Ž .Brain Research 824 1999 18–27

Research report

Gold-thioglucose-induced hypothalamic lesions inhibit metabolic modulationof light-induced circadian phase shifts in mice

Etienne Challet ), Daniel J. Bernard, Fred W. TurekCenter for Circadian Biology and Medicine, Department of Neurobiology and Physiology, Northwestern UniÕersity, 2153 North Campus DriÕe, EÕanston,

IL 60208, USA

Accepted 26 January 1999

Abstract

The circadian clock located in the suprachiasmatic nuclei is entrained by the 24-h variation in light intensity. The clock’s responses tolight can, however, be reduced when glucose availability is decreased. We tested the hypothesis that the ventromedial hypothalamus, akey area in the integration of metabolic and hormonal signals, mediates the metabolic modulation of circadian responses to light by

Ž .injecting C57BLr6J mice with gold-thioglucose 0.6 grkg which damages glucose-receptive neurons, primarily located in theventromedial hypothalamus. Light pulses applied during the mid-subjective night induce phase delays in the circadian rhythm oflocomotor activity in mice kept in constant darkness. As previously observed, light-induced phase delays were significantly attenuated infed mice pre-treated with 500 mgrkg i.p. 2-deoxy-D-glucose and in hypoglycemic mice fasted for 30 h, pre-treated with 5 IUrkg s.c.insulin or saline, compared to control mice fed ad libitum. In contrast, similar metabolic challenges in mice with gold-thioglucose-inducedhypothalamic lesions did not significantly affect light-induced phase delays compared to mice treated with gold-thioglucose and fed adlibitum. These results indicate that destruction of gold-thioglucose-sensitive neurons in the ventromedial hypothalamus prevent metabolicregulation of circadian responses to light during shortage of glucose availability. Therefore, the ventromedial hypothalamus may be acentral site coordinating the metabolic modulation of light-induced phase shifts of the circadian clock. q 1999 Elsevier Science B.V. Allrights reserved.

Keywords: Suprachiasmatic nucleus; Circadian rhythm; Ventromedial hypothalamus; Glucose utilization; Fasting; C57BLr6J mouse

1. Introduction

The daily temporal organization of behavioral andphysiological processes is controlled by a circadian clock

Ž .located in the suprachiasmatic nuclei SCN of the hypo-thalamus. The SCN are primarily entrained to the ambientlight:dark cycle, with the clock being most sensitive to

w xphotic cues during the subjective night 29,44 . The magni-tude of light-induced phase shifts is reduced in situations

w xof decreased glucose availability 12 , including hypo-glycemia and blockade of glucose utilization by 2-deoxy-

Ž .D-glucose 2-DG , a competitive inhibitor of glucose trans-port and phosphorylation. The central mechanisms bywhich metabolic cues modulate photic phase-resetting ofthe SCN are unknown.

) Corresponding author. Center for the Study of Biological Rhythms,School of Medicine, Universite Libre de Bruxelles, 808 Route de Lennik,´B-1070 Brussels, Belgium. Fax: q32-2-555-35-69; E-mail:e-challet@nwu.edu

Although all neurons utilize glucose as an energy sourcew x61 , only a few have their firing rate specifically modified

w xby changes in extracellular glucose concentration 54 .Among such glucose-responsive neurons, those which in-crease their firing rate when extracellular glucose is in-

Ž .creased i.e., glucoreceptor or glucose-receptive neuronsŽ .are primarily located in the ventromedial nuclei VMH of

w xthe hypothalamus 30,53 , and secondarily in the nucleusw xof the solitary tract 41 . Glucose-receptive neurons contain

ATP-sensitive potassium channels that can be inhibited byincreasing extracellular glucose levels to induce cell depo-larization. Likewise, a decrease in extracellular glucoseresults in hyperpolarization and decreased frequency of

w xaction potentials of glucose-receptive neurons 3,62 .The VMH are one of the key brain regions in the

integration of metabolic and hormonal signals leading tow xthe central regulation of glycemia 51,54 and energy

w xmetabolism 4,52 . In particular, the VMH play an impor-tant role for sensing hypoglycemia and 2-DG-inducedcytoglucopenia, and triggering counterregulatory hormonal

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01192-0

( )E. Challet et al.rBrain Research 824 1999 18–27 19

responses, such as the release of catecholamines andw xglucagon 7,8 . The VMH receive monosynaptic, probably

w xexcitatory input from the SCN 10,28,65 . The SCN pro-vide circadian signals to the autonomic nervous systemw x49,50,58 and may also participate in glucose homeostasis,

w xat least via facilitation of VMH activity 49 , and in lipidw xmobilization 5 . The VMH, in turn, have direct and

w xindirect connections back to the SCN 10,31,55 , includingw xexcitatory amino acid projections 42 .

In addition to the reciprocal VMH–SCN connectionsobserved in rats and hamsters, the VMH’s ability to detectand respond to a shortage of cerebral glucose availabilitysuggests that they may be involved in the metabolic modu-lation of the circadian responses of the SCN to light. Totest this hypothesis, the phase resetting responses to a lightpulse after blockade of glucose utilization and hypo-glycemia, induced either by fasting alone or fasting plusinsulin treatment, were determined in mice bearing lesionsof the VMH. Rather than using electrolytic lesions thatdestroy fibers of passage or ibotenic acid lesions that aredifficult to perform in restricted area of the small-sizedmouse brain, mice were injected with gold-thioglucoseŽ .GTG , a glucose analog that causes irreversible damage ofglucose-receptive neurons in the VMH, by acting on neural

w xstructures contiguous with capillaries 9,17 . Prevention ofGTG-induced VMH necrosis by inhibitors of glucose

w xtransport 18 supports the hypothesis that GTG specifi-cally destroys glucose-receptive VMH cells. Moreover,GTG-induced lesions lead to long-term obesity and hyper-

w x w xphagia 6,15,18 , as do ibotenic 60 and electrical lesionsw xof the VMH 4,52 .

2. Materials and methods

2.1. Animals and laboratory conditions

ŽA total of 48 adult male C57BLr6J mice Jackson.Labs, Bar Harbor, ME were housed singly in cages

Ž .equipped with running wheels diameter: 11 cm in aŽ .temperature-controlled room 23"18C with a light:dark

Ž .12:12 h cycle lights on at 0500 h . During daytime, lightŽintensity was about 300 lx at cage level. Food laboratory

.chow, Harlan Teklad, WI and water were available adlibitum, unless otherwise stated. Wheel-running activity

Žwas continuously recorded Chronobiology Kit, Stanford.Software Systems, Stanford, CA . At the end of a 2-week

baseline period, mice were injected i.p. with either 0.6Žmgrg body mass of GTG aurothioglucose, Sigma, St.

