B. GÜNDÜZ, A. KARAKAŞ

727

Turk J Biol

35 (2011) 727-734

© TÜBİTAK

doi:10.3906/biy-1008-63

Th e eff ects of leptin hormone on locomotor activity

in Syrian hamsters (Mesocricetus auratus)

Bülent GÜNDÜZ1, Alper KARAKAŞ

2

1Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale - TURKEY

2Department of Biology, Faculty of Arts and Sciences, Abant İzzet Baysal University, Bolu - TURKEY

Received: 16.08.2010

Abstract: Th e suprachiasmatic nucleus (SCN) generates and controls the circadian rhythms in mammals including

the rhythm of locomotor activity. Leptin is a hormone secreted by adipose tissue that informs the brain about the fat stores. SCN neurons express leptin receptors. Here we investigated the eff ects of 3 diff erent leptin administrations on the locomotor activity of the Syrian hamsters maintained in constant darkness. Animals were intraperitoneally (ip) injected (4 μg/kg), subcutaneously (sc) infused (4 μg/kg), or intra-SCN infused (0.4 μg/kg) with leptin for 3 days at circadian time 10 whereas the controls received saline (0.9% NaCl) at the same time in order to eliminate stress factors. Our results demonstrate that the locomotor activity of the hamsters can be phase advanced by the external leptin administrations. Leptin aff ected the level of phase-shift s in an administration method-dependent manner. Th e biggest phase advance was observed in intra-SCN infusion (P = 0.001), and the smallest was in the ip injection (P = 0.041) group. Th e wheel-turn amounts did not change signifi cantly in the groups before and aft er the leptin administrations (P = 0.233); however, the period lengths increased (P = 0.011) signifi cantly aft er leptin administrations. Th ese results suggest for the fi rst time that

in vivo leptin administrations may change the rhythm of locomotor activity in adult male Syrian hamsters.

Key words: SCN, locomotor activity, leptin, Syrian hamster, Mesocricetus auratus

Leptin hormonunun Suriye hamsterlerinde (Mesocricetus auratus)

lokomotor aktivite üzerine etkileri

Özet: Memelilerde, lokomotor aktivitenin ritmi de dahil olmak üzere, tüm sirkadiyen ritimler, Suprakiyazmatik nukleus (SCN) tarafından oluşturulur ve kontrol edilir. Leptin, yağ dokudan salınan ve vücudun yağ içeriği hakkında beyni bilgilendiren bir hormondur. SCN nöronları leptin reseptörlerini bulundurmaktadır. Bu çalışmada üç farklı metod ile leptin uygulamasının, sürekli karanlıktaki Suriye hamsterlerinin lokomotor aktivitesi üzerine olan etkileri araştırıldı. Hamsterlere, intraperitonal enjeksiyon (Ip) (4 μg/kg), deri altı (Sc) infüzyon (4 μg/kg) ve SCN içine infüzyon (0,4 μg/kg) ile leptin, sirkadiyen zaman ile 10’da, üç gün boyunca uygulandı. Stres faktörünün elimine edilebilmesi için kontrol grubundaki hamsterlere, bu üç uygulama ile serum fi zyolojik (% 0,9 NaCl) verildi. Sonuçlarımız, Suriye hamsterlerinde lokomotor aktivitenin, leptin uygulaması ile öne kayabileceğini göstermektedir. Leptin, uygulanan metoda bağlı olarak, farklı safh a kayma etkileri meydana getirdi. En büyük safh a kayması SCN içine leptin infüzyonu (P = 0,001), en küçük safh a kayması ise intraperitonal leptin enjeksiyonu (P = 0,041) ile gerçekleşti. Tekerlek dönüşlerinin miktarı, gruplarda, leptin uygulaması öncesi ve sonrası arasında anlamlı bir farklılık göstermedi (P = 0,233), ancak, periyod uzunlukları, leptin uygulamaları ile anlamlı olarak yükseldi (P = 0,011). Bu sonuçlar, in vivo leptin uygulaması ile yetişkin erkek

Suriye hamsterlerinde lokomotor aktivite ritminin değişebileceğini gösteren ilk bulgulardır.

