Neuroendocrinology 22: 216-225 (1976)
Effect of Ventromedial and Dorsoniedial Hypothalamic Lesions on Circadian Corticosterone Rhythms1 L.L. Bellin g er 2, L.L. Bern a rd is and V. E. M endel Departments of Surgery and Pathology, State University of New York at Buffalo, Buffalo, N.Y. and Department of Animal Physiology, University of California, Davis, Calif.
Key Words. Circadian rhythm • Corticosterone • Ventromedial and dorsoniedial hypo thalamus • Food intake Abstract. Weanling rats received bilateral electrolytic lesions in the dorsoniedial (DMH) or ventromedial (VMH) hypothalamic areas destroying primarily the dorsoniedial (DMN) or ventromedial (VMN) hypothalamic nuclei. Sham-operated rats served as controls. Lesions in the VMN and DMN, both of which have previously been shown to disrupt normal diurnal feeding rhythms, were also observed to disrupt normal plasma corticoste rone rhythms in the present study. The a.m. values of plasma corticosterone in the DMNlesioned rats were higher than the sham-operated controls. In the p.m.. the values of both VMN- and DMN-lesioned rats were lower than those of the controls but unchanged in comparison to their own a.m. concentrations. This disruption of the normal diurnal plasma corticosterone rhythm persisted for at least 9 post-operative weeks.
Several hypothalamic nuclei have been shown to be involved in rhythmic physiological processes. Experimental destruction of the suprachiasmatic nuclei (SCN) has been observed to alter cyclic drinking and locomotor activity [St e ph a n and Z u c k e r , 1972a, b] and pineal N-acetyltransferase activity [M oore and K lein , 1974]. In addition, M oore and E ic h ler [1972] have reported that the normal diurnal rhythm of adrenal corticosterone is dis rupted after SCN lesions. The ventromedial (VMN) and dorsoniedial (DMN)
Received: May 12th, 1976; revised MS accepted: October 2nd, 1976.
Downloaded by: Kings's College London 137.73.144.138 - 11/10/2017 1:23:12 PM
' This research was supported In part by the National Institute of General Medical Sciences grant 15768. 2 Supported by National Institutes of Health individual postdoctoral fellowship I F22 NS 02625-01-NEURA.
Bellinger/B ernardis/M endel
217
hypothalamic nuclei have also been implicated in cyclic behaviors. Specifi cally, lesions of the VMN and DMN have been observed to disrupt the natural nocturnal feeding patterns of mature [S iegel and S t u c k e y , 1947] and weanling rats [B ern a rd is , 1973]. Corticosterone in rats [C r it c h l o w , 1963] normally peaks prior to the onset of that part of the rat’s activity cycle in which natural feeding and activity reach their peak, i.e. shortly after the onset of the dark period [Le M agnen and D evos , 1970], Recently, K rieger [1974] and M oberg et at. [1975] have reported that when rats are meal-fed during the light period, the natural corticosterone rhythm is altered so that there is a major release of corticosterone prior to meal feeding with a secondary peak occurring at the onset of the dark period. Thus, in rats, corticosterone rhythms are associated with either feeding behavior per se, or more probably, with arousal brought about in conjunction with, or in anticipation of. the onset of feeding under both natural and imposed feeding situations. R ichter [1967] has suggested that the biological clock underlying circadian rhythms in behavior does not appear to reside in any of the endocrine glands. However, S t e ph a n and Z ucker [1972b] have observed that hypophysectomy in ovariectomized rats attenuated the diurnal feeding rhythm compared to ovariectomized, non-hypophysectomized rats. This study investigates whether there is a relationship between cortico sterone patterns and rhythmic feeding patterns in the rat by observing whether VMN and DMN lesions, which are known separately to disrupt diurnal feeding rhythms in rats, also alter diurnal corticosterone patterns.