. Ž .Louis, MO , or saline vehicle ns24 per treatment . Tendays later, mice were transferred to constant darknessŽ .DD where they remained for a period of 20 days. Animalmaintenance in darkness was aided by use of an infrared

Ž .viewer Find-R-Scope, FJW Optical System, Palatine, IL .

2.2. Experimental design

ŽA light pulse was given on the 11th day in DD denoted. ŽDDq10 at circadian time 18 CT18; CT12 is defined as

.the time of activity onset . Some 12 GTG-treated mice and12 control mice were fed ad libitum. On Day DDq10,they received an i.p. injection of 500 mgrkg body mass of

Ž .2-deoxy-D-glucose 2-DG; Sigma or saline at CT17, 1 hŽprior to a sub-saturating 10-min light pulse 50 lx of white

.light; ns6 per group . For light stimulation, individualswere transferred from their own cages to a white chamberŽ .diameter: 11 cm, height: 6 cm inside a photic stimulationdevice. Light intensity was determined using a digitalphotometer. The dose, time of 2-DG injection and lightpulse parameters were chosen according to previous exper-

w ximents 23,52 . Some 12 GTG-treated mice and 12 controlmice were fasted by food removal for 30 h from CT12 onDay DDq9 to CT18 on Day DDq10, a duration of fooddeprivation which has been shown previously to induce

w xhypoglycemia in mice 12 . Half of the fasted mice re-Ž .ceived an s.c. injection of either insulin 5 IUrkg; Sigma

or saline. Injections were administered 30 min prior to aŽ .10-min light pulse 50 lx of white light given at CT18 on

Ž .Day DDq10 ns6 per treatment . The dose and time ofinsulin injections were defined according to previous stud-

w xies 12 , so that severe hypoglycemia occurred at the timeof the light pulse.

2.3. Immunocytochemistry

In order to assess the effectiveness and extent of GTG-induced lesions in the VMH, gliosis was visualized byimmunocytochemistry for glial fibrillary acidic proteinŽ .GFAP , an intracellular protein expressed by astrocytes.After 20 days in DD, mice were killed with CO during2

Ž .the late subjective day i.e., when gut content is low . Micewere weighed and epididymal fat pads mass determined.Mice were then perfused with 4% paraformaldehyde.Brains were postfixed overnight, transferred to a 30%sucrose solution and stored at y808C until 40-mm sectionswere prepared on a cryostat. Sections were then incubatedfor 1 h with 10% normal horse serum in 0.1 M phosphate

Ž .buffered saline PBS; pH 7.4 and for 12 h at 48C with aŽmouse monoclonal anti-GFAP ICN Biochemicals, Costa

.Mesa, CA diluted 1:1000 in PBS containing 0.3% TritonX-100. Sections were washed in PBS and incubated for 1 hwith biotinylated horse anti-mouse immunoglobulinsŽ .Vectastain ABC kit, Vector Labs, CA . Sections werewashed again in PBS and transferred for 1 h to a solutionof ammonium sulfate nickel containing streptavidin–biotincomplex conjugated to horseradish peroxidase. Peroxidasewas visualized by the diaminobenzidine reaction. The brainsections were mounted on gelatin-coated slides.

2.4. Data analysis

To quantify the light pulse-induced phase shifts, a linewas fitted by eye to the onsets of locomotor activity for the

( )E. Challet et al.rBrain Research 824 1999 18–2720

first 10 days before the light pulse. This line was projectedŽ .to the day of the light pulse i.e., Day DDq10 . Similarly,

a line was fitted to the onsets of activity for the 10 daysafter the photic pulse. That line was retroprojected to theday of the pulse. The magnitude of the phase shifts wascalculated as the difference between these two lines. Dailyactivity was defined as the total wheel revolutions per

Ž . Ž .cycle beginning at CT12 . The circadian period t was

2 Žassessed by the x periodogram analysis Chronobiology.Kit software over the 10 days before and after the light

pulse.

2.5. Statistical analysis

Values are means"S.E.M. For a given nutritional stateŽ .fed or fasted , data were analyzed by two-way analyses of

Ž .Fig. 1. Glial fibrillary acidic protein immunoreactive staining in representative coronal sections of suprachiasmatic nuclei SCN , left ventromedialŽ . Ž . Ž .hypothalamic nucleus VMH and left nucleus of the solitary tract NTS in a mouse injected with saline control and a mouse injected with

Ž . Ž .gold-thioglucose GTG . The arrow indicates the GTG-induced increase of GFAP staining in the ventromedial region of the VMH Right middle panel .Scale bars200 mm.

( )E. Challet et al.rBrain Research 824 1999 18–27 21

Table 1Ž .Adiposity index epididymal fat pads mass as % body mass in the

different treatment groups

Ž .Neurotoxin Treatment Adiposity index %aControl Fedrsaline 1.15"0.06aControl Fedr2-DG 1.27"0.12bGTG Fedrsaline 2.02"0.31bGTG Fedr2-DG 2.14"0.41aControl Fastedrsaline 0.94"0.09aControl Fastedrinsulin 0.80"0.08bGTG Fastedrsaline 1.21"0.11bGTG Fastedrinsulin 1.28"0.13

Ž .Data are means"S.E.M. ns6 per group .GTG: gold-thioglucose.2-DG: 2-deoxy-D-glucose.

Ž .For a given nutritional state fed or fasted , groups with no letters inŽ .common are significantly different from one another P -0.05 .

Ž .variance ANOVA to compare the effects of the neuro-Ž .toxin GTG vs. saline and the different metabolic treat-Žments saline vs. 2-DG in fed animals, and saline vs.

.insulin in fasted animals . If significant main effects or aŽ .significant interaction were found P-0.05 , post-hoc

comparisons were performed with the Student–Newman–Keuls test.

3. Results

3.1. Histology

Examination of immunocytochemical staining for GFAPrevealed clear glial proliferation within the ventromedialhypothalamus, especially in the medial region of the VMHand the lateral region of the arcuate nuclei. In the 24GTG-treated mice included in this study, the increase ofGFAP staining in the VMH was evaluated microscopicallyŽ .see an example in Fig. 1 . In keeping with accumulation

w xof gold in the brain by autoradiography 19 , the VMH wasthe brain area presenting the most consistent increase in

Ž .gliosis after GTG treatment Fig. 1 . Staining in otherdiencephalic regions was essentially similar between saline-and GTG-injected mice. For example, in most animals,

w xGFAP immunoreactivity was strong in the SCN 32,45Ž .see Fig. 1 and the preoptic area, and low to moderate inparaventricular hypothalamic nuclei and paraventricular

Ž .thalamic nuclei data not shown . In addition to the VMH,a small increase of gliosis in a few GTG-treated animals

Žwas also apparent in the medulla oblongata i.e., in the.vagal nuclei and nucleus of the solitary tract; Fig. 1 .