Anahtar sözcükler: SCN, lokomotor aktivite, leptin, Suriye hamsteri, Mesocricetus auratus

Th e eff ects of leptin hormone on locomotor activity in Syrian hamsters (Mesocricetus auratus)

728

Introduction

In mammals, the suprachiasmatic nucleus (SCN) is the master biological clock that controls circadian rhythms in a variety of biological processes (1,2). Th ese circadian rhythms disappear by ablation of the SCN region (3) and some of them (e.g., locomotor activity) reappear upon transplantation of fetal SCN tissue (4). Th e daily light-dark cycle is the main environmental cue that entrains the SCN clock to the 24 h (5). Besides light, the phase of the circadian clock can be shift ed by a variety of non-photic factors such as food availability (6), temperature (7), social factors (8), and some hormones (9).

Leptin, a protein secreted by adipocytes, mirrors the body fat content and signals the status of energy stores to the brain (10). Although the release of leptin follows diff erent diurnal rhythms in rats (decrease in the light phase and increase in the dark phase) (11) and in Syrian hamsters (decrease in the dark phase and increase in the light phase) (12), it is directly controlled by the SCN, independent of the other SCN controlled physiological activities in both rats (13) and Syrian hamsters (14). Moreover, it is suggested that leptin may play an important role as a feedback loop between the energy reserves and the hypothalamic centers that control food intake (15). In the hypothalamus, strong immunoactivity for leptin receptors was found in the SCN (16), and the SCN circadian clock, when isolated in vitro, can be phase advanced by leptin in a dose-dependent fashion (9). However, it is not known yet whether leptin hormone phase shift s the locomotor activity of a whole animal when administered in vivo.

Syrian hamsters are very sensitive to environmental changes. Th is species appears thus to be a good model to test the chronobiotic eff ects of leptin hormone. Th e present study aimed to investigate the eff ects of diff erent leptin hormone administrations on the rhythm and the amount of locomotor activity of the Syrian hamster.

Materials and methods

Adult male Syrian hamsters (Mesocricetus auratus: Cricetidae; ~110 g, 120 days of age) were obtained from our laboratory colony maintained at the Abant İzzet Baysal University. Th ey were

exposed from birth to 14:10 L:D (lights off at 2000 h). Animals were maintained in plastic cages (16 × 31 × 42 cm) with pine shavings used as bedding. Food pellets and tap water were accessible ad libitum. Experiments were performed in accordance with Turkish legal requirements, under the license given by the Institutional Animal Care and Use Committee at the Abant İzzet Baysal University. All lighting was provided by cool-white fl uorescent tubes controlled by automatic programmable timers. Ambient temperatures in the animal facilities were held constant at 22 ± 2 °C in air-ventilated rooms.

Th e animals were divided into 3 groups: ip leptin injection, sc leptin infusion and intra-SCN leptin infusion groups. Each group had its own control group that consisted of hamsters having vehicle treatments. Following this, the hamsters were exposed to constant darkness (DD) for 2 weeks. Hamsters were subjected to leptin and saline treatments at circadian time (CT) 10 for 3 days aft er 2 weeks in DD. Finally, locomotor activity was measured for 1 week more aft er the leptin administrations. At the end of the experiment, intra-SCN cannulated hamsters were decapitated following injection with methylene blue through the cannula. Brains were removed and cut with a vibrotome as coronal sections (40 μm). Th ese brain sections were examined under a microscope for the true placement of the cannula.

Before surgery, hamsters were anesthetized subcutaneously with ketamine (20 mg/kg BW, Sigma Chemical Company, MO, USA) and intraperitoneally with pentobarbital (32.5 mg/kg BW). Depth of anesthesia was monitored by frequent testing for the presence of leg fl exion refl exes and active muscle tonus. Aft er awaking from anesthesia, the animals were placed in their cages. We started recording the locomotor activity 2 days aft er the surgery.

