infra-red beam breaks was recorded every 30 min.

Experiment 4: the effects of peripheral injection of GLP-17-36amide, OXMand LiCl on Fos-like immunoreactivity in the hypothalamus andbrainstem of fasted mice

In order to further examine the effects of GLP-17-36amide, OXM andLiCl on neuronal activation, staining for the product of the immediateearly gene c-foswas carried out on hypothalamic and brainstem slices.Mice were fasted overnight (18 h) with ad libitum access to water andinjected intraperitoneally at 8am with GLP-17-36amide (900 nmol/kg),OXM (1400 nmol/kg), lithium chloride (0.15 M 2% bodyweight) or

saline (0.9%) (n=4/group). After 90 min animals were sacrificed andtranscardially perfused with ice-cold PBS containing 4% paraformal-dehyde. Following perfusion-fixation, brains were removed and40 um coronal sections stained for Fos-like immunoreactivity (FLI)as previously described (Dakin et al., 2004). Slides were examined forFLI-positive nuclei using a light microscope. The ARC, PVN, SON,ventromedial hypothalamic nucleus (VMH), central nucleus of theamygdala (CeA) and nucleus of the solitary tract (NTS) were definedwith reference to a standard mouse brain atlas (Franklin and Paxinos,1997). The average number of positively stained nuclei within a givenregion of interest (ROI) was derived from 4 coronal slices that were inregister across all experimental groups, counted by an investigatorblinded to the experimental groups.

Experiment 5: comparison of plasma levels of GLP-17-36amide and OXMachieved following i.p. administration of GLP-17-36amide and OXM in mice

This experiment was performed in order to determine the effectsof peripheral delivery of GLP-17-36amide and OXM on plasma levels ofthese hormones in fasted animals. The transition from a fasted to asatiated state results in co-ordinated changes in a great number ofhormones (Stanley et al., 2005). However, the absolute change inphysiological levels of any individual hormone is not great; rather thesynergistic action of a number of hormones is more significant. Wehave sought to investigate the effects of GLP-17-36amide and OXM inisolation, hence the high, supraphysiological doses used. Thesetherapeutically important doses have been previously shown toreduce food intake in mice (Parkinson et al., 2008). Established in-house radioimmunoassays (RIAs) were used in order to compare thephysiological levels of GLP-17-36amide and OXM in fasted mice withthose resulting from peripheral injection of the two hormones (asused in experiments 2, 3 and 4). C57BL/6 mice were fasted overnight(18 h) and injected i.p. with either saline, GLP-17-36amide (900 nmol/kg), or OXM (1400 nmol/kg) at 8am (n=10/group). Thirty minutesfollowing injection, blood was collected by cardiac puncture andplasma levels of GLP-17-36amide and OXM were assayed as previouslydescribed (Cohen et al., 2003). Briefly, OXM contributes 50% to plasmaOXM-like immunoreactivity (OLI) (Kervran et al., 1987), the remainingcomponent being OXM with a 30-amino acid N-terminal extension(Holst, 1997). The OLI assay (Ghatei et al., 1983) could detect changesof 10 pmol/L (95% confidence limit) with an intra-assay variation of5.7%. The GLP-1 assay (Kreymann et al., 1987) could detect changes of8 pmol/L (95% confidence limit) with an intra-assay variation of 6.1%.The GLP-1 antibody was specific for N-terminal amidated GLP-1 anddid not cross-react with GLP-1-(1–37), GLP-1-(1–36), or GLP-1-(7–37).All samples were assayed in duplicate andwithin one assay in order toeliminate inter-assay variation.

Image analysis

Image processing software (Image J 1.3.1, NIH, USA) was used todefine ROI corresponding to the ARC, VMH, PVN and SON as previouslydescribed (Kuo et al., 2007; Chaudhri et al., 2006), as well as the NTSand area postrema (AP) within the brainstem, with reference to astandard mouse brain atlas (Franklin and Paxinos, 1997) (Supplemen-tary Fig.1). These nuclei are involved in the regulation of energy intakeand have been implicated in themediation of gut peptide effects in theCNS (Stanley et al., 2005). In addition, SI within the central nucleus ofthe amygdala (CeA), a region involved in development of CTA and thenausea response (Rinaman and Dzmura, 2007), was also measured.Inter-animal variation was minimised by scanning male mice that didnot differ significantly in body weight (data not shown). In order tocorrect for changes in the SI due to non-biological factors over thecourse of the scan, the SI of each target area was normalized to the SIof the phantom scanned simultaneously with each mouse (SI of targetarea / SI of phantom). Data are presented as absolute normalized

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values and as percentage increase in SI over baseline values.Baseline acquisitions were those obtained prior to the start of thei.v. Mn2+infusion and the administration of the i.p. injection.Furthermore, to correct for slight variations in the time taken forthe MnCl2 to enter the circulation, the first enhancing time point ofeach scan was defined as the acquisition in which the SI in the 4thventricle was increasedN20% over baseline. The acquisitions werethen realigned in time so that the first enhancing acquisition was inregister across all animals.

