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Gold-thioglucose-induced hypothalamic lesions inhibit metabolic modulation of light-induced circadian phase shifts in mice
Brain Research 824 Ž1999. 18–27
Research report
Gold-thioglucose-induced hypothalamic lesions inhibit metabolic modulation of light-induced circadian phase shifts in mice Etienne Challet ) , Daniel J. Bernard, Fred W. Turek Center 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 to light can, however, be reduced when glucose availability is decreased. We tested the hypothesis that the ventromedial hypothalamus, a key area in the integration of metabolic and hormonal signals, mediates the metabolic modulation of circadian responses to light by injecting C57BLr6J mice with gold-thioglucose Ž0.6 grkg. which damages glucose-receptive neurons, primarily located in the ventromedial hypothalamus. Light pulses applied during the mid-subjective night induce phase delays in the circadian rhythm of locomotor activity in mice kept in constant darkness. As previously observed, light-induced phase delays were significantly attenuated in fed 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-induced hypothalamic lesions did not significantly affect light-induced phase delays compared to mice treated with gold-thioglucose and fed ad libitum. These results indicate that destruction of gold-thioglucose-sensitive neurons in the ventromedial hypothalamus prevent metabolic regulation of circadian responses to light during shortage of glucose availability. Therefore, the ventromedial hypothalamus may be a central site coordinating the metabolic modulation of light-induced phase shifts of the circadian clock. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Suprachiasmatic nucleus; Circadian rhythm; Ventromedial hypothalamus; Glucose utilization; Fasting; C57BLr6J mouse
1. Introduction The daily temporal organization of behavioral and physiological processes is controlled by a circadian clock located in the suprachiasmatic nuclei ŽSCN. of the hypothalamus. The SCN are primarily entrained to the ambient light:dark cycle, with the clock being most sensitive to photic cues during the subjective night w29,44x. The magnitude of light-induced phase shifts is reduced in situations of decreased glucose availability w12x, including hypoglycemia and blockade of glucose utilization by 2-deoxyD-glucose Ž2-DG., a competitive inhibitor of glucose transport and phosphorylation. The central mechanisms by which metabolic cues modulate photic phase-resetting of the 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: [email protected]
Although all neurons utilize glucose as an energy source w61x, only a few have their firing rate specifically modified by changes in extracellular glucose concentration w54x. Among such glucose-responsive neurons, those which increase their firing rate when extracellular glucose is increased Ži.e., glucoreceptor or glucose-receptive neurons. are primarily located in the ventromedial nuclei ŽVMH. of the hypothalamus w30,53x, and secondarily in the nucleus of the solitary tract w41x. Glucose-receptive neurons contain ATP-sensitive potassium channels that can be inhibited by increasing extracellular glucose levels to induce cell depolarization. Likewise, a decrease in extracellular glucose results in hyperpolarization and decreased frequency of action potentials of glucose-receptive neurons w3,62x. The VMH are one of the key brain regions in the integration of metabolic and hormonal signals leading to the central regulation of glycemia w51,54x and energy metabolism w4,52x. In particular, the VMH play an important role for sensing hypoglycemia and 2-DG-induced cytoglucopenia, and triggering counterregulatory hormonal
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 9 2 - 0
E. Challet et al.r Brain Research 824 (1999) 18–27
responses, such as the release of catecholamines and glucagon w7,8x. The VMH receive monosynaptic, probably excitatory input from the SCN w10,28,65x. The SCN provide circadian signals to the autonomic nervous system w49,50,58x and may also participate in glucose homeostasis, at least via facilitation of VMH activity w49x, and in lipid mobilization w5x. The VMH, in turn, have direct and indirect connections back to the SCN w10,31,55x, including excitatory amino acid projections w42x. In addition to the reciprocal VMH–SCN connections observed in rats and hamsters, the VMH’s ability to detect and respond to a shortage of cerebral glucose availability suggests that they may be involved in the metabolic modulation of the circadian responses of the SCN to light. To test this hypothesis, the phase resetting responses to a light pulse after blockade of glucose utilization and hypoglycemia, induced either by fasting alone or fasting plus insulin treatment, were determined in mice bearing lesions of the VMH. Rather than using electrolytic lesions that destroy fibers of passage or ibotenic acid lesions that are difficult to perform in restricted area of the small-sized mouse brain, mice were injected with gold-thioglucose ŽGTG., a glucose analog that causes irreversible damage of glucose-receptive neurons in the VMH, by acting on neural structures contiguous with capillaries w9,17x. Prevention of GTG-induced VMH necrosis by inhibitors of glucose transport w18x supports the hypothesis that GTG specifically destroys glucose-receptive VMH cells. Moreover, GTG-induced lesions lead to long-term obesity and hyperphagia w6,15,18x, as do ibotenic w60x and electrical lesions of the VMH w4,52x.
2. Materials and methods 2.1. Animals and laboratory conditions A total of 48 adult male C57BLr6J mice ŽJackson Labs, Bar Harbor, ME. were housed singly in cages equipped with running wheels Ždiameter: 11 cm. in a temperature-controlled room Ž23 " 18C. with a light:dark 12:12 h cycle Žlights on at 0500 h.. During daytime, light intensity was about 300 lx at cage level. Food Žlaboratory chow, Harlan Teklad, WI. and water were available ad libitum, unless otherwise stated. Wheel-running activity was continuously recorded ŽChronobiology Kit, Stanford Software Systems, Stanford, CA.. At the end of a 2-week baseline period, mice were injected i.p. with either 0.6 mgrg body mass of GTG Žaurothioglucose, Sigma, St. Louis, MO., or saline vehicle Ž n s 24 per treatment.. Ten days later, mice were transferred to constant darkness ŽDD. where they remained for a period of 20 days. Animal maintenance in darkness was aided by use of an infrared viewer ŽFind-R-Scope, FJW Optical System, Palatine, IL..
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2.2. Experimental design A light pulse was given on the 11th day in DD Ždenoted DD q 10. at circadian time 18 ŽCT18; CT12 is defined as the time of activity onset.. Some 12 GTG-treated mice and 12 control mice were fed ad libitum. On Day DD q 10, they received an i.p. injection of 500 mgrkg body mass of 2-deoxy-D-glucose Ž2-DG; Sigma. or saline at CT17, 1 h prior to a sub-saturating 10-min light pulse Ž50 lx of white light; n s 6 per group.. For light stimulation, individuals were transferred from their own cages to a white chamber Ždiameter: 11 cm, height: 6 cm. inside a photic stimulation device. Light intensity was determined using a digital photometer. The dose, time of 2-DG injection and light pulse parameters were chosen according to previous experiments w23,52x. Some 12 GTG-treated mice and 12 control mice were fasted by food removal for 30 h from CT12 on Day DD q 9 to CT18 on Day DD q 10, a duration of food deprivation which has been shown previously to induce hypoglycemia in mice w12x. Half of the fasted mice received an s.c. injection of either insulin Ž5 IUrkg; Sigma. or saline. Injections were administered 30 min prior to a 10-min light pulse Ž50 lx of white light. given at CT18 on Day DD q 10 Ž n s 6 per treatment.. The dose and time of insulin injections were defined according to previous studies w12x, so that severe hypoglycemia occurred at the time of the light pulse. 2.3. Immunocytochemistry In order to assess the effectiveness and extent of GTGinduced lesions in the VMH, gliosis was visualized by immunocytochemistry for glial fibrillary acidic protein ŽGFAP., an intracellular protein expressed by astrocytes. After 20 days in DD, mice were killed with CO 2 during the late subjective day Ži.e., when gut content is low.. Mice were 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 sections were prepared on a cryostat. Sections were then incubated for 1 h with 10% normal horse serum in 0.1 M phosphate buffered saline ŽPBS; pH 7.4. and for 12 h at 48C with a mouse monoclonal anti-GFAP ŽICN Biochemicals, Costa Mesa, CA. diluted 1:1000 in PBS containing 0.3% Triton X-100. Sections were washed in PBS and incubated for 1 h with biotinylated horse anti-mouse immunoglobulins ŽVectastain ABC kit, Vector Labs, CA.. Sections were washed again in PBS and transferred for 1 h to a solution of ammonium sulfate nickel containing streptavidin–biotin complex conjugated to horseradish peroxidase. Peroxidase was visualized by the diaminobenzidine reaction. The brain sections were mounted on gelatin-coated slides. 2.4. Data analysis To quantify the light pulse-induced phase shifts, a line was fitted by eye to the onsets of locomotor activity for the
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E. Challet et al.r Brain Research 824 (1999) 18–27
first 10 days before the light pulse. This line was projected to the day of the light pulse Ži.e., Day DD q 10.. Similarly, a line was fitted to the onsets of activity for the 10 days after the photic pulse. That line was retroprojected to the day of the pulse. The magnitude of the phase shifts was calculated as the difference between these two lines. Daily activity was defined as the total wheel revolutions per cycle Žbeginning at CT12.. The circadian period Žt . was
assessed by the x 2 periodogram analysis ŽChronobiology 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 state Žfed or fasted., data were analyzed by two-way analyses of
Fig. 1. Glial fibrillary acidic protein immunoreactive staining in representative coronal sections of suprachiasmatic nuclei ŽSCN., left ventromedial hypothalamic nucleus ŽVMH. and left nucleus of the solitary tract ŽNTS. in a mouse injected with saline Žcontrol. and a mouse injected with gold-thioglucose ŽGTG.. The arrow indicates the GTG-induced increase of GFAP staining in the ventromedial region of the VMH ŽRight middle panel.. Scale bar s 200 mm.