Th e methods of cannulation and infusion were based on Carter and Goldman (17). Briefl y, the skin at the back of the neck was shaved, the area was sterilized with 70% ethanol, and a small incision was made to allow insertion of a subcutaneous cannula for leptin or vehicle infusion. Th e cannula consisted of a 60 cm length of polyethylene tubing (PE 20). Only 1 cm of the tubing remained beneath the skin of the animal; the remainder was for transport purpose only. At the distal end of the cannula, 5 cm of stainless steel

B. GÜNDÜZ, A. KARAKAŞ

729

tubing was fi xed to prevent the cannula from being

chewed. Th e tubing was attached to a disk of nylon

mesh, saturated with nitrofurazone that prevented

the nylon mesh sticking to the animal’s skin. Th e

cannula was then secured by a purse-string suture.

Newskin adhesive was applied to the incision area

to prevent any contamination. Th e entire cannula

assembly was sterilized with 70% ethanol before use.

Th e cannula was implanted between the 2 SCN.

Hamsters were anesthetized and fi xed in a stereotaxic

instrument (Stoelting Co., IL, USA) and a hole was

opened at the skull by a dental drill; a 22-gauge

stainless steel guide cannula 313-G/Spc (Plastics One

Inc., VA, USA) was implanted aseptically into the

SCN region (coordinates anterior +0.6  mm; at the

midline and ventral −7.8  mm; relative to bregma).

Th e guide cannula was secured in place by dental

cement (Dental Products of Turkey, İstanbul) affi xed

to 2 mounting screws. A stainless steel dummy

cannula was used to occlude the guide cannula

when not in use. Each cannulated hamster was

then kept individually for a week to recover from

surgery. At the end of the experiment, intra-SCN

cannulated hamsters were sacrifi ced and their brains

were histologically examined. Th e results of the

improperly cannulated hamsters were not included

in the calculations.

Th e same procedures were applied in both

subcutaneous and intra-SCN infusions. A plastic

syringe guard was secured above the cage. A small

hole was drilled in the syringe guard through which

the cannula was passed and loosely knotted above to

prevent the distal cannula from becoming too slack.

Th e proximal end of the cannula was attached to the

syringe fi lled with vehicle or leptin solutions. Leptin

solutions were made in saline (0.9% NaCl). Stock

solutions were kept at 4 °C prior to use. Fresh working

solutions were made by diluting stock with sterile

saline to the desired concentration. Th e syringes

were mounted onto an infusion pump modifi ed to

deliver fl uid to 10 animals simultaneously through 10

separate Hamilton syringes. Th is pump was controlled

by an electronic timer so that infusions of controlled

duration can be administered at specifi c times of the

day. Th e animals received 0.1 mL solution including

4 μg/kg leptin hormone for subcutaneous and 0.01

mL solution including 0.4 μg/kg leptin hormone

for intra-SCN infusions. Th e control animals were infused only with saline (0.9% NaCl). Th e duration of the infusions was 30 min each.

Th en 0.1 mL solution including leptin (4μg/kg) hormone was injected intraperitoneally into the animals with a 22-gauge needle. Th e same amount of saline (0.9% NaCl) was injected into the control animals.