Statistical analysis

All data are presented as mean±standard error (sem). Differencesin SI profile between the ROI in all experimental groups and

Fig. 1. T1-weighted MEMRI signal intensity profiles in fasted or ad libitum fed mice. Time cour(A) ARC, (B) PVN, (C) SON, (D) VMH, (E) AP and (F) NTS, in fasted mice or ad libitum fed mic⁎⁎⁎=pb0.001 fed vs. fasted, NPE – normalised percentage enhancement; n=4–5/group. Theindicates the duration of the i.v. infusion. Statistical differences by GEE. Data are presented

cumulative food intake as measured by the CLAMS metabolic cageswere analysed using generalized estimating equation (GEE) curveanalysis and the Mann-Whitney-U test in commercial statisticalsoftware (Stata 9.1, Statacorp, College Station, TX). Two-way analysisof variance with the Bonferroni correction for multiple comparisonswas used to analyze FLI cell counts across treatment groups.

Results

Experiment 1: comparison of Mn2+uptake in the hypothalamus andbrainstem of fasted and ad libitum fed mice

Significant increases in Mn2+accumulation were recorded in fasted,compared to ad libitum fed, animals in theARC, PVNand SON(Figs.1A–C,

se of normalised T1-weighted MEMRI SI change after i.v. MnCl2 infusion recorded in thee receiving i.p. injection of vehicle (saline 0.9%) (n=4–5/group). ⁎=pb0.05, ⁎⁎=pb0.01,arrow indicates the start of i.v. MnCl2 infusion and bolus i.p. injection, the hatched baras mean±sem.

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Table 1). In contrast, a significant decrease in SIwas recorded in theAP infasted compared to ad libitum fed animals (Fig. 1E; Table 1). Nodifferences were recorded between the SI profiles of fed and fastedanimals in either the VMH or NTS, indicating region specific changes inMn2+accumulation (Figs. 1D, F).

Experiment 2: the effect of peripheral administration of LiCl, GLP-17-36amide and OXM on Mn2+uptake in the hypothalamus and brainstem offasted mice

Intraperitoneal administration of OXM resulted in a significantreduction in SI in the ARC (pb0.01) and SON (pb0.001) compared tofasted controls (Figs. 2A, C; Table 1). In the AP, OXM was responsiblefor an increase in SI (pb0.05) (Fig. 2E; Table 1). GLP-17-36amide injectionresulted in a significant reduction in SI in the PVN (pb0.01) and asignificant increase in SI in both the VMH (pb0.05) and AP (pb0.01)compared to fasted controls (Figs. 3B, D, E, Table 1). Peripheralinjection of lithium chloride produced a significant increase in SIwithin the VMH (pb0.01) compared to saline injected fasted controls(Fig. 3D; Table 1). There was a lower penetration of manganese ionsand subsequently a reduced SI enhancement in the NTS (Figs. 1F, 2F)and CeA (Fig. 3F) compared to other regions of the hypothalamus.

Experiment 3: the effect of intraperitoneal GLP-17-36amide, OXM and LiCladministration on food intake and ambulatory activity in fasted mice asmeasured by CLAMS metabolic cages

The anorexigenic effects of GLP-17-36amide, OXM and LiCl can be seenin Fig. 4. Thedifference in cumulative food intakebetweenGLP-17-36amide

and saline-injected animals became statistically significant at 50 minpost injection (Fig. 4A). Peripheral injection of OXM resulted in astatistically significant reduction in cumulative food intake at 55 minpost injection (Fig. 4B). Compared to GLP-17-36amide and OXM thesignificant reduction in food intake inducedby lithiumchloride injectionoccurred more rapidly, becoming statistically significant at 20 min postinjection and remaining so for the duration of the study (Fig. 4C). Therewas no significant effect of GLP-17-36amide or OXM administration onambulatory activity. However, injection of LiCl resulted in a significantreduction in ambulatory activity compared to saline injected animals,beginning 30 min post injection (Ambulatory count (XAMB): Saline:339±25; LiCl: 99±30; GLP-17-36amide: 429±27; OXM: 348±73, pb0.01LiCl vs. all groups) and remaining reduced for the duration of the study(Supplementary Fig. 2).