Ž .Fig. 2. Double-plotted daily wheel-running activity of four mice kept in constant darkness and fed ad libitum. Upper panels Records from mice receivingŽ .an i.p. injection of saline at CT17 followed 1 h later by a light pulse 50 lx of white light lasting 10 min . Mice previously treated with saline or GTG are

Ž . Ž . Ž . Ž .shown in panels A and B , respectively. Lower panels Records from mice receiving an i.p. injection of 500 mgrkg 2-deoxy-D-glucose 2-DG at CT17Ž . Ž . Ž .followed by a light pulse as above . Mice previously treated with saline or GTG are shown in panels C and D , respectively. For each actogram, the two

lines are fitted lines to the nocturnal activity onsets before and after the light pulse. Arrows denote day of treatment. Time of injection is indicated by acircle.

( )E. Challet et al.rBrain Research 824 1999 18–2722

3.2. Metabolic changes

GTG treatment leads to obesity after long-term condi-Žtions of ad libitum feeding i.e., after several months; e.g.,

w x.Ref. 15 . In the present study, to rule out any simpleeffect of huge lipid fuel reserves, the GTG-treated micewere given a light pulse 20 days after the injection of GTGand killed 10 days after the light pulse. Nevertheless, toassess subtle metabolic changes between groups, we deter-mined an adiposity index by expressing epididymal fat

w xpads as a percentage of body mass 13 . As shown in Table1, the adiposity index in fed mice was significantly larger

Žin GTG-injected than in saline-injected mice 2.08"0.25Ž . .vs. 1.21"0.07%, respectively; F 1,20 s10.6, P-0.01 ,

Ž .regardless of the injection saline or 2-DG . In mice previ-ously fasted, the adiposity index was also larger in GTG-

Žinjected than in control mice 1.24"0.08 vs. 0.87"Ž . .0.06%, respectively; F 1,20 s12.6, P-0.01 , regardless

Ž .of the injection saline or insulin .

3.3. Light-induced behaÕioral phase shifts and circadianperiod

In mice fed ad libitum throughout the experiment,Ž .regardless of the injection saline or 2-DG , mean phase

delays induced by a light pulse at CT18 were significantlysmaller in control mice compared to GTG-treated miceŽ Ž . .F 1,20 s5.8, P-0.05; Figs. 2 and 4 . Regardless of the

Ž .lesion GTG or saline , mean light-induced phase delayswere significantly smaller in mice injected with saline

Ž Ž .compared to those injected with 2-DG F 1,20 s12.9,.P-0.01; Figs. 2 and 4 . Moreover, there was a significant

Ž Ž . .interaction F 1,20 s13.3, P-0.01; Fig. 4 between theŽ . Žeffects of lesion GTG or saline and injection saline or

.2-DG . Post-hoc analysis indicated that in individuals in-jected with saline, the light-induced phase delays in controlmice fed ad libitum were similar to those in GTG-treatedanimals fed ad libitum. After blockade of glucose utiliza-tion, however, phase delays were significantly reduced in

Žcontrol mice compared to GTG-treated mice Figs. 2 and.4 .

In mice fasted prior to the light pulse, the phase delaysinduced by a light pulse at CT18 were significantly smaller

Ž Ž .in control mice compared to GTG-treated mice F 1,20 s.32.0, P-0.001; Figs. 3 and 4 . The effect of the injection

Ž .saline or insulin and the interaction GTG= injectionŽ . Ž Ž .saline or insulin were not significant F 1,20 s0.3 andŽ . .F 1,20 s0.1, respectively, P)0.1; Figs. 3 and 4 .

Using a two-way ANOVA with repeated measures, theŽ .circadian period t was analyzed in fed animals across

Fig. 3. Double-plotted daily wheel-running activity of four mice kept in constant darkness and fasted for 30 h, from CT12 the day before to the end of theŽ . Ž .light pulse. Upper panels Records from mice receiving a s.c. injection of saline 30 min prior to a light pulse 50 lx of white light lasting 10 min given at

Ž . Ž . Ž .CT18. Mice previously treated with saline or GTG are shown in panels A and B , respectively. Lower panels Records from mice receiving a s.c.Ž . Ž . Ž .injection of 5 IUrkg insulin 30 min prior to a light pulse as above . Mice previously treated with saline or GTG are shown in panels C and D ,

respectively. For each actogram, the two lines are fitted lines to the nocturnal activity onsets before and after the light pulse. Arrows denote day oftreatment. Time of injection is indicated by a circle.

( )E. Challet et al.rBrain Research 824 1999 18–27 23

Ž .Fig. 4. Phase shifts negative values are delays in circadian activityŽrhythm of mice housed in constant darkness after a light pulse 50 lx of

.white light lasting 10 min given at CT18. Half the mice were previouslyŽ .treated with saline or GTG GTG-treated . Mice that were fed ad libitum

over the experiment received an i.p. injection of 500 mgrkg 2-deoxy-D-Ž . Ž .glucose 2-DG or saline at CT17 Fed groups . Mice that were fasted

Ži.e., food was removed for 30 h, from CT12 the day before to the end of.the light pulse received an s.c. injection of 5 IUrkg insulin or saline 30

Ž . Žmin prior to the light pulse Fasted groups . Means"S.E.M. ns6 per.group . Groups with no letters in common are significantly different from

Ž .one another P -0.05 .

Žthe four treatment groups controlrsaline, controlr2-DG,.GTGrsaline or GTGr2-DG for the 10-day period before

or after the light pulse. The circadian period did not differŽ Ž . .among the treatment groups F 3,20 s0.4, P)0.1 , nor

Ž Ž .did it change following the light pulse F 1,20 s0.1,.P)0.1 . In all fed groups, t was close to 23.7"0.08 h

Ž .see Fig. 2 for examples . A similar analysis was per-formed in fasted mice across the four treatment groupsŽcontrolrsaline, controlrinsulin, GTGrsaline or GTGrin-

.sulin and the 10-day period before or after the light pulse.Again, the circadian period did not differ among the

Ž Ž . .treatment groups F 3,20 s1.6, P)0.1 , or followingŽ Ž . .the light pulse F 1,20 s0.1, P)0.1 . In all fasted

Žgroups, t was close to 23.7"0.07 h see Fig. 3 for.examples .