E. Challet et al.r Brain Research 824 (1999) 18–27 Table 1 Adiposity index Žepididymal fat pads mass as % body mass. in the different treatment groups Neurotoxin
Treatment
Adiposity index Ž%.
Control Control GTG GTG Control Control GTG GTG
Fedrsaline Fedr2-DG Fedrsaline Fedr2-DG Fastedrsaline Fastedrinsulin Fastedrsaline Fastedrinsulin
1.15"0.06 a 1.27"0.12 a 2.02"0.31b 2.14"0.41b 0.94"0.09 a 0.80"0.08 a 1.21"0.11b 1.28"0.13 b
Data are means"S.E.M. Ž ns6 per group.. GTG: gold-thioglucose. 2-DG: 2-deoxy-D-glucose. For a given nutritional state Žfed or fasted., groups with no letters in common are significantly different from one another Ž P - 0.05..
variance ŽANOVA. to compare the effects of the neurotoxin ŽGTG vs. saline. and the different metabolic treatments Žsaline vs. 2-DG in fed animals, and saline vs. insulin in fasted animals.. If significant main effects or a significant interaction were found Ž P - 0.05., post-hoc comparisons were performed with the Student–Newman– Keuls test.
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3. Results
3.1. Histology Examination of immunocytochemical staining for GFAP revealed clear glial proliferation within the ventromedial hypothalamus, especially in the medial region of the VMH and the lateral region of the arcuate nuclei. In the 24 GTG-treated mice included in this study, the increase of GFAP staining in the VMH was evaluated microscopically Žsee an example in Fig. 1.. In keeping with accumulation of gold in the brain by autoradiography w19x, the VMH was the brain area presenting the most consistent increase in gliosis after GTG treatment ŽFig. 1.. Staining in other diencephalic regions was essentially similar between salineand GTG-injected mice. For example, in most animals, GFAP immunoreactivity was strong in the SCN w32,45x Žsee Fig. 1. and the preoptic area, and low to moderate in paraventricular hypothalamic nuclei and paraventricular thalamic nuclei Ždata not shown.. In addition to the VMH, a small increase of gliosis in a few GTG-treated animals was also apparent in the medulla oblongata Ži.e., in the vagal nuclei and nucleus of the solitary tract; Fig. 1..
Fig. 2. Double-plotted daily wheel-running activity of four mice kept in constant darkness and fed ad libitum. ŽUpper panels. Records from mice receiving an i.p. injection of saline at CT17 followed 1 h later by a light pulse Ž50 lx of white light lasting 10 min.. Mice previously treated with saline or GTG are shown in panels ŽA. and ŽB., respectively. ŽLower panels. Records from mice receiving an i.p. injection of 500 mgrkg 2-deoxy-D-glucose Ž2-DG. at CT17 followed by a light pulse Žas above.. Mice previously treated with saline or GTG are shown in panels ŽC. and ŽD., 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 a circle.
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E. Challet et al.r Brain Research 824 (1999) 18–27
3.2. Metabolic changes GTG treatment leads to obesity after long-term conditions of ad libitum feeding Ži.e., after several months; e.g., Ref. w15x.. In the present study, to rule out any simple effect of huge lipid fuel reserves, the GTG-treated mice were given a light pulse 20 days after the injection of GTG and killed 10 days after the light pulse. Nevertheless, to assess subtle metabolic changes between groups, we determined an adiposity index by expressing epididymal fat pads as a percentage of body mass w13x. As shown in Table 1, the adiposity index in fed mice was significantly larger in GTG-injected than in saline-injected mice Ž2.08 " 0.25 vs. 1.21 " 0.07%, respectively; F Ž1,20. s 10.6, P - 0.01., regardless of the injection Žsaline or 2-DG.. In mice previously fasted, the adiposity index was also larger in GTGinjected than in control mice Ž1.24 " 0.08 vs. 0.87 " 0.06%, respectively; F Ž1,20. s 12.6, P - 0.01., regardless of the injection Žsaline or insulin.. 3.3. Light-induced behaÕioral phase shifts and circadian period In mice fed ad libitum throughout the experiment, regardless of the injection Žsaline or 2-DG., mean phase
delays induced by a light pulse at CT18 were significantly smaller in control mice compared to GTG-treated mice Ž F Ž1,20. s 5.8, P - 0.05; Figs. 2 and 4.. Regardless of the lesion ŽGTG or saline., mean light-induced phase delays were significantly smaller in mice injected with saline compared to those injected with 2-DG Ž F Ž1,20. s 12.9, P - 0.01; Figs. 2 and 4.. Moreover, there was a significant interaction Ž F Ž1,20. s 13.3, P - 0.01; Fig. 4. between the effects of lesion ŽGTG or saline. and injection Žsaline or 2-DG.. Post-hoc analysis indicated that in individuals injected with saline, the light-induced phase delays in control mice fed ad libitum were similar to those in GTG-treated animals fed ad libitum. After blockade of glucose utilization, however, phase delays were significantly reduced in control mice compared to GTG-treated mice ŽFigs. 2 and 4.. In mice fasted prior to the light pulse, the phase delays induced by a light pulse at CT18 were significantly smaller in control mice compared to GTG-treated mice Ž F Ž1,20. s 32.0, P - 0.001; Figs. 3 and 4.. The effect of the injection Žsaline or insulin. and the interaction GTG = injection Žsaline or insulin. were not significant Ž F Ž1,20. s 0.3 and F Ž1,20. s 0.1, respectively, P ) 0.1; Figs. 3 and 4.. Using a two-way ANOVA with repeated measures, the circadian period Žt . 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 the light pulse. ŽUpper panels. Records from mice receiving a s.c. injection of saline 30 min prior to a light pulse Ž50 lx of white light lasting 10 min. given at CT18. Mice previously treated with saline or GTG are shown in panels ŽA. and ŽB., respectively. ŽLower panels. Records from mice receiving a s.c. injection of 5 IUrkg insulin 30 min prior to a light pulse Žas above.. Mice previously treated with saline or GTG are shown in panels ŽC. and ŽD., 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 a circle.