Experiment 4: the effects of peripheral injection of GLP-17-36amide, OXMand LiCl on Fos-like immunoreactivity in the hypothalamus andbrainstem of fasted mice

There were no statistically significant changes in FLI in response toi.p. administration of either OXM or GLP-17-36amide compared to salineinjected controls, in the ARC, PVN, VMH, SON, CeA or NTS(Supplementary Fig. 3). Peripheral injection of lithium chloride

Table 1Comparison of changes in SI over time by GEE following administration of GLP-1, OXM,LiCl or vehicle in fasted mice or vehicle injected ad libitum fed mice

Region of interest

ARC PVN SON VMH AP NTS

Fasted vs. ad libitum fed control 0.001 0.002 b0.001 0.40 0.032 0.81OXM vs. Fasted controls 0.002 0.12 b0.001 0.22 0.04 0.69GLP-17-36amide vs. fasted controls 0.68 0.022 0.62 0.031 0.002 0.75Lithium chloride vs. fasted controls 0.661 0.1219 0.774 0.0032 0.456 0.72

The values represent p values generated by GEE from statistical comparison of the SIprofiles of fasted mice administered GLP-1 (900 nmol/kg), OXM (1400 nmol/kg), LiCl(100 μl of 3M solution) or vehicle control (0.9% saline) or ad libitum fed miceadministered vehicle control (0.9% saline).

resulted in significantly increased FLI in the PVN, SON and CeA (PVNFLI count: Saline: 70±12; LiCl: 141±11, pb0.01; SON FLI count: Saline:35±3; LiCl: 60±6, pb0.05; CeA FLI count: Saline: 18±5; LiCl: 45±2,pb0.05) (Supplementary Fig. 3).

Experiment 5: comparison of plasma levels of GLP-17-36amide and OXMachieved following i.p. administration of GLP-17-36amide and OXM in mice

A significant increase in the levels of both GLP-1 and OXM wererecorded 30min after intraperitoneal injection of GLP-17-36amide (PlasmaGLP-1: Fasted state: 1.0±0.01 pM/L, Fasted and i.p. GLP-17-36amide: 37.58±5.21 pM/L; pb0.001 vs. fasted state) and OXM (Plasma OXM: Fastedstate: 1.1±0.08pM/L, Fasted and i.p. OXM: 13.78±3.63 pM/L;pb0.001 vs.fasted state) (Supplementary Fig. 4).

Discussion

Gut peptides released from the GI tract mediate their effects in partbymodifying the activity of two subpopulations of neurons within theARC nucleus. One population co-expresses the neurotransmittersneuropeptide Y (NPY) and agouti-related peptide (AgRP) andpromotes food intake while a second population, co-expressing pro-opiomelanocortin (POMC) and cocaine-and amphetamine-regulatedtranscript (CART), inhibits food intake (Schwartz et al., 2000). Inkeeping with previous MEMRI studies we demonstrated an increasedrate of Mn2+accumulation in the ARC, PVN and SON in fasted versus adlibitum fed animals (Kuo et al., 2006, 2007; Chaudhri et al., 2006).Whilst MEMRI analysis is able to generate a quantitative measure ofthe effect of a stimulus, it is important to note that functional MRIparadigms such as MEMRI and BOLD (Blood oxygen level dependent)-based MRI currently lack the ability to discriminate between theactivities of different populations of neuron within the same region(Silva et al., 2004). The observation of an increase in SI may indicateactivation of either stimulatory or inhibitory neurons. Similarly, anincrease in activity in one subset of neurons counterbalanced by adecrease in another would result in no detectable effect in SI. This isespecially relevant to neuronal activity within the hypothalamus,where the aforementioned populations of orexigenic and anorexi-genic neurons coexist.