3.4. QuantitatiÕe analysis of wheel-running actiÕity

A two-way ANOVA with repeated measures was usedto compare the number of wheel revolutions on Day

Ž .DDq9 i.e., prior to the light pulse vs. Day DDq10Ž .i.e., the day when a light pulse was applied in fed mice.The total number of wheel revolutions did not differ

Žsignificantly by treatment controlrsaline, GTGrsaline,Ž . .controlr2-DG or GTGr2-DG: F 3,20 s1.9, P)0.1 , or

Ž Ž .by the day DDq9 vs. DDq10: F 1,20 s0.7, P)0.1;.Table 2 . In fasted mice, the total number of wheel revolu-

Žtions was significantly affected by the day DDq9 vs.Ž . .DDq10: F 1,20 s33.0, P-0.01 , but not by the treat-

Žment controlrsaline, GTGrsaline, controlrinsulin orŽ . .GTGrinsulin: F 3,20 s0.1, P)0.1 . No other compar-

isons were significantly different.The number of wheel revolutions was determined be-

Ž .tween CT6 and CT12 on Day DDq9 Table 2 . In fedmice, afternoon activity was not affected significantly by

Ž Ž . .the neurotoxin GTG or saline: F 1,20 s1.7, P)0.1 , orŽ Ž . .the injection saline or 2-DG: F 1,20 s0.2, P)0.1 .

Similarly, afternoon activity in fasted mice was not af-Žfected significantly by the neurotoxin GTG or saline:

Ž . . ŽF 1,20 s0.1, P)0.1 , or the injection saline or 2-DG:Ž . .F 1,20 s0.1, P)0.1 .

In order to assess subtle differences in the wheel-run-ning activity prior to the light pulse that may interfere with

w xthe circadian responses to light 40,57 , the number ofwheel revolutions was also determined between CT12 and

Table 2Number of wheel revolutions in the different treatment groups

Neurotoxin Treatment Daily activity Daily activity Afternoon act. Early noct. act.on Day DDq9 on Day DDq10 on Day DDq9 on Day DDq10

Control Fedrsaline 21845"1871 20746"1335 1226"517 13428"1950Control Fedr2-DG 21638"4239 16837"2599 1588"639 12050"2526GTG Fedrsaline 16561"2324 21327"3494 1200"420 11367"1567GTG Fedr2-DG 14427"3939 11290"1975 373"151 8598"2035Control Fastedrsaline 23074"4733 16802"2476 2030"692 12409"2191Control Fastedrinsulin 25041"6270 13822"2919 3844"2065 11865"2586GTG Fastedrsaline 22452"5823 14440"3812 3746"2132 9064"2349GTG Fastedrinsulin 25820"3209 15436"1467 2448"501 12364"1731

Ž .Data are means"S.E.M. ns6 per group .GTG: gold-thioglucose.2-DG: 2-deoxy-D-glucose.

Ž .Daily Activity: total wheel revolutions performed per day, beginning at CT circadian time 12.Afternoon Act.: wheel revolutions between CT6 and CT12.Early Noct. Act.: wheel revolutions between CT12 and CT18, prior to the light pulse started at CT18.Day DDq9: day when mice were either fed or fasted.Day DDq10: day on which a light pulse was administered.

Ž . Ž .For a given nutritional state fed or fasted , no groups in a given column were significantly different from one another P)0.05 .Ž .There was a reduction in the total number of wheel revolutions in fasted mice between DDq9 and DDq10 P-0.01 .

( )E. Challet et al.rBrain Research 824 1999 18–2724

ŽCT18 on Day DDq10 i.e., during the 6 circadian hours.before the light pulse; Table 2 . Here again, there were no

Žsignificant effects of the neurotoxin GTG or saline:Ž . . ŽF 1,20 s1.8, P)0.1 and of the injection saline or

Ž . .2-DG: F 1,20 s1.0, P)0.1 on early nocturnal activityof mice fed ad libitum. In fasted mice, nocturnal activityprior to the light pulse was not modified significantly by

Ž Ž . .the neurotoxin GTG or saline: F 1,20 s0.4, P)0.1 , orŽ Ž . .the injection saline or insulin: F 1,20 s0.4, P)0.1 .

None of the possible interactions in the comparisons ofwheel running activity were significant.

4. Discussion

The present study confirms that photic phase resettingof the circadian clock is reduced in intact mice whenglucose availability is decreased. This effect is observedwhether glucose utilization is blocked or animals are fasted,

w xwith or without added insulin-induced hypoglycemia 12 .In addition, the present report demonstrates that destruc-tion of glucose-receptive neurons with the neurotoxin GTGblocks these altered circadian responses to light.

4.1. Lesions induced by GTG treatment

Given that morphology and function of peripheral tis-sues are not altered during the development of obesity in

w xGTG-injected mice 15,34 , the observed effects after GTGinjection are likely to be due to changes in the centralnervous system. Because some responses of the circadianclock to light were different in control mice compared toGTG-injected mice, one can speculate that GTG treatmentaffects directly SCN neurons. Due to high levels of GFAPimmunoreactivity in the SCN of control mice, it was notpossible to detect a change in the SCN following GTGtreatment. While GTG treatment impairs attenuation oflight-induced phase shifts in response to metabolic chal-lenges, it does not affect the phase-angle of photic entrain-

w xment in mice fed ad libitum 11 . In addition, the free-run-ning periods of the circadian rhythm of locomotor activityand the light-induced behavioral phase delays are similarin both GTG- and saline-injected mice fed ad libitumŽ .present study . To specifically rule out direct effects ofGTG in the SCN, further studies are needed in rodentsreceiving GTG in the SCN without damaging the VMH.Taken together, however, the available data suggest thatSCN function in mice with free access to food is notaltered by GTG injection.

Most SCN neurons in vitro do not display changes inw xfiring rate when extracellular glucose is modified 24 , an

effect typical of glucose-receptive neurons as defined byw xOomura 54 . Conversely, about 20% of VMH neurons

increase firing rate when extracellular glucose is increasedw x54 . In the present study, the most extensive damagefollowing GTG administration was observed in the VMH.