Before the start of the experiment, 40 hamsters were kept under conditions of 14:10 LD cycle for 10 days. Th irty hamsters (10 animals per group; 5 for control and 5 for leptin) that showed a distinct circadian rhythm of locomotor activity were selected for the experiment. Locomotor activity was measured for running-wheel activity under a constant temperature (22 ± 2 °C). Animals were exposed to constant darkness. Th e number of wheel revolutions per 10-min interval was automatically recorded and stored on a hard disk by a computer. Th e stored results were analyzed by the program Vital View Data Acquisition Soft ware (Mini Mitter Company, Inc. Bend, OR USA). Th e activity was shown by the double plotted actograms by the program Acti View Soft ware (Mini Mitter Company). Th e analysis of circadian locomotor activity rhythm was performed by the combination of visual inspection and computerization of actograms and frequency histograms. Circadian period was determined by chi-square periodogram analysis. Phase shift s were defi ned as the diff erence between the predicted and observed onset times on the day following the manipulation. Th e predicted onset was determined from an eye-fi t line drawn through the last 5 activity onsets in DD prior to the manipulation. Th is line was extended to the fi rst postadministration day to give the predicted onset for that day. Th e observed onset time was determined from an eye-fi t line drawn through the fi rst onsets in DD following the manipulation. Lines were drawn by 2 experienced observers who were blind to the experimental groups. Th e wheel-turn revolutions used were the mean of 24 h.

Data were analyzed by one-way analysis of variance (ANOVA) using SPSS (SPSS Inc., Los Angeles, CA, USA, Ver. 11.0). When the test indicated a signifi cant diff erence (P < 0.05), Tukey’s post hoc test was performed. Data are presented as mean ± SEM aft er back transforming from ANOVA results.

Th e eff ects of leptin hormone on locomotor activity in Syrian hamsters (Mesocricetus auratus)

730

Results and discussion

Figure 1 is the representative actogram of hamsters

that were subjected to 4 applications. Figure 2 shows

the onset of activity (A) and average wheel turns of

hamsters (B).

Figure 1A shows a representative actogram from

a control hamster that was injected with saline. Since

the actograms for the controls of sc and intra-SCN

infusions were similar to those for the saline injected

animals, they were not presented. Th e mean period

length was 23.50 ± 0.12 h and the average wheel turn

was 112.23 ± 8.1 in saline injected controls.

Figure 1B shows a representative actogram from

a hamster that was ip injected with 4 μg/kg leptin

hormone. Leptin injection phase advanced the onset

of activity (1.12 ± 0.15 h) (F = 2.451, P = 0.041, df =

5) (Figure 2A). Th e mean period length was 24.22 ±

0.12 h in leptin injected hamsters.

Figure 1C shows a representative actogram from

a hamster that was sc infused with 4 μg/kg leptin.

Subcutaneous infusion phase advanced the onset of

activity (2.15 ± 0.20 h) (F = 3.825, P = 0.012, df =

8) (Figure 2A). Th e mean period length was 24:00 ±

0.22 h in sc saline infused controls and was 24.35 ±

0.18 h in leptin infused hamsters.

Figure 1D shows a representative actogram from

a hamster that was intra-SCN infused with 0.4 μg/kg

leptin. Intra-SCN infusion phase advanced the onset

of activity (2.48 ± 0.24 h) (F = 10.221, P = 0.001, df =

8) (Figure 2A). Th e mean period length was 23.52 ±

0.20 h in saline infused controls and was 24.48 ± 0.10

h in leptin infused hamsters.

Figure 2B shows the average wheel turns of the

hamsters that received ip injection [control (102 ±

15), leptin (98 ± 13)], sc infusion [control (99 ± 10),

leptin (104 ± 11)], and intra-SCN infusion [control

(98 ± 17), leptin (99 ± 15)] in DD. Th e activity levels

were similar in the 3 groups before and aft er the

leptin administrations (F = 1.632, P = 0.233, df = 5).

Figure 3 shows a photograph of a coronal section

from the SCN region of the cannulated hamster.

We have previously demonstrated that the SCN

may directly regulate the diurnal changes in plasma

leptin levels but the precise mechanisms had to be

elucidated (14). Here, we hypothesized that leptin

hormone may aff ect locomotion. We tested this hypothesis by administrating leptin hormone in 3 diff erent ways followed by measuring locomotor activity. Th e following principal fi nding emerged: administration of leptin hormone phase advances the locomotor activity of the Syrian hamsters and bigger phase shift s in the locomotor activity onsets occur by the administration of leptin hormone directly to the SCN area. Th is is the fi rst report of the in vivo eff ects of leptin hormone on the rhythm of locomotor activity.