E. Challet et al.r Brain Research 824 (1999) 18–27
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treatment groups Ž F Ž3,20. s 1.6, P ) 0.1., or following the light pulse Ž F Ž1,20. s 0.1, P ) 0.1.. In all fasted groups, t was close to 23.7 " 0.07 h Žsee Fig. 3 for examples.. 3.4. QuantitatiÕe analysis of wheel-running actiÕity
Fig. 4. Phase shifts Žnegative values are delays. in circadian activity rhythm of mice housed in constant darkness after a light pulse Ž50 lx of white light lasting 10 min. given at CT18. Half the mice were previously treated with saline or GTG ŽGTG-treated.. Mice that were fed ad libitum over the experiment received an i.p. injection of 500 mgrkg 2-deoxy-Dglucose Ž2-DG. or saline at CT17 ŽFed 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 min prior to the light pulse ŽFasted groups.. Means"S.E.M. Ž ns6 per group.. Groups with no letters in common are significantly different from one another Ž P - 0.05..
the four treatment groups Žcontrolrsaline, controlr2-DG, GTGrsaline or GTGr2-DG. for the 10-day period before or after the light pulse. The circadian period did not differ among the treatment groups Ž F Ž3,20. s 0.4, P ) 0.1., nor did it change following the light pulse Ž F Ž1,20. s 0.1, P ) 0.1.. In all fed groups, t was close to 23.7 " 0.08 h Žsee Fig. 2 for examples.. A similar analysis was performed in fasted mice across the four treatment groups Žcontrolrsaline, controlrinsulin, GTGrsaline or GTGrinsulin. and the 10-day period before or after the light pulse. Again, the circadian period did not differ among the
A two-way ANOVA with repeated measures was used to compare the number of wheel revolutions on Day DD q 9 Ži.e., prior to the light pulse. vs. Day DD q 10 Ži.e., the day when a light pulse was applied. in fed mice. The total number of wheel revolutions did not differ significantly by treatment Žcontrolrsaline, GTGrsaline, controlr2-DG or GTGr2-DG: F Ž3,20. s 1.9, P ) 0.1., or by the day ŽDD q 9 vs. DD q 10: F Ž1,20. s 0.7, P ) 0.1; Table 2.. In fasted mice, the total number of wheel revolutions was significantly affected by the day ŽDD q 9 vs. DD q 10: F Ž1,20. s 33.0, P - 0.01., but not by the treatment Žcontrolrsaline, GTGrsaline, controlrinsulin or GTGrinsulin: F Ž3,20. s 0.1, P ) 0.1.. No other comparisons were significantly different. The number of wheel revolutions was determined between CT6 and CT12 on Day DD q 9 ŽTable 2.. In fed mice, afternoon activity was not affected significantly by the neurotoxin ŽGTG or saline: F Ž1,20. s 1.7, P ) 0.1., or the injection Žsaline or 2-DG: F Ž1,20. s 0.2, P ) 0.1.. Similarly, afternoon activity in fasted mice was not affected significantly by the neurotoxin ŽGTG or saline: F Ž1,20. s 0.1, P ) 0.1., or the injection Žsaline or 2-DG: F Ž1,20. s 0.1, P ) 0.1.. In order to assess subtle differences in the wheel-running activity prior to the light pulse that may interfere with the circadian responses to light w40,57x, the number of wheel revolutions was also determined between CT12 and
Table 2 Number of wheel revolutions in the different treatment groups Neurotoxin
Treatment
Daily activity on Day DD q 9
Daily activity on Day DD q 10
Afternoon act. on Day DD q 9
Early noct. act. on Day DD q 10
Control Control GTG GTG Control Control GTG GTG
Fedrsaline Fedr2-DG Fedrsaline Fedr2-DG Fastedrsaline Fastedrinsulin Fastedrsaline Fastedrinsulin
21845" 1871 21638" 4239 16561" 2324 14427" 3939 23074" 4733 25041" 6270 22452" 5823 25820" 3209
20746" 1335 16837" 2599 21327" 3494 11290" 1975 16802" 2476 13822" 2919 14440" 3812 15436" 1467
1226 " 517 1588 " 639 1200 " 420 373 " 151 2030 " 692 3844 " 2065 3746 " 2132 2448 " 501
13428" 1950 12050" 2526 11367" 1567 8598 " 2035 12409" 2191 11865" 2586 9064 " 2349 12364" 1731
Data are means " S.E.M. Ž n s 6 per group.. GTG: gold-thioglucose. 2-DG: 2-deoxy-D-glucose. Daily Activity: total wheel revolutions performed per day, beginning at CT Žcircadian 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 DD q 9: day when mice were either fed or fasted. Day DD q 10: day on which a light pulse was administered. For a given nutritional state Žfed or fasted., no groups in a given column were significantly different from one another Ž P ) 0.05.. There was a reduction in the total number of wheel revolutions in fasted mice between DD q 9 and DD q 10 Ž P - 0.01..
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E. Challet et al.r Brain Research 824 (1999) 18–27
CT18 on Day DD q 10 Ži.e., during the 6 circadian hours before the light pulse; Table 2.. Here again, there were no significant effects of the neurotoxin ŽGTG or saline: F Ž1,20. s 1.8, P ) 0.1. and of the injection Žsaline or 2-DG: F Ž1,20. s 1.0, P ) 0.1. on early nocturnal activity of mice fed ad libitum. In fasted mice, nocturnal activity prior to the light pulse was not modified significantly by the neurotoxin ŽGTG or saline: F Ž1,20. s 0.4, P ) 0.1., or the injection Žsaline or insulin: F Ž1,20. s 0.4, P ) 0.1.. None of the possible interactions in the comparisons of wheel running activity were significant.
4. Discussion The present study confirms that photic phase resetting of the circadian clock is reduced in intact mice when glucose availability is decreased. This effect is observed whether glucose utilization is blocked or animals are fasted, with or without added insulin-induced hypoglycemia w12x. In addition, the present report demonstrates that destruction of glucose-receptive neurons with the neurotoxin GTG blocks these altered circadian responses to light. 4.1. Lesions induced by GTG treatment Given that morphology and function of peripheral tissues are not altered during the development of obesity in GTG-injected mice w15,34x, the observed effects after GTG injection are likely to be due to changes in the central nervous system. Because some responses of the circadian clock to light were different in control mice compared to GTG-injected mice, one can speculate that GTG treatment affects directly SCN neurons. Due to high levels of GFAP immunoreactivity in the SCN of control mice, it was not possible to detect a change in the SCN following GTG treatment. While GTG treatment impairs attenuation of light-induced phase shifts in response to metabolic challenges, it does not affect the phase-angle of photic entrainment in mice fed ad libitum w11x. In addition, the free-running periods of the circadian rhythm of locomotor activity and the light-induced behavioral phase delays are similar in both GTG- and saline-injected mice fed ad libitum Žpresent study.. To specifically rule out direct effects of GTG in the SCN, further studies are needed in rodents receiving GTG in the SCN without damaging the VMH. Taken together, however, the available data suggest that SCN function in mice with free access to food is not altered by GTG injection. Most SCN neurons in vitro do not display changes in firing rate when extracellular glucose is modified w24x, an effect typical of glucose-receptive neurons as defined by Oomura w54x. Conversely, about 20% of VMH neurons increase firing rate when extracellular glucose is increased w54x. In the present study, the most extensive damage following GTG administration was observed in the VMH.