This is best exemplified by changes in both anti-GFAPŽ . Ž .staining Fig. 1 and adiposity Table 1 . Limited gliosis

was also observed in the nucleus of the solitary tract insome GTG-treated mice. With higher doses than the onewe used, the nucleus of the solitary tract and dorsal vagal

w xnuclei can be heavily damaged by the GTG treatment 56 .Gold autoradiography after GTG treatment has been usedby Debons and colleagues to localize where GTG acts inthe mouse brain, and these authors found some labeled

w xgold in the dorsal hindbrain 19 . This technique, however,does not provide information on the extent of cell damage.To specifically rule out an involvement of these brainstemnuclei in the metabolic modulation of circadian responsesto light, further studies are needed in rodents with localchemical lesions of the nucleus of the solitary tract androrvagal nuclei. Nevertheless, the present results taken to-gether suggest that the GTG-induced inhibition ofmetabolic modulation of photic phase shifting may bemediated by the VMH.

4.2. ActiÕity feedback on the circadian clock

Acute changes of locomotor activity can modulate thew xregulation of circadian rhythmicity in hamsters 46,63 and

w xmice 21,36 . In the present study, no significant changesin the number of wheel revolutions were detected betweenGTG- and saline-injected mice prior to the light pulse. For

Ž .example, the decrease in total daily activity after afasting period was similar in both GTG- and saline-

Ž .injected animals Table 2 . Therefore, it seems unlikelythat the observed effects on the light-induced phase shiftsare mediated by changes in the level of physical activitythat can interfere with photic phase-resetting in hamsters

w xfed ad libitum 40,57 . As we have hypothesized previ-w xously 12 , the altered phase-shifting responses to light

during shortage of glucose availability may be due tometabolic signals.

4.3. Blockade of glucose utilization by 2-DG

Peripheral injection of 2-DG blocks local glucose uti-w xlization in most brain areas, including the SCN 61 . In

w14 xresponse to C -DG treatment, there is an increase ofblockade of glucose utilization in the SCN during thesubjective day and a decrease during the subjective night

Ž w x.in vivo e.g., Ref. 29 . In addition, the phase of theŽrhythmic firing rate in a slice of SCN can be altered i.e.,

.delayed temporarily by decreasing the availability of glu-w xcose in the bathing solution 24 . Considering a direct

effect of 2-DG in the SCN, one may expect that light-induced phase delays would be greater after 2-DG-induced cytoglucopenia. On the contrary, there was anattenuation of light-induced phase delays in control micepretreated with 2-DG. Furthermore, blockade of glucoseutilization in GTG-treated mice did not reduce the light-induced phase delays. Assuming that the SCN function is

( )E. Challet et al.rBrain Research 824 1999 18–27 25

Ž .not impaired by GTG in mice fed ad libitum see above , adirect effect of cytoglucopenia in the SCN would havebeen as effective in GTG-treated as in control mice. Thus,2-DG-induced effects on SCN appear to be indirect.

The counterregulatory hormonal responses to cytoglu-w xcopenia have been shown to be mediated by the VMH 8

Ž .see Section 1 . In keeping with the hypothesis that theVMH play a major role in sensing 2-DG-induced cytoglu-

w xcopenia, rats with electrolytic lesions of the VMH 27 orw xGTG-treated mice 6 do not increase food intake after

2-DG treatment, although these mice remain sensitive tow xthe inhibitory effect of cholecystokinin 6 . Accordingly,

GTG treatment reduces and delays 2-DG-induced hyper-w xglycemia in mice 48 . In the present experiment, we found

that GTG treatment also impairs the 2-DG dependentattenuation of light-induced phase delays. A glucose injec-tion prior to a light pulse, that increases plasma glucose,

w xdoes not alter the photic phase-resetting 12 . Therefore, itis unlikely that the 2-DG-induced rise in glycemia per seplays a critical role in the reduced phase-shifting effects oflight after blockade of cerebral glucose utilization.

4.4. Hypoglycemia during fasting, with or without insulininjection

The attenuated light-induced phase shifts in controlfasted mice were counteracted in fasted GTG-treated mice,with or without insulin injection prior to a light pulse. Thisdifference may occur because GTG treatment impairs fast-ing- or insulin-induced hypoglycemia, leading to similarcircadian responses to those in control mice fed ad libitum.Previous studies, however, have shown that damage to theVMH does not prevent fasting-induced decreases in plasma

w xglucose in rats with electrical lesions of the VMH 4,52 orw xin mice treated with GTG 15 .

In addition to blockade of glucose utilization and fast-ing, calorie restriction is another situation of negativeenergy balance, usually associated with chronic hypo-

w xglycemia 25,38 . Calorie restriction can phase shift circa-dian rhythms and modify the phase angle of photic entrain-

w xment in rodents 11,13,14 . This altered photic regulationof circadian rhythmicity, that implies SCN involvement,

w xcan be blocked by ibotenic lesions of the VMH in rats 13w xand GTG treatment in mice 11 . Taken together, these

data indicate that the loss of effects of fasting, 2-DGinjection and calorie restriction on the light-induced phaseshifts in GTG-treated mice may be due to an impairment

Ž .in the integration detection of metabolic signals by theVMH that would, when glucose availability is decreased,impact on the SCN function in control mice.

4.5. Metabolic and hormonal signals modulating VMHneurons actiÕity

The metabolic cues that modulate the photic phase-resetting of the SCN are not yet clearly defined. One likelycandidate is a decrease in intracellular glucose availability

Ž .or an altered rate of glucose utilization occurring duringfasting and after 2-DG injection. Such a decrease in glu-cose availability modifies the activity of VMH neuronsw x Ž .54 and may, in turn, affect SCN function present study .Fasting and 2-DG treatment, however, are also associatedwith elevation of plasma ketone bodies and free fatty acidsw x22,52 , and these increases are not impaired by elec-

w xtrolytic lesions of the VMH 4,52 . The changes in adipos-ity we observed among groups of mice are consistent withexpected mobilization of fatty acids during previous fast-

Ž .ing Table 1 . Interestingly, free fatty acids applied elec-trophoretically can affect the firing rate of VMH neuronsw x54 . Therefore, the effects of metabolic cues on photicphase-shifting may be mediated, in part, by changes in freefatty acid levels. Further studies using specific blockers offatty acid oxidation will be useful for testing this hypothe-sis.

Insulin plays a key role in the control of energy utiliza-w xtion 33 and, therefore, may mediate some of the observed

effects. Furthermore, insulin applied during the subjectivew xday inhibits the firing rate of SCN cells in vitro 59 .

w xInsulinemia is unchanged after 2-DG treatment 2,23 , isw xdecreased after fasting 15 , and is increased after insulin

w xinjection 33 . Despite these varied responses to differentmetabolic challenges, reduced circadian responses are ob-

Ž .served in all three situations present study . Moreover,neither electrolytic lesions of the VMH, nor GTG treat-ment affect the typical changes in insulinemia in response

Ž w x.to fasting i.e., hypoinsulinemia; see Refs. 4,15 andŽ w x.2-DG injection i.e., normoinsulinemia; see Ref. 52 . In

spite of the fact that insulin can modulate firing rate ofw xVMH neurons 54 , this hormone does not appear to be

involved critically in the reduced phase shifting effects oflight during metabolic challenges.