It has been shown that external administration of leptin resulted in a signifi cant increase in physical activity in rats (18,19) and in marsupials (20). However, our results here show that, fi rst, leptin administrated animals demonstrated similar locomotor activity with controls in the running wheel and that it does not depend on the type of administration. Second, we questioned whether the pattern of leptin administration could aff ect the phase shift in hamsters. We demonstrated that leptin phase advances the locomotor activity of the Syrian hamsters, suggesting that exogenous leptin may evoke the SCN activity. Since the rhythm of the locomotor activity is driven by the SCN, an early onset of the SCN due to the direct administration of the leptin hormone to that area could have a phase advancing eff ect.

We can speculate on the potential mechanisms through which leptin alters locomotor activity and why this eff ect is much altered in intra-SCN infusions. Th erefore, when hypothesizing how SCN sensitivity is altered, we can focus on the passage of leptin into the brain. Leptin is a satiety polypeptide and is too large (16 kDa) to cross the blood-brain barrier. It is only able to enter the brain by a saturable transporter (21) located at both endothelial and epithelial (blood-CSF barrier) arms of the blood-brain barrier. Th erefore, the passage of leptin from this barrier is rate-limited. We examined whether the administration-dependent changes are associated with a diff erent locomotor activity. We found that intra-SCN infusions which eliminated the blood-brain barrier were much more eff ective than sc infusions or ip injections. Intra-SCN infusions caused bigger phase shift s in the locomotor activity onsets. Th is protocol enables leptin access to a wide range of SCN regions that are involved in rhythmic functions.

B. GÜNDÜZ, A. KARAKAŞ

731

A B

C D

Figure 1. Representative actograms from control and leptin administered hamsters. A. Control. B. 4 μg/kg ip

leptin C. 4 μg/kg sc leptin. D. 0.4 μg/kg intra-SCN leptin. dd indicates constant darkness. * indicates

the time when 3-day saline and leptin administrations starts. Vertical black lines indicate the onset

of activities. Note the presence of the phase advances in B, C, and D.

Th e eff ects of leptin hormone on locomotor activity in Syrian hamsters (Mesocricetus auratus)

732

Th e relation between the circadian clock and

leptin in Syrian hamsters has not been studied

extensively. In theory, these 2 systems might interact

in such a way that the circadian clock regulates

leptin, and leptin regulates the clock, or both. It is

interesting to compare the leptin curve with the

rhythm of endogenous leptin release. In rodents,

plasma leptin levels rise aft er feeding begins, which

usually is at lights off (CT12), and leptin levels peak

about 6 h later (13). Because leptin transport into the

brain appears to be rate-limited (22), there probably

is a delay between leptin increase in plasma and in

the brain. Th us, it is tempting to speculate that the

time when brain leptin levels peak may coincide with

when the SCN clock becomes insensitive to phase-

resetting by leptin. Th us, circadian clock sensitivity

to leptin levels may be down-regulated at the time

when leptin levels normally peak. On the other hand,

our animal model is hamster and peak level of leptin

appears at midday (12,23). Th is means that leptin

peak may coincide with the time when the SCN

becomes sensitive. Moreover, we used intra-SCN

leptin infusion. Th ese results suggest that the type of

leptin administration may play a role in modulating

circadian clock phase. Th erefore, we conclude that

the SCN may be directly sensitive to phase-resetting

by leptin. Kalsbeek et al. (13) have shown that the

SCN controls the diurnal rhythm of plasma leptin

concentrations directly or indirectly of other clock-

controlled rhythms (cortisol, feeding) in the rat.

Th ey show that ablation of the SCN by thermal lesion

completely eliminated the diurnal rhythmicity of

circulating plasma leptin concentrations.