This is best exemplified by changes in both anti-GFAP staining ŽFig. 1. and adiposity ŽTable 1.. Limited gliosis was also observed in the nucleus of the solitary tract in some GTG-treated mice. With higher doses than the one we used, the nucleus of the solitary tract and dorsal vagal nuclei can be heavily damaged by the GTG treatment w56x. Gold autoradiography after GTG treatment has been used by Debons and colleagues to localize where GTG acts in the mouse brain, and these authors found some labeled gold in the dorsal hindbrain w19x. This technique, however, does not provide information on the extent of cell damage. To specifically rule out an involvement of these brainstem nuclei in the metabolic modulation of circadian responses to light, further studies are needed in rodents with local chemical lesions of the nucleus of the solitary tract andror vagal nuclei. Nevertheless, the present results taken together suggest that the GTG-induced inhibition of metabolic modulation of photic phase shifting may be mediated by the VMH. 4.2. ActiÕity feedback on the circadian clock Acute changes of locomotor activity can modulate the regulation of circadian rhythmicity in hamsters w46,63x and mice w21,36x. In the present study, no significant changes in the number of wheel revolutions were detected between GTG- and saline-injected mice prior to the light pulse. For example, the decrease in total Ždaily. activity after a fasting period was similar in both GTG- and salineinjected animals ŽTable 2.. Therefore, it seems unlikely that the observed effects on the light-induced phase shifts are mediated by changes in the level of physical activity that can interfere with photic phase-resetting in hamsters fed ad libitum w40,57x. As we have hypothesized previously w12x, the altered phase-shifting responses to light during shortage of glucose availability may be due to metabolic signals. 4.3. Blockade of glucose utilization by 2-DG Peripheral injection of 2-DG blocks local glucose utilization in most brain areas, including the SCN w61x. In response to w14 Cx-DG treatment, there is an increase of blockade of glucose utilization in the SCN during the subjective day and a decrease during the subjective night in vivo Že.g., Ref. w29x.. In addition, the phase of the rhythmic firing rate in a slice of SCN can be altered Ži.e., delayed. temporarily by decreasing the availability of glucose in the bathing solution w24x. Considering a direct effect of 2-DG in the SCN, one may expect that lightinduced phase delays would be greater after 2-DGinduced cytoglucopenia. On the contrary, there was an attenuation of light-induced phase delays in control mice pretreated with 2-DG. Furthermore, blockade of glucose utilization in GTG-treated mice did not reduce the lightinduced phase delays. Assuming that the SCN function is
E. Challet et al.r Brain Research 824 (1999) 18–27
not impaired by GTG in mice fed ad libitum Žsee above., a direct effect of cytoglucopenia in the SCN would have been 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 cytoglucopenia have been shown to be mediated by the VMH w8x Žsee Section 1.. In keeping with the hypothesis that the VMH play a major role in sensing 2-DG-induced cytoglucopenia, rats with electrolytic lesions of the VMH w27x or GTG-treated mice w6x do not increase food intake after 2-DG treatment, although these mice remain sensitive to the inhibitory effect of cholecystokinin w6x. Accordingly, GTG treatment reduces and delays 2-DG-induced hyperglycemia in mice w48x. In the present experiment, we found that GTG treatment also impairs the 2-DG dependent attenuation of light-induced phase delays. A glucose injection prior to a light pulse, that increases plasma glucose, does not alter the photic phase-resetting w12x. Therefore, it is unlikely that the 2-DG-induced rise in glycemia per se plays a critical role in the reduced phase-shifting effects of light after blockade of cerebral glucose utilization. 4.4. Hypoglycemia during fasting, with or without insulin injection The attenuated light-induced phase shifts in control fasted mice were counteracted in fasted GTG-treated mice, with or without insulin injection prior to a light pulse. This difference may occur because GTG treatment impairs fasting- or insulin-induced hypoglycemia, leading to similar circadian responses to those in control mice fed ad libitum. Previous studies, however, have shown that damage to the VMH does not prevent fasting-induced decreases in plasma glucose in rats with electrical lesions of the VMH w4,52x or in mice treated with GTG w15x. In addition to blockade of glucose utilization and fasting, calorie restriction is another situation of negative energy balance, usually associated with chronic hypoglycemia w25,38x. Calorie restriction can phase shift circadian rhythms and modify the phase angle of photic entrainment in rodents w11,13,14x. This altered photic regulation of circadian rhythmicity, that implies SCN involvement, can be blocked by ibotenic lesions of the VMH in rats w13x and GTG treatment in mice w11x. Taken together, these data indicate that the loss of effects of fasting, 2-DG injection and calorie restriction on the light-induced phase shifts in GTG-treated mice may be due to an impairment in the integration Ždetection. of metabolic signals by the VMH that would, when glucose availability is decreased, impact on the SCN function in control mice. 4.5. Metabolic and hormonal signals modulating VMH neurons actiÕity The metabolic cues that modulate the photic phaseresetting of the SCN are not yet clearly defined. One likely candidate is a decrease in intracellular glucose availability
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Žor an altered rate of glucose utilization. occurring during fasting and after 2-DG injection. Such a decrease in glucose availability modifies the activity of VMH neurons w54x and may, in turn, affect SCN function Žpresent study.. Fasting and 2-DG treatment, however, are also associated with elevation of plasma ketone bodies and free fatty acids w22,52x, and these increases are not impaired by electrolytic lesions of the VMH w4,52x. The changes in adiposity we observed among groups of mice are consistent with expected mobilization of fatty acids during previous fasting ŽTable 1.. Interestingly, free fatty acids applied electrophoretically can affect the firing rate of VMH neurons w54x. Therefore, the effects of metabolic cues on photic phase-shifting may be mediated, in part, by changes in free fatty acid levels. Further studies using specific blockers of fatty acid oxidation will be useful for testing this hypothesis. Insulin plays a key role in the control of energy utilization w33x and, therefore, may mediate some of the observed effects. Furthermore, insulin applied during the subjective day inhibits the firing rate of SCN cells in vitro w59x. Insulinemia is unchanged after 2-DG treatment w2,23x, is decreased after fasting w15x, and is increased after insulin injection w33x. Despite these varied responses to different metabolic challenges, reduced circadian responses are observed in all three situations Žpresent study.. Moreover, neither electrolytic lesions of the VMH, nor GTG treatment affect the typical changes in insulinemia in response to fasting Ži.e., hypoinsulinemia; see Refs. w4,15x. and 2-DG injection Ži.e., normoinsulinemia; see Ref. w52x.. In spite of the fact that insulin can modulate firing rate of VMH neurons w54x, this hormone does not appear to be involved critically in the reduced phase shifting effects of light during metabolic challenges. Leptin, another hormone involved in the regulation of energy metabolism w1x, has been shown to inhibit the activity of glucose-receptive neurons in the VMH w62x. To our knowledge, the effect of 2-DG on leptin release has not yet been studied in vivo. In vitro, however, 2-DG causes an inhibition of leptin release from cultured rat adipocytes w47x. Fasting decreases plasma leptin w1x and increases leptin receptor mRNA expression in the VMH w35x. Taken together, these data suggest that, at least during fasting, lowered plasma leptin may somehow disinhibit VMH neurons. Whether this effect plays a role in the metabolic modulation of the circadian responses to light remains to be determined. 4.6. Direct Õs. indirect connections from the VMH to the SCN The present results suggest that the integration of metabolic and hormonal cues by the VMH plays a role in the altered circadian responses to light during metabolic challenges. As mentioned in Section 1, anatomical studies revealed that the VMH send a small number of fibers to the SCN, some of which are excitatory amino acidergic
26
E. Challet et al.r Brain Research 824 (1999) 18–27
w42x. The critical pathway for photic entrainment of the SCN clock is the retino-hypothalamic tract that conveys photic cues from the retina w20,44x. A large body of experimental data indicates that glutamate release from retino-hypothalamic terminals is the neurochemical substrate mediating photic signals to the clock w20x. Given that several subtypes of NMDA, non-NMDA and metabotropic receptors are found in the SCN w20x, it is possible that distinct, possibly conflicting, glutamatergic signals Ži.e., photic from the retina or ‘metabolic’ from the VMH. activate different post-synaptic glutamatergic receptor subtypes. Besides direct Žmonosynaptic. modulation of SCN function by the VMH, indirect pathways from the VMH to the SCN may also be involved in the metabolic modulation of SCN function. For example, the paraventricular thalamic nuclei, which receive a large projection from the VMH w16x, send direct glutamatergic fibers to the SCN w42x. The intergeniculate leaflets, which receive a projection from the VMH w64x, send a dense projection releasing neuropeptide Y and g-aminobutyric acid in the SCN w43x. The geniculo-hypothalamic terminals are considered to play a pivotal role in conveying several non-photic cues to the SCN w26,37,39,46,66x, including putative ‘metabolic’ cues w14x. Because lesions of the intergeniculate leaflets induce an almost complete disappearance of neuropeptide Y-immunoreactive fibers and terminals in the SCN of rodents w14,26,37,39x, an increase of neuropeptide Y in the SCN most likely reflects geniculate activation. The intergeniculate leaflets may also convey to the SCN some ‘metabolic’ cues integrated by the VMH, because 2-DG injection increases the level of neuropeptide Y in the SCN w2x. In conclusion, both physiological and hodological data give support to the hypothesis that the VMH integrate metabolic cues and modulate the photic regulation of circadian rhythmicity by means of direct andror indirect connections to the SCN.