Leptin, another hormone involved in the regulation ofw xenergy metabolism 1 , has been shown to inhibit the

w xactivity of glucose-receptive neurons in the VMH 62 . Toour knowledge, the effect of 2-DG on leptin release hasnot yet been studied in vivo. In vitro, however, 2-DGcauses an inhibition of leptin release from cultured rat

w x w xadipocytes 47 . Fasting decreases plasma leptin 1 andincreases leptin receptor mRNA expression in the VMHw x35 . Taken together, these data suggest that, at least duringfasting, lowered plasma leptin may somehow disinhibitVMH neurons. Whether this effect plays a role in themetabolic modulation of the circadian responses to lightremains to be determined.

4.6. Direct Õs. indirect connections from the VMH to theSCN

The present results suggest that the integration ofmetabolic and hormonal cues by the VMH plays a role inthe altered circadian responses to light during metabolicchallenges. As mentioned in Section 1, anatomical studiesrevealed that the VMH send a small number of fibers tothe SCN, some of which are excitatory amino acidergic

( )E. Challet et al.rBrain Research 824 1999 18–2726

w x42 . The critical pathway for photic entrainment of theSCN clock is the retino-hypothalamic tract that conveys

w xphotic cues from the retina 20,44 . A large body ofexperimental data indicates that glutamate release fromretino-hypothalamic terminals is the neurochemical sub-

w xstrate mediating photic signals to the clock 20 . Given thatseveral subtypes of NMDA, non-NMDA and metabotropic

w xreceptors are found in the SCN 20 , it is possible thatŽdistinct, possibly conflicting, glutamatergic signals i.e.,

.photic from the retina or ‘metabolic’ from the VMHactivate different post-synaptic glutamatergic receptor sub-types.

Ž .Besides direct monosynaptic modulation of SCN func-tion by the VMH, indirect pathways from the VMH to theSCN may also be involved in the metabolic modulation ofSCN function. For example, the paraventricular thalamicnuclei, which receive a large projection from the VMHw x w x16 , send direct glutamatergic fibers to the SCN 42 . Theintergeniculate leaflets, which receive a projection from

w xthe VMH 64 , send a dense projection releasing neuropep-w xtide Y and g-aminobutyric acid in the SCN 43 . The

geniculo-hypothalamic terminals are considered to play apivotal role in conveying several non-photic cues to the

w xSCN 26,37,39,46,66 , including putative ‘metabolic’ cuesw x14 . Because lesions of the intergeniculate leaflets inducean almost complete disappearance of neuropeptide Y-im-munoreactive fibers and terminals in the SCN of rodentsw x14,26,37,39 , an increase of neuropeptide Y in the SCNmost likely reflects geniculate activation. The intergenicu-late leaflets may also convey to the SCN some ‘metabolic’cues integrated by the VMH, because 2-DG injection

w xincreases the level of neuropeptide Y in the SCN 2 .In conclusion, both physiological and hodological data

give support to the hypothesis that the VMH integratemetabolic cues and modulate the photic regulation ofcircadian rhythmicity by means of direct andror indirectconnections to the SCN.

Acknowledgements

The present research was supported by NIH HD-28048,HD-09885 and F32-MH-11493. We wish to thank Dr.Joaquın Recio for helpful discussions and comments, and´Susan Losee-Olson for expert technical assistance.

References

w x1 R.S. Ahima, D. Prabakaran, C. Mantzoros, D.Q. Qu, B. Lowell, E.Maratos-Flier, J.S. Flier, Role of leptin in the neuroendocrine re-

Ž .sponse to fasting, Nature 382 1996 250–252.w x2 A. Akabayashi, C.T. Zaia, I. Silva, H.J. Chae, S.F. Leibowitz,

Neuropeptide Y in the arcuate nucleus is modulated by alterations inŽ .glucose utilization, Brain Res. 621 1993 343–348.

w x3 M.L.J. Ashford, P.R. Boden, J.M. Treherne, Glucose-induced excita-tion of hypothalamic neurones is mediated by ATP-sensitive Kq

Ž .channels, Pflug. Arch. Eur. J. Physiol. 415 1990 479–483.w x4 H. Balkan, G. Van Dijk, J.H. Strubbe, J.E. Bruggink, A.B. Steffens,

Exercise-induced sympathetic FFA mobilization in VMH-lesionedŽ .rats is normalized by fasting, Am. J. Physiol. 262 1992 R981–R985.

w x5 M. Bamshad, V.T. Aoki, M.G. Adkison, W.S. Warren, T.J. Bart-ness, Central nervous system origins of the sympathetic nervous

Ž .system outflow to white adipose tissue, Am. J. Physiol. 275 1998R291–R299.

w x6 H.T. Bergen, N. Monkman, C.V. Mobbs, Injection with goldthioglucose impairs sensitivity to glucose: evidence that glucose-re-sponsive neurons are important for long-term regulation of body

Ž .weight, Brain Res. 734 1996 332–336.w x7 M.A. Borg, R.S. Sherwin, W.P. Borg, W.V. Tamborlane, G.I.

Shulman, Local ventromedial hypothalamus glucose perfusion blockscounterregulation during systemic hypoglycemia in awake rats, J.