In contrast to the hypothalamus, analysis of the area postrema (AP)revealed significant reductions in SI in fasted compared to ad libitumfed animals. Similar to the ARC, the AP is a circumventricular organwith an incomplete BBB at its location and represents a receptive sitefor humoral factors regulating feeding behaviour (Price et al., 2008).Here we demonstrated that sufficient penetration of manganesefollowing tail-vein infusion occurs at this site, enabling significantdifferences in SI to be detected using the MEMRI technique. The AP isalso involved in transmitting the appetite regulating effects of manygut derived peptides via connections to the hypothalamus (Abbott etal., 2005; Date et al., 2002; Lorenz and Goldman, 1982; Baggio et al.,2004). The increased activity we have recorded in the AP of ad libitumfed animals mirrors the feeding-induced c-Fos expression that occursin this region following a number of post-prandial processes,including vagal stimulation induced by gastric distension, gastricemptying and gastric acid secretion (Fraser et al., 1995; Glatzle et al.,2001; Timofeeva et al., 2002; Zhang et al., 2003; Holzer, 2003).

Consistent with their function as anorexigenic signals, the admin-istration of GLP-17-36amide and OXM to fasted animals resulted in asignificant increase in SI within the AP, generating a profile similar tothat of an ad libitum fed animal. In addition to their anorexigenic effects,GLP-17-36amide and OXM have also been shown to affect gastricemptying, gastric acid secretion and gastric motility (Tolessa et al.,1998;Wettergren et al.,1997). It is therefore possible that the increase inSI we have recorded in the brainstem following GLP-17-36amide and OXMadministration may therefore be a composite of various post-prandialevents. However, it should be noted that the doses of GLP-17-36amide and

Fig. 2. T1-weighted MEMRI signal intensity profiles after peripheral administration of GLP-17-36amide, OXM or vehicle in fasted mice. Time course of normalised T1-weighted MEMRISI change after i.v. MnCl2 infusion recorded in the (A) ARC, (B) PVN, (C) SON, (D) VMH, (E) AP, (F) NTS, in fasted mice receiving i.p. GLP-17-36amide (900 nmol/kg), OXM (1400 nmol/kg)or vehicle (saline 0.9%) (n=4–5/group).●=pb0.05, ●●=pb0.01, ●●●= pb0.001: GLP-17-36amide vs. fasted,■= pb0.05, ■■■=pb0.001 OXM vs. fasted; NPE–normalised percentageenhancement; n=4–5/group. The arrow indicates the start of i.v. MnCl2 infusion and bolus i.p. injection, the hatched bar indicates the duration of the i.v. infusion. Statisticaldifferences by GEE. Data are presented as mean±sem.

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OXM used here resulted in supraphysiological plasma levels of thehormones and therefore do not reflect the endogenous changes in GLP-1 and OXM secretion that occur in the fasted state. In agreement with aprevious MEMRI study (Chaudhri et al., 2006), the data presented heresuggest that OXM alters neuronal activity within the ARC and SON,whereas GLP-17-36amide acts via the VMH and PVN. That two relatedpeptides, thought to act via the same receptor, are responsible fordifferent patterns of activation lends support to the notion that OXMmay be working via an as yet unidentified receptor (Fehmann et al.,1994; Dakin et al., 2004).

As discussed above, theMEMRI technique suffers form a number oflimitations, for example in the interpretation of a change in SI.Approaches such as measurement of FLI may overcome these

shortcomings of MEMRI by allowing identification of individualneuronal populations through the use of dual-staining techniques.However, whilst FLI measurement is a well-established and effectivemeans of determining neuronal activation, there are instances wherethe same experimental paradigm has produced conflicting resultsusing this technique. For instance, studies indicating no effect offasting upon the hypothalamic expression of c-Fos (Hewson andDickson, 2000; Timofeeva et al., 2002; Timofeeva and Richard, 2001)contrast with other studies where a difference was detected (Riedigeret al., 2004; Miller et al., 2004; Gautron et al., 2005). In addition, theeffects of peripherally administered GLP-17-36amide on neuronal activitywithin theARC are contentious,with some authors reportingno effect ofthe peptide on ARC c-Fos expression (Baggio et al., 2004; Dakin et al.,

Fig. 3. T1-weighted MEMRI signal intensity profiles after peripheral administration of lithium chloride or vehicle in fasted mice. Time course of normalised T1-weighted MEMRI SIchange after i.v. MnCl2 infusion recorded in the (A) ARC, (B) PVN, (C) SON, (D) VMH, (E) AP and (F) CeA, in fasted mice receiving i.p. injections of lithium chloride (LiCl) (0.15 M at2% of bwt) or saline control (saline 0.9%) (n=4–5/group). ♦=pb0.01 LiCl vs. fasted control; NPE–normalised percentage enhancement; n=4–5/group. The arrow indicates the startof i.v. MnCl2 infusion and bolus i.p. injection, the hatched bar indicates the duration of the i.v. infusion. Statistical differences by GEE and Mann-Whitney U test. Data are presentedas mean±sem.