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Research report
Gold-thioglucose-induced hypothalamic lesions inhibit metabolic modulation of light-induced circadian phase shifts in mice Etienne Challet ) , Daniel J. Bernard, Fred W. Turek Center 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 to light can, however, be reduced when glucose availability is decreased. We tested the hypothesis that the ventromedial hypothalamus, a key area in the integration of metabolic and hormonal signals, mediates the metabolic modulation of circadian responses to light by injecting C57BLr6J mice with gold-thioglucose Ž0.6 grkg. which damages glucose-receptive neurons, primarily located in the ventromedial hypothalamus. Light pulses applied during the mid-subjective night induce phase delays in the circadian rhythm of locomotor activity in mice kept in constant darkness. As previously observed, light-induced phase delays were significantly attenuated in fed 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-induced hypothalamic lesions did not significantly affect light-induced phase delays compared to mice treated with gold-thioglucose and fed ad libitum. These results indicate that destruction of gold-thioglucose-sensitive neurons in the ventromedial hypothalamus prevent metabolic regulation of circadian responses to light during shortage of glucose availability. Therefore, the ventromedial hypothalamus may be a central site coordinating the metabolic modulation of light-induced phase shifts of the circadian clock. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Suprachiasmatic nucleus; Circadian rhythm; Ventromedial hypothalamus; Glucose utilization; Fasting; C57BLr6J mouse
1. Introduction The daily temporal organization of behavioral and physiological processes is controlled by a circadian clock located in the suprachiasmatic nuclei ŽSCN. of the hypothalamus. The SCN are primarily entrained to the ambient light:dark cycle, with the clock being most sensitive to photic cues during the subjective night w29,44x. The magnitude of light-induced phase shifts is reduced in situations of decreased glucose availability w12x, including hypoglycemia and blockade of glucose utilization by 2-deoxyD-glucose Ž2-DG., a competitive inhibitor of glucose transport and phosphorylation. The central mechanisms by which metabolic cues modulate photic phase-resetting of the 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: [email protected]
Although all neurons utilize glucose as an energy source w61x, only a few have their firing rate specifically modified by changes in extracellular glucose concentration w54x. Among such glucose-responsive neurons, those which increase their firing rate when extracellular glucose is increased Ži.e., glucoreceptor or glucose-receptive neurons. are primarily located in the ventromedial nuclei ŽVMH. of the hypothalamus w30,53x, and secondarily in the nucleus of the solitary tract w41x. Glucose-receptive neurons contain ATP-sensitive potassium channels that can be inhibited by increasing extracellular glucose levels to induce cell depolarization. Likewise, a decrease in extracellular glucose results in hyperpolarization and decreased frequency of action potentials of glucose-receptive neurons w3,62x. The VMH are one of the key brain regions in the integration of metabolic and hormonal signals leading to the central regulation of glycemia w51,54x and energy metabolism w4,52x. In particular, the VMH play an important role for sensing hypoglycemia and 2-DG-induced cytoglucopenia, and triggering counterregulatory hormonal
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 9 2 - 0
E. Challet et al.r Brain Research 824 (1999) 18–27
responses, such as the release of catecholamines and glucagon w7,8x. The VMH receive monosynaptic, probably excitatory input from the SCN w10,28,65x. The SCN provide circadian signals to the autonomic nervous system w49,50,58x and may also participate in glucose homeostasis, at least via facilitation of VMH activity w49x, and in lipid mobilization w5x. The VMH, in turn, have direct and indirect connections back to the SCN w10,31,55x, including excitatory amino acid projections w42x. In addition to the reciprocal VMH–SCN connections observed in rats and hamsters, the VMH’s ability to detect and respond to a shortage of cerebral glucose availability suggests that they may be involved in the metabolic modulation of the circadian responses of the SCN to light. To test this hypothesis, the phase resetting responses to a light pulse after blockade of glucose utilization and hypoglycemia, induced either by fasting alone or fasting plus insulin treatment, were determined in mice bearing lesions of the VMH. Rather than using electrolytic lesions that destroy fibers of passage or ibotenic acid lesions that are difficult to perform in restricted area of the small-sized mouse brain, mice were injected with gold-thioglucose ŽGTG., a glucose analog that causes irreversible damage of glucose-receptive neurons in the VMH, by acting on neural structures contiguous with capillaries w9,17x. Prevention of GTG-induced VMH necrosis by inhibitors of glucose transport w18x supports the hypothesis that GTG specifically destroys glucose-receptive VMH cells. Moreover, GTG-induced lesions lead to long-term obesity and hyperphagia w6,15,18x, as do ibotenic w60x and electrical lesions of the VMH w4,52x.
2. Materials and methods 2.1. Animals and laboratory conditions A total of 48 adult male C57BLr6J mice ŽJackson Labs, Bar Harbor, ME. were housed singly in cages equipped with running wheels Ždiameter: 11 cm. in a temperature-controlled room Ž23 " 18C. with a light:dark 12:12 h cycle Žlights on at 0500 h.. During daytime, light intensity was about 300 lx at cage level. Food Žlaboratory chow, Harlan Teklad, WI. and water were available ad libitum, unless otherwise stated. Wheel-running activity was continuously recorded ŽChronobiology Kit, Stanford Software Systems, Stanford, CA.. At the end of a 2-week baseline period, mice were injected i.p. with either 0.6 mgrg body mass of GTG Žaurothioglucose, Sigma, St. Louis, MO., or saline vehicle Ž n s 24 per treatment.. Ten days later, mice were transferred to constant darkness ŽDD. where they remained for a period of 20 days. Animal maintenance in darkness was aided by use of an infrared viewer ŽFind-R-Scope, FJW Optical System, Palatine, IL..
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2.2. Experimental design A light pulse was given on the 11th day in DD Ždenoted DD q 10. at circadian time 18 ŽCT18; CT12 is defined as the time of activity onset.. Some 12 GTG-treated mice and 12 control mice were fed ad libitum. On Day DD q 10, they received an i.p. injection of 500 mgrkg body mass of 2-deoxy-D-glucose Ž2-DG; Sigma. or saline at CT17, 1 h prior to a sub-saturating 10-min light pulse Ž50 lx of white light; n s 6 per group.. For light stimulation, individuals were transferred from their own cages to a white chamber Ždiameter: 11 cm, height: 6 cm. inside a photic stimulation device. Light intensity was determined using a digital photometer. The dose, time of 2-DG injection and light pulse parameters were chosen according to previous experiments w23,52x. Some 12 GTG-treated mice and 12 control mice were fasted by food removal for 30 h from CT12 on Day DD q 9 to CT18 on Day DD q 10, a duration of food deprivation which has been shown previously to induce hypoglycemia in mice w12x. Half of the fasted mice received an s.c. injection of either insulin Ž5 IUrkg; Sigma. or saline. Injections were administered 30 min prior to a 10-min light pulse Ž50 lx of white light. given at CT18 on Day DD q 10 Ž n s 6 per treatment.. The dose and time of insulin injections were defined according to previous studies w12x, so that severe hypoglycemia occurred at the time of the light pulse. 2.3. Immunocytochemistry In order to assess the effectiveness and extent of GTGinduced lesions in the VMH, gliosis was visualized by immunocytochemistry for glial fibrillary acidic protein ŽGFAP., an intracellular protein expressed by astrocytes. After 20 days in DD, mice were killed with CO 2 during the late subjective day Ži.e., when gut content is low.. Mice were 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 sections were prepared on a cryostat. Sections were then incubated for 1 h with 10% normal horse serum in 0.1 M phosphate buffered saline ŽPBS; pH 7.4. and for 12 h at 48C with a mouse monoclonal anti-GFAP ŽICN Biochemicals, Costa Mesa, CA. diluted 1:1000 in PBS containing 0.3% Triton X-100. Sections were washed in PBS and incubated for 1 h with biotinylated horse anti-mouse immunoglobulins ŽVectastain ABC kit, Vector Labs, CA.. Sections were washed again in PBS and transferred for 1 h to a solution of ammonium sulfate nickel containing streptavidin–biotin complex conjugated to horseradish peroxidase. Peroxidase was visualized by the diaminobenzidine reaction. The brain sections were mounted on gelatin-coated slides. 2.4. Data analysis To quantify the light pulse-induced phase shifts, a line was fitted by eye to the onsets of locomotor activity for the
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first 10 days before the light pulse. This line was projected to the day of the light pulse Ži.e., Day DD q 10.. Similarly, a line was fitted to the onsets of activity for the 10 days after the photic pulse. That line was retroprojected to the day of the pulse. The magnitude of the phase shifts was calculated as the difference between these two lines. Daily activity was defined as the total wheel revolutions per cycle Žbeginning at CT12.. The circadian period Žt . was
assessed by the x 2 periodogram analysis ŽChronobiology 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 state Žfed or fasted., data were analyzed by two-way analyses of
Fig. 1. Glial fibrillary acidic protein immunoreactive staining in representative coronal sections of suprachiasmatic nuclei ŽSCN., left ventromedial hypothalamic nucleus ŽVMH. and left nucleus of the solitary tract ŽNTS. in a mouse injected with saline Žcontrol. and a mouse injected with gold-thioglucose ŽGTG.. The arrow indicates the GTG-induced increase of GFAP staining in the ventromedial region of the VMH ŽRight middle panel.. Scale bar s 200 mm.