Ž .Clin. Invest. 99 1997 361–365.w x8 W.P. Borg, R.S. Sherwin, M.J. During, M.A. Borg, G.I. Shulman,

Local ventromedial hypothalamus glucopenia triggers counterregula-Ž .tory hormone release, Diabetes 44 1995 180–184.

w x9 D.F. Brown, J.M. Viles, An ultrastructural study of gold thioglucoselesion formation in the ventromedial hypothalamus: subnecrotic gold

Ž .thioglucose dose, J. Neuropathol. Exp. Neurol. 45 1986 20–27.w x10 N.S. Canteras, R.B. Simerly, L.W. Swanson, Organization of projec-

tions from the ventromedial nucleus of the hypothalamus: a Phaseo-lus Õulgaris-leucoagglutinin study in the rat, J. Comp. Neurol. 348Ž .1994 41–79.

w x11 E. Challet, D.J. Bernard, F.W. Turek, Lesions of glucose-responsiveneurons impair synchronizing effects of calorie restriction in mice,

Ž .Brain Res. 801 1998 244–250.w x12 E. Challet, S. Losee-Olson, F.W. Turek, Reduced glucose availabil-

ity attenuates circadian responses to light in mice, Am. J. Physiol.Ž .276 1999 in press.

w x13 E. Challet, P. Pevet, N. Lakhdar-Ghazal, A. Malan, Ventromedial´nuclei of the hypothalamus are involved in the phase-advance oftemperature and activity rhythms in food-restricted rats fed during

Ž .daytime, Brain Res. Bull. 43 1997 209–218.w x14 E. Challet, P. Pevet, A. Malan, Intergeniculate leaflet lesion and´

daily rhythms in food-restricted rats fed during daytime, Neurosci.Ž .Lett. 216 1996 214–218.

w x15 C. Chen, P.F. Williams, G.J. Cooney, I.D. Caterson, J.R. Turtle, Theeffects of fasting and refeeding on liver glycogen synthase and

Ž .phosphorylase in obese and lean mice, Horm. Metab. Res. 24 1992161–166.

w x16 S. Chen, H.S. Su, Afferent connections of the thalamic paraventricu-lar and parataenial nuclei in the rat: a retrograde tracing study with

Ž .iontophoretic application of Fluoro-Gold, Brain Res. 522 19901–6.

w x17 A.F. Debons, I. Krimsky, A. From, E. Siclari, M.L. Maayan, K.Fani, F.A. Jimenez, Action of gold thioglucose on pericapillary

Ž .structures in the ventromedial hypothalamus, J. Pathol. 129 197973–81.

w x18 A.F. Debons, I. Krimsky, M.L. Maayan, K. Fani, F.A. Jimenez,Ž .Gold thioglucose obesity syndrome, Fed. Proc. 36 1977 143–147.

w x19 A.F. Debons, L. Silver, E.P. Cronkite, H.A. Johnson, G. Brecher, D.Tenzer, I.L. Schwartz, Localization of gold in mouse brain in

Ž .relation to gold thioglucose obesity, Am. J. Physiol. 202 1962743–750.

w x20 F.J.P. Ebling, The role of glutamate in the photic regulation of theŽ .suprachiasmatic nucleus, Prog. Neurobiol. 50 1996 109–132.

w x21 D.M. Edgar, W.C. Dement, Regularly scheduled voluntary exerciseŽ .synchronizes the mouse circadian clock, Am. J. Physiol. 261 1991

R928–R933.w x22 C. Escobar, M. Dıaz-Munoz, F. Encinas, R. Aguilar-Roblero, Persis-´ ˜

tence of metabolic rhythmicity during fasting and its entrainment byŽ .restricted feeding schedules in rats, Am. J. Physiol. 274 1998

R1309–R1316.w x23 K. Haito, S. Kagawa, A. Takeda, S. Shimizu, K. Mimura, A.

Matsuoka, Elevation of plasma triglyceride levels due to 2-deoxy-D-Ž .glucose in conscious rats, Life Sci. 35 1984 1821–1827.

( )E. Challet et al.rBrain Research 824 1999 18–27 27

w x24 A.C. Hall, R.M. Hoffmaster, E.L. Stern, M.E. Harrington, D. Bickar,Suprachiasmatic nucleus neurons are glucose sensitive, J. Biol.

Ž .Rhythms 12 1997 388–400.w x25 K.D. Hunsicker, B.J. Mullen, R.J. Martin, Effect of starvation or

restriction on self-selection of macronutrients in rats, Physiol. Be-Ž .hav. 51 1992 325–330.

w x26 R.F. Johnson, L. Smale, R.Y. Moore, L.P. Morin, Lateral geniculatelesions block circadian phase-shift responses to a benzodiazepine,

Ž .Proc. Natl. Acad. Sci. U.S.A. 85 1988 5301–5304.w x27 B.M. King, B.A. Stamoutsos, S.P. Grossman, Delayed response to

2-deoxy-D-glucose in hypothalamic obese rats, Pharmacol. Biochem.Ž .Behav. 8 1978 259–262.

w x28 H. Kita, S. Shibata, Y. Oomura, K. Ohki, Excitatory effects of thesuprachiasmatic nucleus on the ventromedial nucleus in the rat

Ž .hypothalamic slice, Brain Res. 235 1982 137–141.w x29 D.C. Klein, R.Y. Moore, S.M. Reppert, Suprachiasmatic Nucleus:

The Mind’s Clock, Oxford Univ. Press, New York, 1991.w x30 L.M. Kow, D.W. Pfaff, Actions of feeding-relevant agents on

hypothalamic glucose-responsive neurons in vitro, Brain Res. Bull.Ž .15 1985 509–513.

w x31 M.S. Krieger, L.C.A. Conrad, D. Pfaff, An autoradiographic studyof the efferent connections of the ventromedial nucleus of the

Ž .hypothalamus, J. Comp. Neurol. 183 1979 785–816.w x32 M. Lavialle, J. Serviere, Circadian fluctuations in GFAP distribution`

Ž .in the Syrian hamster suprachiasmatic nucleus, NeuroReport 4 19931243–1246.

w x33 B.E. Levin, A.C. Sullivan, Glucose, insulin and sympathoadrenalŽ .activation, J. Auton. Nerv. Syst. 20 1987 233–242.

w x34 R.A. Liebelt, J.H. Perry, Action of gold thioglucose in the centralŽ .nervous system, in: C.F. Code Ed. , Handbook of Physiology,

Section 6: Alimentary Canal, Vol. 1: Control of Food and WaterIntake, Am. Physiol. Soc., 1967, pp. 271–286.

w x35 S. Lin, X.F. Huang, Fasting increases leptin receptor mRNA expres-Ž .sion in lean but not obese obrob mouse brain, NeuroReport 8

Ž .1997 3625–3629.w x36 E.G. Marchant, R.E. Mistlberger, Entrainment and phase shifting of

circadian rhythms in mice by forced treadmill running, Physiol.Ž .Behav. 60 1996 657–663.

w x37 E.G. Marchant, N.V. Watson, R.E. Mistlberger, Both neuropeptideY and serotonin are necessary for entrainment of circadian rhythms

Ž .in mice by daily treadmill running schedules, J. Neurosci. 17 19977974–7987.

w x38 E.J. Masoro, R.J.M. McCarter, M.S. Katz, C.A. McMahan, Dietaryrestriction alters characteristics of glucose fuel use, J. Gerontol. Biol.