Weanling male Sprague-Dawley rats (Sprague-Dawley, Madison, Wise.) were caged individually in a light-cycle controlled (L:D, 12:12 with lights on at 06.00 h) and tem perature controlled (23 C) room. They were given lab chow (Charles River Rat and Mouse Formula) and tap water ad libitum. At the age of 34 days (corticosterone rhythm appears at approximately 22 days of age, according to Levin and L evine [1975]). the rats were anesthetized with hexobarbital sodium (Evipal, Winthrop, 14 mg/100 g b.w.) B.w. and naso-anal length were measured and the rats were inserted into a stereotaxic instrument (Baltimore Instrument Co.) and bilateral lesions were placed with a direct anodal current of 1.0 mA that flowed for 10 sec. Stainless steel wires, 0.25 mm in diameter, telescoped into 24-gauge hypodermic needles and spar varnish-coated except for about 0.2 mm at the tip, served as electrodes. The coordinates have been previously established [Bernardis and S kelton, 1965; Bernardis, 1970a]. The rats were divided into 3 groups: group 1 received lesions in the ventromedial hypothalamus (VMH), group 2 received lesions in the
Downloaded by: Kings's College London 137.73.144.138 - 11/10/2017 1:23:12 PM
Materials and Methods
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Belli nger/B ernardis/M endel
dorsoniedial hypothalamus (DMH) and group 3 consisted of sham-operated controls with the electrode inserted into either the VMH or DMH, but without current flow. Food intake was recorded. 20 days after the operation, the 3 groups were divided with one-half of the animals of each group bled by heart puncture under light ether anesthesia at 08.00 h and the remaining half being similarly bled at 18.00 h. Only samples obtained within 90 sec of the animals' removal from the cage were analyzed for corticosterone (see below). Two weeks later, the animals were again bled. This time, those animals that were previously bled at 08.00 h were now' bled at 18.00 h, and vice versa. After another 19 days, the animals were again bled in a manner reverse to the previous bleeding schedule. Finally, 18 days after the 3rd bleeding, the sampling sequence was reversed again and the blood was collected after decapitation. Thus, each animal was bled twice at 08.00 and twice at 18.00 h. After the blood was collected, it was chilled until centrifugation; the plasma was stored at -20 C until assayed for corticosterone [G ivner and Rochefort, 1965]. For statistical analysis, all 08.00 h hormone values collected over the 4 bleeding periods of the individual groups w'erc pooled, as were the 18.00 h values. The 4 individual collection periods of each group were also analyz.ed to determine if the observed patterns varied between periods. Four days before the last bleeding, the animals were lightly anesthetized to accurately assess b.w. and naso-anal length and to compute the obesity or Lee Index. After sacrifice, the brains were removed and placed in 10% buffered formalin for sub sequent histological work-up and analysis. This yielded the following populations: group 1 (VMN-lesioncd), 6 rats; group 2 (DMN-lesioned), 12 rats and group 3 (sham-operated), 12 rats. The data collected from this population were used for statistical analysis. The data of this experiment were analyzed using analysis of variance, Duncan’s mul tiple range test or Student’s /-test.
Figure 1 shows the lesion placement in 2 rats representative of group 1 (VMN lesioned) and group 2 (DMN - lesioned rats). Table I shows that the animals used in this study are proper representations of the weanling rat ventromedial and dorsomedial hypothalamic syndromes [B ern a rd is et al., 1975], The sham-operated rats showed a normal corticosterone rhythm with the 18.00 h values significantly higher than the 08.00 h concentrations (fig. 2). However, VMN and DMN lesions apparently disrupted the normal diurnal pattern of corticosterone release (fig. 2). When the morning values of plasma corticosterone of the control, VMN-lesioned and DMN-lesioned groups were compared by analysis of variance, a significant difference was found (F (2,42) = 13.39; p
Effect of Ventromedial and Dorsoniedial Hypothalamic Lesions on Circadian Corticosterone Rhythms1 L.L. Bellin g er 2, L.L. Bern a rd is and V. E. M endel Departments of Surgery and Pathology, State University of New York at Buffalo, Buffalo, N.Y. and Department of Animal Physiology, University of California, Davis, Calif.
Key Words. Circadian rhythm • Corticosterone • Ventromedial and dorsoniedial hypo thalamus • Food intake Abstract. Weanling rats received bilateral electrolytic lesions in the dorsoniedial (DMH) or ventromedial (VMH) hypothalamic areas destroying primarily the dorsoniedial (DMN) or ventromedial (VMN) hypothalamic nuclei. Sham-operated rats served as controls. Lesions in the VMN and DMN, both of which have previously been shown to disrupt normal diurnal feeding rhythms, were also observed to disrupt normal plasma corticoste rone rhythms in the present study. The a.m. values of plasma corticosterone in the DMNlesioned rats were higher than the sham-operated controls. In the p.m.. the values of both VMN- and DMN-lesioned rats were lower than those of the controls but unchanged in comparison to their own a.m. concentrations. This disruption of the normal diurnal plasma corticosterone rhythm persisted for at least 9 post-operative weeks.
Several hypothalamic nuclei have been shown to be involved in rhythmic physiological processes. Experimental destruction of the suprachiasmatic nuclei (SCN) has been observed to alter cyclic drinking and locomotor activity [St e ph a n and Z u c k e r , 1972a, b] and pineal N-acetyltransferase activity [M oore and K lein , 1974]. In addition, M oore and E ic h ler [1972] have reported that the normal diurnal rhythm of adrenal corticosterone is dis rupted after SCN lesions. The ventromedial (VMN) and dorsoniedial (DMN)
Received: May 12th, 1976; revised MS accepted: October 2nd, 1976.
Downloaded by: Kings's College London 137.73.144.138 - 11/10/2017 1:23:12 PM
' This research was supported In part by the National Institute of General Medical Sciences grant 15768. 2 Supported by National Institutes of Health individual postdoctoral fellowship I F22 NS 02625-01-NEURA.