E. Challet et al.r Brain Research 824 (1999) 18–27 Table 1 Adiposity index Žepididymal fat pads mass as % body mass. in the different treatment groups Neurotoxin
Treatment
Adiposity index Ž%.
Control Control GTG GTG Control Control GTG GTG
Fedrsaline Fedr2-DG Fedrsaline Fedr2-DG Fastedrsaline Fastedrinsulin Fastedrsaline Fastedrinsulin
1.15"0.06 a 1.27"0.12 a 2.02"0.31b 2.14"0.41b 0.94"0.09 a 0.80"0.08 a 1.21"0.11b 1.28"0.13 b
Data are means"S.E.M. Ž ns6 per group.. GTG: gold-thioglucose. 2-DG: 2-deoxy-D-glucose. For a given nutritional state Žfed or fasted., groups with no letters in common are significantly different from one another Ž P - 0.05..
variance ŽANOVA. to compare the effects of the neurotoxin ŽGTG vs. saline. and the different metabolic treatments Žsaline vs. 2-DG in fed animals, and saline vs. insulin in fasted animals.. If significant main effects or a significant interaction were found Ž P - 0.05., post-hoc comparisons were performed with the Student–Newman– Keuls test.
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3. Results
3.1. Histology Examination of immunocytochemical staining for GFAP revealed clear glial proliferation within the ventromedial hypothalamus, especially in the medial region of the VMH and the lateral region of the arcuate nuclei. In the 24 GTG-treated mice included in this study, the increase of GFAP staining in the VMH was evaluated microscopically Žsee an example in Fig. 1.. In keeping with accumulation of gold in the brain by autoradiography w19x, the VMH was the brain area presenting the most consistent increase in gliosis after GTG treatment ŽFig. 1.. Staining in other diencephalic regions was essentially similar between salineand GTG-injected mice. For example, in most animals, GFAP immunoreactivity was strong in the SCN w32,45x Žsee Fig. 1. and the preoptic area, and low to moderate in paraventricular hypothalamic nuclei and paraventricular thalamic nuclei Ždata not shown.. In addition to the VMH, a small increase of gliosis in a few GTG-treated animals was also apparent in the medulla oblongata Ži.e., in the vagal nuclei and nucleus of the solitary tract; Fig. 1..
Fig. 2. Double-plotted daily wheel-running activity of four mice kept in constant darkness and fed ad libitum. ŽUpper panels. Records from mice receiving an i.p. injection of saline at CT17 followed 1 h later by a light pulse Ž50 lx of white light lasting 10 min.. Mice previously treated with saline or GTG are shown in panels ŽA. and ŽB., respectively. ŽLower panels. Records from mice receiving an i.p. injection of 500 mgrkg 2-deoxy-D-glucose Ž2-DG. at CT17 followed by a light pulse Žas above.. Mice previously treated with saline or GTG are shown in panels ŽC. and ŽD., 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 a circle.
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3.2. Metabolic changes GTG treatment leads to obesity after long-term conditions of ad libitum feeding Ži.e., after several months; e.g., Ref. w15x.. In the present study, to rule out any simple effect of huge lipid fuel reserves, the GTG-treated mice were given a light pulse 20 days after the injection of GTG and killed 10 days after the light pulse. Nevertheless, to assess subtle metabolic changes between groups, we determined an adiposity index by expressing epididymal fat pads as a percentage of body mass w13x. As shown in Table 1, the adiposity index in fed mice was significantly larger in GTG-injected than in saline-injected mice Ž2.08 " 0.25 vs. 1.21 " 0.07%, respectively; F Ž1,20. s 10.6, P - 0.01., regardless of the injection Žsaline or 2-DG.. In mice previously fasted, the adiposity index was also larger in GTGinjected than in control mice Ž1.24 " 0.08 vs. 0.87 " 0.06%, respectively; F Ž1,20. s 12.6, P - 0.01., regardless of the injection Žsaline or insulin.. 3.3. Light-induced behaÕioral phase shifts and circadian period In mice fed ad libitum throughout the experiment, regardless of the injection Žsaline or 2-DG., mean phase
delays induced by a light pulse at CT18 were significantly smaller in control mice compared to GTG-treated mice Ž F Ž1,20. s 5.8, P - 0.05; Figs. 2 and 4.. Regardless of the lesion ŽGTG or saline., mean light-induced phase delays were significantly smaller in mice injected with saline compared to those injected with 2-DG Ž F Ž1,20. s 12.9, P - 0.01; Figs. 2 and 4.. Moreover, there was a significant interaction Ž F Ž1,20. s 13.3, P - 0.01; Fig. 4. between the effects of lesion ŽGTG or saline. and injection Žsaline or 2-DG.. Post-hoc analysis indicated that in individuals injected with saline, the light-induced phase delays in control mice fed ad libitum were similar to those in GTG-treated animals fed ad libitum. After blockade of glucose utilization, however, phase delays were significantly reduced in control mice compared to GTG-treated mice ŽFigs. 2 and 4.. In mice fasted prior to the light pulse, the phase delays induced by a light pulse at CT18 were significantly smaller in control mice compared to GTG-treated mice Ž F Ž1,20. s 32.0, P - 0.001; Figs. 3 and 4.. The effect of the injection Žsaline or insulin. and the interaction GTG = injection Žsaline or insulin. were not significant Ž F Ž1,20. s 0.3 and F Ž1,20. s 0.1, respectively, P ) 0.1; Figs. 3 and 4.. Using a two-way ANOVA with repeated measures, the circadian period Žt . 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 the light pulse. ŽUpper panels. Records from mice receiving a s.c. injection of saline 30 min prior to a light pulse Ž50 lx of white light lasting 10 min. given at CT18. Mice previously treated with saline or GTG are shown in panels ŽA. and ŽB., respectively. ŽLower panels. Records from mice receiving a s.c. injection of 5 IUrkg insulin 30 min prior to a light pulse Žas above.. Mice previously treated with saline or GTG are shown in panels ŽC. and ŽD., 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 a circle.
E. Challet et al.r Brain Research 824 (1999) 18–27
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treatment groups Ž F Ž3,20. s 1.6, P ) 0.1., or following the light pulse Ž F Ž1,20. s 0.1, P ) 0.1.. In all fasted groups, t was close to 23.7 " 0.07 h Žsee Fig. 3 for examples.. 3.4. QuantitatiÕe analysis of wheel-running actiÕity
Fig. 4. Phase shifts Žnegative values are delays. in circadian activity rhythm of mice housed in constant darkness after a light pulse Ž50 lx of white light lasting 10 min. given at CT18. Half the mice were previously treated with saline or GTG ŽGTG-treated.. Mice that were fed ad libitum over the experiment received an i.p. injection of 500 mgrkg 2-deoxy-Dglucose Ž2-DG. or saline at CT17 ŽFed 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 min prior to the light pulse ŽFasted groups.. Means"S.E.M. Ž ns6 per group.. Groups with no letters in common are significantly different from one another Ž P - 0.05..