Ž .Sci. 47 1992 B202–B208.w x39 E.S. Maywood, E. Smith, S.J. Hall, M.H. Hastings, A thalamic

contribution to arousal-induced, non-photic entrainment of the circa-Ž .dian clock of the Syrian hamster, Eur. J. Neurosci. 9 1997 1739–

1747.w x40 R.E. Mistlberger, M.C. Antle, Behavioral inhibition of light-induced

circadian phase resetting is phase and serotonin dependent, BrainŽ .Res. 786 1998 31–38.

w x41 Y. Mizuno, Y. Oomura, Glucose responding neurons in the nucleusŽ .tractus solitarius of the rat: in vitro study, Brain Res. 307 1984

109–116.w x42 M.M. Moga, R.Y. Moore, Putative excitatory amino acid projections

Ž .to the suprachiasmatic nucleus in the rat, Brain Res. 743 1996171–177.

w x43 R.Y. Moore, J.P. Card, Intergeniculate leaflet: an anatomically andfunctionally distinct subdivision of the lateral geniculate complex, J.

Ž .Comp. Neurol. 344 1994 403–430.w x Ž .44 L.P. Morin, The circadian visual system, Brain Res. Rev. 67 1994

102–127.w x45 L.P. Morin, R.F. Johnson, R.Y. Moore, Two brain nuclei controlling

circadian rhythms are identified by GFAP immunoreactivity inŽ .hamsters and rats, Neurosci. Lett. 99 1989 55–60.

w x46 N. Mrosovsky, Locomotor activity and non-photic influences onŽ .circadian clocks, Biol. Rev. 71 1996 343–372.

w x47 W.M. Mueller, F.M. Gregoire, K.L. Stanhope, C.V. Mobbs, T.M.Mizuno, C.H. Warden, J.S. Stern, P.J. Havel, Evidence that glucosemetabolism regulates leptin secretion from cultured rat adipocytes,

Ž .Endocrinology 139 1998 551–558.w x48 E.E. Muller, D. Cocchi, A. Forni, A central site for the hyper-¨

glycemic action of 2-deoxy-D-glucose in mouse and rat, Life Sci. 10Ž .1971 1057–1067.

w x49 K. Nagai, T. Fujii, S. Inoue, Y. Takamura, H. Nakagawa, Electricalstimulation of the suprachiasmatic nucleus of the hypothalamus

Ž .causes hyperglycemia, Horm. Metab. Res. 20 1988 37–39.w x50 K. Nagai, N. Nagai, K. Shimizu, S. Chun, H. Nakagawa, A. Niijima,

SCN output drives the autonomic nervous system, with specialreference to the autonomic function related to the regulation ofglucose metabolism, in: R.M. Buijs, A. Kalsbeek, H.J. Romijn,

Ž .C.M.A. Pennartz, M. Mirmiran Eds. , Prog. Brain Res., Vol. 111,Elsevier, Amsterdam, 1996, pp. 253–272.

w x51 A. Niijima, Neural mechanisms in the control of blood glucoseŽ .concentration, J. Nutr. 119 1989 833–840.

w x52 Y. Nishizawa, G.A. Bray, Ventromedial hypothalamic lesions andŽ .the mobilization of fatty acids, J. Clin. Invest. 61 1978 714–721.

w x53 T. Ono, H. Nishino, M. Fukuda, K. Sasaki, K.I. Muramoto, Y.Oomura, Glucoresponsive neurons in rat ventromedial hypothalamic

Ž .tissue slices in vitro, Brain Res. 232 1982 494–499.w x54 Y. Oomura, Glucose as a regulator of neuronal activity, Adv. Metab.

Ž .Disord. 10 1983 31–65.w x55 G.E. Pickard, The afferent connections of the suprachiasmatic nu-

cleus of the Golden hamster with emphasis on the retinohypothala-Ž .mic projection, J. Comp. Neurol. 211 1982 65–83.

w x56 T.L. Powley, J.C. Prechtl, Gold thioglucose selectively damagesŽ .dorsal vagal nuclei, Brain Res. 367 1986 192–200.

w x57 M.R. Ralph, N. Mrosovsky, Behavioral inhibition of circadian re-Ž .sponses to light, J. Biol. Rhythms 7 1992 353–359.

w x58 T. Sakaguchi, M. Takahashi, G.A. Bray, Diurnal changes in sympa-thetic activity: relation to food intake and to insulin injected into the

Ž .ventromedial or suprachiasmatic nucleus, J. Clin. Invest. 82 1988282–286.

w x59 S. Shibata, S. Liou, S. Ueki, Y. Oomura, Inhibitory action of insulinon suprachiasmatic nucleus neurons in rat hypothalamic slice prepa-

Ž .rations, Physiol. Behav. 36 1986 79–81.w x60 N. Shimizu, Y. Oomura, C.R. Plata-Salaman, M. Morimoto, Hyper-

phagia and obesity in rats with bilateral ibotenic acid-induced le-sions of the ventromedial hypothalamic nucleus, Brain Res. 416Ž .1987 153–156.

w x61 L. Sokoloff, M. Reivich, C. Kennedy, M.H. Des Rosiers, C.S.w14 xPatlak, K.D. Pettigrew, O. Sakurada, M. Shinohara, The C de-

oxyglucose method for the measurement of local cerebral glucoseutilization: theory, procedure, and normal values in the conscious

Ž .and anesthetized albino rat, J. Neurochem. 28 1977 897–916.w x62 D. Spanswick, M.A. Smith, V.E. Groppi, S.D. Logan, M.L.J. Ash-

ford, Leptin inhibits hypothalamic neurons by activation of ATP-Ž .sensitive potassium channels, Nature 390 1997 521–525.

w x63 O. Van Reeth, F.W. Turek, Stimulated activity mediates phase shiftsin the hamster circadian clock induced by dark pulses or benzodi-

Ž .azepines, Nature 339 1989 49–51.w x64 N. Vrang, J.D. Mikkelsen, Afferent connections of the intergenicu-

late leaflet of the Golden hamster: a combined retrograde andŽ .anterograde tracing study, Soc. Neurosci. Abstr. 22 1996 1139.

w x65 A.G. Watts, L.W. Swanson, S. Sanchez-Watts, Efferent projectionsof the suprachiasmatic nucleus: I. studies using anterograde transportof Phaseolus Õulgaris leucoagglutinin in the rat, J. Comp. Neurol.

Ž .258 1987 204–229.w x66 C. Wickland, F.W. Turek, Lesions of the thalamic intergeniculate

leaflet block activity-induced phase shifts in the circadian activityŽ .rhythm of the golden hamster, Brain Res. 660 1994 293–300.