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2004; Tang-Christensen et al., 2001), whilst others have described anincrease, albeit indirectly mediated via the brainstem and vagus (Abbottet al., 2005).

These differences underline the difficulties associated with inter-preting changes in indirect markers of neuronal activity and in suchinstances MEMRI offers an effective alternative producing a repro-ducible response in both cases. However, MEMRI is probably bestregarded as complimentary tomore traditional techniques rather thanas a replacement. For example, there is concordance between c-Fosand MEMRI data supporting a role for the AP in the control of feeding.Where MEMRI and c-Fos data are discordant, as in the hypothalamus,further investigation using other approaches such as CNS microinjec-tion or electrophysiological studies would be appropriate.

The c-Fos data presented here suggests that neither GLP-17-36amide

nor OXM alters FLI in hypothalamic or brainstem nuclei. However, in

order to replicate theMEMRI protocol we havemeasured the effects ofGLP-17-36amide and OXM on a fasted background and therefore reliedupon the gut hormones altering fasting induced changes in FLI.Previous studies demonstrating a significant effect of these peptideson FLI within the CNS have been carried out on an ad libitum fedbackground (Tang-Christensen et al., 2001; Baggio et al., 2004; Dakinet al., 2004). The aforementioned ambiguity in FLI expression in adlibitum fed compared to fasted animals may explainwhy no significantchanges were recorded in FLI expression following GLP-17-36amide orOXM administration.

Consistent with previous MEMRI data, we recorded no effect offasting or OXM administration on SI in the VMH (Chaudhri et al.,2006). There is some debate as to the role of this structure in thephysiological control of food intake (King, 2006) and there is a notableabsence of increased SI in the VMH of fasted animals compared to

Fig. 4. The effects on cumulative food intake of i.p. injection of GLP-17-36amide, OXM orLiCl in fastedmice. Cumulative food intake in fastedmice after i.p. injection of (A) GLP-1(B) OXM or (C) LiCl as measured by the CLAMS system. The line covers the time periodwhere a significant difference in cumulative food intake exists between peptide/lithiumchloride and saline injected animals. GLP-17-36amide dose 900 nmol/kg; OXM dose1400 nmol/kg; LiCl dose 0.15 M at 2% of bwt; Saline – 0.9%. n=8/12 per group. Dataanalysed by the Mann-Whitney U test. Data are presented as mean±sem.

1029J.R.C. Parkinson et al. / NeuroImage 44 (2009) 1022–1031

other hypothalamic nuclei. Interestingly, GLP-17-36amide administra-tion caused a robust increase in SI within this region and it is possiblethat GLP-17-36amide activated neuronal circuits that control functionsother than appetite. Central administration of GLP-17-36amide into thelateral ventricle elicits symptoms of visceral illness and conditionedtaste aversion (CTA) and produces a pattern of c-Fos expression thatclosely resembles that of LiCl (Kinzig et al., 2002; Thiele et al., 1998).Furthermore, pharmacological antagonists of the GLP-1R block theeffects of LiCl in rats (Lachey et al., 2005), suggesting a link between

the aversive effects of both LiCl and GLP-1. Here we show peripheraladministration of GLP-17-36amide results in a hypothalamic SI profilecomparable to that of LiCl, with a significant increase in VMH SI and atrend towards a reduced activity within the PVN. Whilst the GLP-1R isnot thought to be expressed in the VMH, the numerous neuronalconnections between this region and other sites within the hypotha-lamus and brainstem which express GLP-1R could be responsible forthe increased SI (Shughrue et al., 1996; Wei and Mojsov, 1995).

The similarity between the CTA-inducing effects of GLP-17-36amide

and LiCl and their SI profiles suggests a possible role for the VMH intransmitting the effects of visceral illness that occur following deliveryof both of these agents. This is further supported by the SI profile ofOXM, which, whilst causing an anorexigenic response of similarmagnitude, does not induce nausea and vomiting to the same extentas GLP-1 (Wynne et al., 2005; Cohen et al., 2003) and in our study doesnot induce an increase in SI within the VMH. In addition to the AP andPVN, the NTS, CeA and parabrachial nucleus have also been implicatedin transmitting the CTA response (Rinaman and Dzmura, 2007).However, the insufficient penetrance of manganese across the BBB toboth the NTS and CeA resulted in a reduced baseline SI within theseregions and is likely to have prevented the detection of any significantdifferences in SI between experimental groups. A modified MEMRIprotocol in which sufficient time is allocated for manganese to reachthese regions following i.v. infusion may allow differences in SI to bedetected.