the four treatment groups Žcontrolrsaline, controlr2-DG, GTGrsaline or GTGr2-DG. for the 10-day period before or after the light pulse. The circadian period did not differ among the treatment groups Ž F Ž3,20. s 0.4, P ) 0.1., nor did it change following the light pulse Ž F Ž1,20. s 0.1, P ) 0.1.. In all fed groups, t was close to 23.7 " 0.08 h Žsee Fig. 2 for examples.. A similar analysis was performed in fasted mice across the four treatment groups Žcontrolrsaline, controlrinsulin, GTGrsaline or GTGrinsulin. and the 10-day period before or after the light pulse. Again, the circadian period did not differ among the
A two-way ANOVA with repeated measures was used to compare the number of wheel revolutions on Day DD q 9 Ži.e., prior to the light pulse. vs. Day DD q 10 Ži.e., the day when a light pulse was applied. in fed mice. The total number of wheel revolutions did not differ significantly by treatment Žcontrolrsaline, GTGrsaline, controlr2-DG or GTGr2-DG: F Ž3,20. s 1.9, P ) 0.1., or by the day ŽDD q 9 vs. DD q 10: F Ž1,20. s 0.7, P ) 0.1; Table 2.. In fasted mice, the total number of wheel revolutions was significantly affected by the day ŽDD q 9 vs. DD q 10: F Ž1,20. s 33.0, P - 0.01., but not by the treatment Žcontrolrsaline, GTGrsaline, controlrinsulin or GTGrinsulin: F Ž3,20. s 0.1, P ) 0.1.. No other comparisons were significantly different. The number of wheel revolutions was determined between CT6 and CT12 on Day DD q 9 ŽTable 2.. In fed mice, afternoon activity was not affected significantly by the neurotoxin ŽGTG or saline: F Ž1,20. s 1.7, P ) 0.1., or the injection Žsaline or 2-DG: F Ž1,20. s 0.2, P ) 0.1.. Similarly, afternoon activity in fasted mice was not affected significantly by the neurotoxin ŽGTG or saline: F Ž1,20. s 0.1, P ) 0.1., or the injection Žsaline or 2-DG: F Ž1,20. s 0.1, P ) 0.1.. In order to assess subtle differences in the wheel-running activity prior to the light pulse that may interfere with the circadian responses to light w40,57x, the number of wheel revolutions was also determined between CT12 and
Table 2 Number of wheel revolutions in the different treatment groups Neurotoxin
Treatment
Daily activity on Day DD q 9
Daily activity on Day DD q 10
Afternoon act. on Day DD q 9
Early noct. act. on Day DD q 10
Control Control GTG GTG Control Control GTG GTG
Fedrsaline Fedr2-DG Fedrsaline Fedr2-DG Fastedrsaline Fastedrinsulin Fastedrsaline Fastedrinsulin
21845" 1871 21638" 4239 16561" 2324 14427" 3939 23074" 4733 25041" 6270 22452" 5823 25820" 3209
20746" 1335 16837" 2599 21327" 3494 11290" 1975 16802" 2476 13822" 2919 14440" 3812 15436" 1467
1226 " 517 1588 " 639 1200 " 420 373 " 151 2030 " 692 3844 " 2065 3746 " 2132 2448 " 501
13428" 1950 12050" 2526 11367" 1567 8598 " 2035 12409" 2191 11865" 2586 9064 " 2349 12364" 1731
Data are means " S.E.M. Ž n s 6 per group.. GTG: gold-thioglucose. 2-DG: 2-deoxy-D-glucose. Daily Activity: total wheel revolutions performed per day, beginning at CT Žcircadian 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 DD q 9: day when mice were either fed or fasted. Day DD q 10: day on which a light pulse was administered. For a given nutritional state Žfed or fasted., no groups in a given column were significantly different from one another Ž P ) 0.05.. There was a reduction in the total number of wheel revolutions in fasted mice between DD q 9 and DD q 10 Ž P - 0.01..
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E. Challet et al.r Brain Research 824 (1999) 18–27
CT18 on Day DD q 10 Ži.e., during the 6 circadian hours before the light pulse; Table 2.. Here again, there were no significant effects of the neurotoxin ŽGTG or saline: F Ž1,20. s 1.8, P ) 0.1. and of the injection Žsaline or 2-DG: F Ž1,20. s 1.0, P ) 0.1. on early nocturnal activity of mice fed ad libitum. In fasted mice, nocturnal activity prior to the light pulse was not modified significantly by the neurotoxin ŽGTG or saline: F Ž1,20. s 0.4, P ) 0.1., or the injection Žsaline or insulin: F Ž1,20. s 0.4, P ) 0.1.. None of the possible interactions in the comparisons of wheel running activity were significant.
4. Discussion The present study confirms that photic phase resetting of the circadian clock is reduced in intact mice when glucose availability is decreased. This effect is observed whether glucose utilization is blocked or animals are fasted, with or without added insulin-induced hypoglycemia w12x. In addition, the present report demonstrates that destruction of glucose-receptive neurons with the neurotoxin GTG blocks these altered circadian responses to light. 4.1. Lesions induced by GTG treatment Given that morphology and function of peripheral tissues are not altered during the development of obesity in GTG-injected mice w15,34x, the observed effects after GTG injection are likely to be due to changes in the central nervous system. Because some responses of the circadian clock to light were different in control mice compared to GTG-injected mice, one can speculate that GTG treatment affects directly SCN neurons. Due to high levels of GFAP immunoreactivity in the SCN of control mice, it was not possible to detect a change in the SCN following GTG treatment. While GTG treatment impairs attenuation of light-induced phase shifts in response to metabolic challenges, it does not affect the phase-angle of photic entrainment in mice fed ad libitum w11x. In addition, the free-running periods of the circadian rhythm of locomotor activity and the light-induced behavioral phase delays are similar in both GTG- and saline-injected mice fed ad libitum Žpresent study.. To specifically rule out direct effects of GTG in the SCN, further studies are needed in rodents receiving GTG in the SCN without damaging the VMH. Taken together, however, the available data suggest that SCN function in mice with free access to food is not altered by GTG injection. Most SCN neurons in vitro do not display changes in firing rate when extracellular glucose is modified w24x, an effect typical of glucose-receptive neurons as defined by Oomura w54x. Conversely, about 20% of VMH neurons increase firing rate when extracellular glucose is increased w54x. In the present study, the most extensive damage following GTG administration was observed in the VMH.