Bellinger/B ernardis/M endel
217
hypothalamic nuclei have also been implicated in cyclic behaviors. Specifi cally, lesions of the VMN and DMN have been observed to disrupt the natural nocturnal feeding patterns of mature [S iegel and S t u c k e y , 1947] and weanling rats [B ern a rd is , 1973]. Corticosterone in rats [C r it c h l o w , 1963] normally peaks prior to the onset of that part of the rat’s activity cycle in which natural feeding and activity reach their peak, i.e. shortly after the onset of the dark period [Le M agnen and D evos , 1970], Recently, K rieger [1974] and M oberg et at. [1975] have reported that when rats are meal-fed during the light period, the natural corticosterone rhythm is altered so that there is a major release of corticosterone prior to meal feeding with a secondary peak occurring at the onset of the dark period. Thus, in rats, corticosterone rhythms are associated with either feeding behavior per se, or more probably, with arousal brought about in conjunction with, or in anticipation of. the onset of feeding under both natural and imposed feeding situations. R ichter [1967] has suggested that the biological clock underlying circadian rhythms in behavior does not appear to reside in any of the endocrine glands. However, S t e ph a n and Z ucker [1972b] have observed that hypophysectomy in ovariectomized rats attenuated the diurnal feeding rhythm compared to ovariectomized, non-hypophysectomized rats. This study investigates whether there is a relationship between cortico sterone patterns and rhythmic feeding patterns in the rat by observing whether VMN and DMN lesions, which are known separately to disrupt diurnal feeding rhythms in rats, also alter diurnal corticosterone patterns.
Weanling male Sprague-Dawley rats (Sprague-Dawley, Madison, Wise.) were caged individually in a light-cycle controlled (L:D, 12:12 with lights on at 06.00 h) and tem perature controlled (23 C) room. They were given lab chow (Charles River Rat and Mouse Formula) and tap water ad libitum. At the age of 34 days (corticosterone rhythm appears at approximately 22 days of age, according to Levin and L evine [1975]). the rats were anesthetized with hexobarbital sodium (Evipal, Winthrop, 14 mg/100 g b.w.) B.w. and naso-anal length were measured and the rats were inserted into a stereotaxic instrument (Baltimore Instrument Co.) and bilateral lesions were placed with a direct anodal current of 1.0 mA that flowed for 10 sec. Stainless steel wires, 0.25 mm in diameter, telescoped into 24-gauge hypodermic needles and spar varnish-coated except for about 0.2 mm at the tip, served as electrodes. The coordinates have been previously established [Bernardis and S kelton, 1965; Bernardis, 1970a]. The rats were divided into 3 groups: group 1 received lesions in the ventromedial hypothalamus (VMH), group 2 received lesions in the
Downloaded by: Kings's College London 137.73.144.138 - 11/10/2017 1:23:12 PM
Materials and Methods
218
Belli nger/B ernardis/M endel
dorsoniedial hypothalamus (DMH) and group 3 consisted of sham-operated controls with the electrode inserted into either the VMH or DMH, but without current flow. Food intake was recorded. 20 days after the operation, the 3 groups were divided with one-half of the animals of each group bled by heart puncture under light ether anesthesia at 08.00 h and the remaining half being similarly bled at 18.00 h. Only samples obtained within 90 sec of the animals' removal from the cage were analyzed for corticosterone (see below). Two weeks later, the animals were again bled. This time, those animals that were previously bled at 08.00 h were now' bled at 18.00 h, and vice versa. After another 19 days, the animals were again bled in a manner reverse to the previous bleeding schedule. Finally, 18 days after the 3rd bleeding, the sampling sequence was reversed again and the blood was collected after decapitation. Thus, each animal was bled twice at 08.00 and twice at 18.00 h. After the blood was collected, it was chilled until centrifugation; the plasma was stored at -20 C until assayed for corticosterone [G ivner and Rochefort, 1965]. For statistical analysis, all 08.00 h hormone values collected over the 4 bleeding periods of the individual groups w'erc pooled, as were the 18.00 h values. The 4 individual collection periods of each group were also analyz.ed to determine if the observed patterns varied between periods. Four days before the last bleeding, the animals were lightly anesthetized to accurately assess b.w. and naso-anal length and to compute the obesity or Lee Index. After sacrifice, the brains were removed and placed in 10% buffered formalin for sub sequent histological work-up and analysis. This yielded the following populations: group 1 (VMN-lesioncd), 6 rats; group 2 (DMN-lesioned), 12 rats and group 3 (sham-operated), 12 rats. The data collected from this population were used for statistical analysis. The data of this experiment were analyzed using analysis of variance, Duncan’s mul tiple range test or Student’s /-test.
Figure 1 shows the lesion placement in 2 rats representative of group 1 (VMN lesioned) and group 2 (DMN - lesioned rats). Table I shows that the animals used in this study are proper representations of the weanling rat ventromedial and dorsomedial hypothalamic syndromes [B ern a rd is et al., 1975], The sham-operated rats showed a normal corticosterone rhythm with the 18.00 h values significantly higher than the 08.00 h concentrations (fig. 2). However, VMN and DMN lesions apparently disrupted the normal diurnal pattern of corticosterone release (fig. 2). When the morning values of plasma corticosterone of the control, VMN-lesioned and DMN-lesioned groups were compared by analysis of variance, a significant difference was found (F (2,42) = 13.39; p