Despite the similarities in hypothalamic SI profile followingperipheral GLP-17-36amide and LiCl administration we have recorded anumber of differences between the two agents. Firstly, the robustincrease in c-Fos expression in the PVN, SON and CeA we haverecorded after LiCl administration in the fasted state were notduplicated by GLP-17-36amide. All of these regions are thought to beinvolved in the transmission of visceral information contributing tothe formation of a CTA (Sakai and Yamamoto, 1999; Rinaman andDzmura, 2007; Olszewski et al., 2000) but were not activated byGLP-17-36amide in our fasted paradigm. Secondly, the MEMRI dataindicate the effects of LiCl on SI within the AP are immediate buttransient, with a notable increase recorded 6 min following injectionbut only lasting 8 min. Contrary to this rapid effect, the increase inAP SI induced by both GLP-1-36amide and OXM occurs at a later timepoint (40 min) post injection and once established, the increase issustained for the duration of the scan.

Signal intensity profiles represent a marker for neuronal activa-tion based upon the accumulation of manganese within a given ROI.The increase and subsequent decrease in SI recorded in the APfollowing LiCl injection implies a transport of manganese firstly intoand then, unusually, out of this region and previous MEMRI studiescarried out by our laboratory have not recorded a SI profile of thisunique biphasic nature (Chaudhri et al., 2006; Kuo et al., 2005, 2006,2007). Interestingly, the immediate and profound effects of LiCl onfood intake and ambulatory activity reflect the initial peak in SIrecorded in the AP, whilst the comparatively delayed anorexigeniceffects of GLP-17-36amide and OXM are also replicated in thecorresponding AP SI profiles. Therefore, the temporal alterations inSI within the AP appear to correlate with subsequent changes in foodintake.

MEMRI data is reliant upon the speed at which Mn2+ions are ableto penetrate into specific brain regions. The significant effects of OXMand GLP-17-36amide injection on SI profiles within hypothalamic ROImanifest between 15–30 min, whilst changes in AP SI do not developuntil at least 40 min post injection. The feeding studies carried out inthe metabolic cages suggest the anorexigenic action of both peptidesbegins to take effect around 30 min post-injection. Ultimately, thetime-point at which GLP-17-36amide and OXM purportedly induce areduction in food intake is reliant upon a time-course separation inmanganese accumulation between peptide and saline injectedcontrols. As such, it is perhaps unwise to try and draw comparisons

1030 J.R.C. Parkinson et al. / NeuroImage 44 (2009) 1022–1031

as to the temporal nature of the effects of GLP-17-36amide and OXMupon these regions until further work investigating the nature ofmanganese transport within the brainstem and hypothalamus hasbeen carried out.

In summary, we demonstrated that MEMRI is capable ofmeasuring differences in SI simultaneously in both the hypothalamusand brainstem. The differential patterns of activation followinginjection of GLP-17-36amide and OXM suggesting that these peptidesdo not manifest their effects via the same neuronal pathways.Furthermore, the hypothalamic pattern of signal intensity generatedby GLP-17-36amide, closely matches that generated by peripheralinjection of LiCl, suggesting the anorexigenic effects of GLP-17-36amide

may be in part transmitted via nausea circuits.

Acknowledgments

The Department of Integrative Science is funded by programmegrants from theMRC (G7811974) andWellcome Trust (072643/Z/03/Z)and by an EU FP6 Integrated Project Grant LSHM-CT-2003-503041. W.S.D is supported by a Department of Health Clinician Scientist Award.We are also grateful for support from the NIHR Biomedical ResearchCentre funding scheme and an Integrative Mammalian Biology (IMB)Capacity building award. We are grateful to the United Kingdom (UK)Medical Research Council and the UK Biotechnology BiologicalSciences Research Council for financial support. We also acknowledgethe Biological Imaging Centre (BIC), Imperial College London and theWellcome Trust for the use of the MR facilities. We thank also ManuelSegundo Taquias Vergara por inspiratio.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.neuroimage.2008.09.047.

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