This is best exemplified by changes in both anti-GFAP staining ŽFig. 1. and adiposity ŽTable 1.. Limited gliosis was also observed in the nucleus of the solitary tract in some GTG-treated mice. With higher doses than the one we used, the nucleus of the solitary tract and dorsal vagal nuclei can be heavily damaged by the GTG treatment w56x. Gold autoradiography after GTG treatment has been used by Debons and colleagues to localize where GTG acts in the mouse brain, and these authors found some labeled gold in the dorsal hindbrain w19x. This technique, however, does not provide information on the extent of cell damage. To specifically rule out an involvement of these brainstem nuclei in the metabolic modulation of circadian responses to light, further studies are needed in rodents with local chemical lesions of the nucleus of the solitary tract andror vagal nuclei. Nevertheless, the present results taken together suggest that the GTG-induced inhibition of metabolic modulation of photic phase shifting may be mediated by the VMH. 4.2. ActiÕity feedback on the circadian clock Acute changes of locomotor activity can modulate the regulation of circadian rhythmicity in hamsters w46,63x and mice w21,36x. In the present study, no significant changes in the number of wheel revolutions were detected between GTG- and saline-injected mice prior to the light pulse. For example, the decrease in total Ždaily. activity after a fasting period was similar in both GTG- and salineinjected animals ŽTable 2.. Therefore, it seems unlikely that the observed effects on the light-induced phase shifts are mediated by changes in the level of physical activity that can interfere with photic phase-resetting in hamsters fed ad libitum w40,57x. As we have hypothesized previously w12x, the altered phase-shifting responses to light during shortage of glucose availability may be due to metabolic signals. 4.3. Blockade of glucose utilization by 2-DG Peripheral injection of 2-DG blocks local glucose utilization in most brain areas, including the SCN w61x. In response to w14 Cx-DG treatment, there is an increase of blockade of glucose utilization in the SCN during the subjective day and a decrease during the subjective night in vivo Že.g., Ref. w29x.. In addition, the phase of the rhythmic firing rate in a slice of SCN can be altered Ži.e., delayed. temporarily by decreasing the availability of glucose in the bathing solution w24x. Considering a direct effect of 2-DG in the SCN, one may expect that lightinduced phase delays would be greater after 2-DGinduced cytoglucopenia. On the contrary, there was an attenuation of light-induced phase delays in control mice pretreated with 2-DG. Furthermore, blockade of glucose utilization in GTG-treated mice did not reduce the lightinduced phase delays. Assuming that the SCN function is
E. Challet et al.r Brain Research 824 (1999) 18–27
not impaired by GTG in mice fed ad libitum Žsee above., a direct effect of cytoglucopenia in the SCN would have been 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 cytoglucopenia have been shown to be mediated by the VMH w8x Žsee Section 1.. In keeping with the hypothesis that the VMH play a major role in sensing 2-DG-induced cytoglucopenia, rats with electrolytic lesions of the VMH w27x or GTG-treated mice w6x do not increase food intake after 2-DG treatment, although these mice remain sensitive to the inhibitory effect of cholecystokinin w6x. Accordingly, GTG treatment reduces and delays 2-DG-induced hyperglycemia in mice w48x. In the present experiment, we found that GTG treatment also impairs the 2-DG dependent attenuation of light-induced phase delays. A glucose injection prior to a light pulse, that increases plasma glucose, does not alter the photic phase-resetting w12x. Therefore, it is unlikely that the 2-DG-induced rise in glycemia per se plays a critical role in the reduced phase-shifting effects of light after blockade of cerebral glucose utilization. 4.4. Hypoglycemia during fasting, with or without insulin injection The attenuated light-induced phase shifts in control fasted mice were counteracted in fasted GTG-treated mice, with or without insulin injection prior to a light pulse. This difference may occur because GTG treatment impairs fasting- or insulin-induced hypoglycemia, leading to similar circadian responses to those in control mice fed ad libitum. Previous studies, however, have shown that damage to the VMH does not prevent fasting-induced decreases in plasma glucose in rats with electrical lesions of the VMH w4,52x or in mice treated with GTG w15x. In addition to blockade of glucose utilization and fasting, calorie restriction is another situation of negative energy balance, usually associated with chronic hypoglycemia w25,38x. Calorie restriction can phase shift circadian rhythms and modify the phase angle of photic entrainment in rodents w11,13,14x. This altered photic regulation of circadian rhythmicity, that implies SCN involvement, can be blocked by ibotenic lesions of the VMH in rats w13x and GTG treatment in mice w11x. Taken together, these data indicate that the loss of effects of fasting, 2-DG injection and calorie restriction on the light-induced phase shifts in GTG-treated mice may be due to an impairment in the integration Ždetection. of metabolic signals by the VMH that would, when glucose availability is decreased, impact on the SCN function in control mice. 4.5. Metabolic and hormonal signals modulating VMH neurons actiÕity The metabolic cues that modulate the photic phaseresetting of the SCN are not yet clearly defined. One likely candidate is a decrease in intracellular glucose availability
25
Žor an altered rate of glucose utilization. occurring during fasting and after 2-DG injection. Such a decrease in glucose availability modifies the activity of VMH neurons w54x and may, in turn, affect SCN function Žpresent study.. Fasting and 2-DG treatment, however, are also associated with elevation of plasma ketone bodies and free fatty acids w22,52x, and these increases are not impaired by electrolytic lesions of the VMH w4,52x. The changes in adiposity we observed among groups of mice are consistent with expected mobilization of fatty acids during previous fasting ŽTable 1.. Interestingly, free fatty acids applied electrophoretically can affect the firing rate of VMH neurons w54x. Therefore, the effects of metabolic cues on photic phase-shifting may be mediated, in part, by changes in free fatty acid levels. Further studies using specific blockers of fatty acid oxidation will be useful for testing this hypothesis. Insulin plays a key role in the control of energy utilization w33x and, therefore, may mediate some of the observed effects. Furthermore, insulin applied during the subjective day inhibits the firing rate of SCN cells in vitro w59x. Insulinemia is unchanged after 2-DG treatment w2,23x, is decreased after fasting w15x, and is increased after insulin injection w33x. Despite these varied responses to different metabolic challenges, reduced circadian responses are observed in all three situations Žpresent study.. Moreover, neither electrolytic lesions of the VMH, nor GTG treatment affect the typical changes in insulinemia in response to fasting Ži.e., hypoinsulinemia; see Refs. w4,15x. and 2-DG injection Ži.e., normoinsulinemia; see Ref. w52x.. In spite of the fact that insulin can modulate firing rate of VMH neurons w54x, this hormone does not appear to be involved critically in the reduced phase shifting effects of light during metabolic challenges. Leptin, another hormone involved in the regulation of energy metabolism w1x, has been shown to inhibit the activity of glucose-receptive neurons in the VMH w62x. To our knowledge, the effect of 2-DG on leptin release has not yet been studied in vivo. In vitro, however, 2-DG causes an inhibition of leptin release from cultured rat adipocytes w47x. Fasting decreases plasma leptin w1x and increases leptin receptor mRNA expression in the VMH w35x. Taken together, these data suggest that, at least during fasting, lowered plasma leptin may somehow disinhibit VMH neurons. Whether this effect plays a role in the metabolic modulation of the circadian responses to light remains to be determined. 4.6. Direct Õs. indirect connections from the VMH to the SCN The present results suggest that the integration of metabolic and hormonal cues by the VMH plays a role in the altered circadian responses to light during metabolic challenges. As mentioned in Section 1, anatomical studies revealed that the VMH send a small number of fibers to the SCN, some of which are excitatory amino acidergic
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E. Challet et al.r Brain Research 824 (1999) 18–27
w42x. The critical pathway for photic entrainment of the SCN clock is the retino-hypothalamic tract that conveys photic cues from the retina w20,44x. A large body of experimental data indicates that glutamate release from retino-hypothalamic terminals is the neurochemical substrate mediating photic signals to the clock w20x. Given that several subtypes of NMDA, non-NMDA and metabotropic receptors are found in the SCN w20x, it is possible that distinct, possibly conflicting, glutamatergic signals Ži.e., photic from the retina or ‘metabolic’ from the VMH. activate different post-synaptic glutamatergic receptor subtypes. Besides direct Žmonosynaptic. modulation of SCN function by the VMH, indirect pathways from the VMH to the SCN may also be involved in the metabolic modulation of SCN function. For example, the paraventricular thalamic nuclei, which receive a large projection from the VMH w16x, send direct glutamatergic fibers to the SCN w42x. The intergeniculate leaflets, which receive a projection from the VMH w64x, send a dense projection releasing neuropeptide Y and g-aminobutyric acid in the SCN w43x. The geniculo-hypothalamic terminals are considered to play a pivotal role in conveying several non-photic cues to the SCN w26,37,39,46,66x, including putative ‘metabolic’ cues w14x. Because lesions of the intergeniculate leaflets induce an almost complete disappearance of neuropeptide Y-immunoreactive fibers and terminals in the SCN of rodents w14,26,37,39x, an increase of neuropeptide Y in the SCN most likely reflects geniculate activation. The intergeniculate leaflets may also convey to the SCN some ‘metabolic’ cues integrated by the VMH, because 2-DG injection increases the level of neuropeptide Y in the SCN w2x. In conclusion, both physiological and hodological data give support to the hypothesis that the VMH integrate metabolic cues and modulate the photic regulation of circadian rhythmicity by means of direct andror indirect connections to the SCN.
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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 w1x R.S. Ahima, D. Prabakaran, C. Mantzoros, D.Q. Qu, B. Lowell, E. Maratos-Flier, J.S. Flier, Role of leptin in the neuroendocrine response to fasting, Nature 382 Ž1996. 250–252. w2x A. Akabayashi, C.T. Zaia, I. Silva, H.J. Chae, S.F. Leibowitz, Neuropeptide Y in the arcuate nucleus is modulated by alterations in glucose utilization, Brain Res. 621 Ž1993. 343–348. w3x M.L.J. Ashford, P.R. Boden, J.M. Treherne, Glucose-induced excitation of hypothalamic neurones is mediated by ATP-sensitive Kq channels, Pflug. Arch. Eur. J. Physiol. 415 Ž1990. 479–483. w4x H. Balkan, G. Van Dijk, J.H. Strubbe, J.E. Bruggink, A.B. Steffens,
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