A

Groups Groups

Ph

ase

Ad

van

ce (

h)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Ip Inj. Sc Inf. Intra SCN Inf.

a

b

c

B a

bb

Ip Inj. Sc Inf. Intra SCN Inf.

Mea

n W

hee

l T

urn

/Day

0

10

20

30

40

50

60

70

80

90

100

110

120a

a aaa

a

Figure 2. Phase advances (h) by the leptin administrations (A) and the mean wheel turns of the hamsters (B). In Figure 2B, open bars

represent the controls, striated bars represent the experimental groups. Diff erent letters indicate the statistically signifi cant

diff erence among the groups (P < 0.05).

Figure 3. Th e photograph of a coronal section (40 μm) from

SCN region of the hypothalamus (200×). Methylene

blue is seen in between the right and left SCN regions.

SCN: Suprachiasmatic nucleus, Ox: Optic chiasm,

Pvn: Paraventricular nucleus, 3v: Th ird ventricle.

B. GÜNDÜZ, A. KARAKAŞ

733

Many hypothalamic sites express leptin receptors; however, our knowledge about the role of these diff erent target sites in the regulation of locomotor activity is very limited. Because SCN is part of a network of hypothalamic nuclei that homeostatically regulate the time of feeding (24), the SCN may impose a daily rhythm on physical activity and energy expenditure in part through the actions of leptin in those regions (SCN, PVN, etc.) (25,26). Leptin receptors have been reported in the SCN of rat (14). Th e SCN provides its most intense output to the dorsomedial nucleus of the hypothalamus (DMH) (27), which is necessary for locomotor activity. Th is is an especially intriguing possibility given the links between clock gene defi ciencies and obesity (28), and given the decreased amplitude of the daily rhythm in energy expenditure in obese objects (29). In addition, our data support a model whereby leptin eff ects are segregated by action on diff erent neuroanatomical regions in the brain; the SCN may represent a leptin target that modulates locomotor activity.

Hormones can infl uence additional aspects of the clock besides the onset of rhythmicity. For example, in some species of mammals and birds it has been shown that melatonin and steroid hormones infl uence tau, the length of the clock’s free-running period (30,31). Our studies suggest that leptin does have similar function in Syrian hamsters because leptin treatments did produce signifi cant increase in tau.

Leptin administrations did not change the wheel-turn activity signifi cantly. Th erefore, the behavioral and physiological eff ects of leptin administration can be separated into at least 2 independent neural pathways, one of which modulates simultaneously, but independently, PVN cells (to stimulate) and one or more that do not (to aff ect locomotor behavior, body weight). Interestingly, leptin’s advance of locomotor activity in hamsters appears

be a species-specifi c action. In addition, hamsters displayed signifi cantly advanced locomotor activity when given as intra-SCN infusion. Th ough it may seem counterproductive to activate one aspect of locomotion while concurrently inhibiting another, under normal conditions, endogenous leptin might not simultaneously aff ect multiple areas of the brain (as it is with intra-SCN infusions). Th us, leptin’s regulation of locomotor activity and body weight/food intake may be reproduced both spatially and temporally. Future studies selectively infusing leptin into discrete brain regions implicated in the control of locomotive behaviors and eliminating SCN will begin to address this critical issue of where and how leptin aff ects locomotor behavior and/or food intake/body weight.

In summary, the data presented in the present paper support a distributed model in which leptin signals to hypothalamic region to generate an overall behavior. While previous work has established the hypothalamus as a critical brain region in response to leptin (9,14), the inclusion of SCN suggests that leptin-mediated modulation of locomotor activity provides a novel neural pathway.

Acknowledgement

Financial support for this study was provided by TÜBİTAK (SBAG-2740).

Corresponding author:

Bülent GÜNDÜZ

Department of Biology, Faculty of Arts and Sciences,

Çanakkale Onsekiz Mart University,

Çanakkale - TURKEY

E-mail: bgunduzbio@comu.edu.tr

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