Diurnal rhythm of galanin-like immunoreactivity in the paraventricular and suprachiasmatic nuclei and other hypothalamic areas

Peptides, Vol. 15, No. 8, pp. 1437-1444, 1994 Copyright © 1994ElsevierScienceLtd Printed in the USA.All rightsreserved 0196-9781/94 $6.00 + .00

Pergamon 0196-9781(94)00127-8

Diurnal Rhythm of Galanin-Like Immunoreactivity in the Paraventricular and Suprachiasmatic Nuclei and Other Hypothalamic Areas A K I R A A K A B A Y A S H I , * C. T. B. V. ZAIA,* J A M E S I. K O E N I G , t S T E V E N M. GABRIEL,J; I V A N SILVA* A N D S A R A H F. L E I B O W l T Z .1

*The Rockefeller University, New York, N Y 10021, -Hmmunobiology Research Institute, Annandale, NJ 08801, and ~:Mount Sinai School of Medicine, New York, N Y 10029 Received 21 J a n u a r y 1994 AKABAYASHI, A., C. T. B. V. ZAIA, J. I. KOEN1G, S. M. GABRIEL, I. SILVA AND S. F. LEIBOWITZ. Diurnal rhythm of galanin-like immunoreactivityin theparaventricularand suprachiasmaticnucleiand otherhypothalamicareas. PEPTIDES 15(8) 1437-1444, 1994.--The peptide galanin (GAL), when injected into the rat hypothalamus, is known to stimulate feedingbehavior and affect the secretion of various hormones, including insulin and the adrenal steroid, corticosterone. To determine whether endogenous peptide levels shift in relation to natural rhythms of feeding and circulating hormone levels, rats were sacrificed at different times of the light/dark cycle, and their GAL levels were measured, via radioimmunoassay, in medial hypothalamic dissectionsand micropunchedhypothalamicareas. The resultssuggestthe existenceof two distinct diurnal rhythms for hypothalamic GAL. One rhythm, detected exclusivelyin the area of the SCN, is characterized by bimodal peaks of GAL, threefold higher than basal peptide levels, around the onset of the dark and light periods. The second rhythm shows a single peak of GAL towards the middle of the nocturnal feeding cycle, specificallybetween the third and sixth hour. This latter rhythm is evident in the dorsal region of the medial hypothalamus, localized specificallyto the lateral portion of the PVN, Moreover, it is inversely related to circulating insulin but unrelated to the adrenal steroids, suggestinga possible association between this pancreatic hormone and GAL in the PVN. Hypothalamus

Galanin

Insulin

Circadian

THE neuropeptide galanin (GAL), first isolated from porcine intestine (42), has been shown to exist in high concentrations within the brain (30). Galanin in the rat consists of an amidated 29 amino acid chain. Within the brain, high concentrations of GAL peptide and mRNA have been detected in the hypothalamus where a high density of putative high-affinity GAL receptors exists ( 18,31 ). The physiological function of neuronal GAL in the hypothalamus remains to be established. It has been suggested to be endocrine in nature, with GAL reducing circulating levels of adrenocorticotropin (ACTH), corticosterone (CORT), and insulin ( 15,19,45), while stimulating the release of growth hormone and prolactin (20,32). Hypothalamic GAL has also been linked to feeding behavior, which is stimulated by injections of GAL (5,22,23,36), as well as by GAL's first 16 N-terminal amino acids (8). In animals maintained on separate macronutrient diets, this effect of GAL is found to be nutrient specific, associated with a

preferential increase in fat ingestion and little or no change in the consumption of protein or carbohydrate (44). These findings clearly differentiate the endocrine and behavioral effects of GAL from those of another peptide, neuropeptide Y (NPY), which also potentiates feeding behavior (7,41). Injections of this peptide preferentially increase carbohydrate intake (40), and its endogenous levels are positively correlated with natural appetite for carbohydrate (17). Moreover, NPY stimulates, rather than inhibits, the release of CORT (28,51), insulin (1), and vasopressin (28). In an effort to understand the physiological functions of these and other peptides, studies of diurnal rhythms have been particularly helpful. Investigations of NPY levels suggest that neurons containing this peptide may be specifically active at the onset of the natural feeding cycle, the dark period for the rat. The evidence demonstrates that hypothalamic levels of NPY peak around dark onset and then decline over the next 2-3 h

J Requests for reprints should be addressed to Dr. Sarah Leibowitz, The Rockefeller University, 1230 York Avenue, New York, NY 10021.

1437

1438

AKABAYASHI ET AL.

(2,16). This temporal pattern is associated with that of circulating CORT, which rises and peaks around dark onset and is positively correlated with NPY levels (2,27). Moreover, this rise in NPY and CORT levels is additionally linked to the ingestion of carbohydrate, which predominates in the early clark period (38,39). With regard to GAL, there are no studies of its diurnal rhythm. If its physiological actions do, in fact, differ from those of NPY, the rhythms of these two peptides across the feeding cycle may also be expected to differ. Based on GAL's endocrine actions, which involve an inhibitory effect on CORT and insulin release (15,19,45), this peptide may peak at a time when circulating hormone levels are declining. Moreover, if endogenous GAL is normally linked to the ingestion of fat rather than carbohydrate, its rhythm may be associated with the animal's natural shift in fat intake, which rises a few hours after dark onset and peaks during the middle of the natural feeding cycle (38). Thus, the objective of this study was to examine the diurnal rhythm of GAL in different hypothalamic sites and determine whether its rhythm in specific areas coincides with shifts in circulating hormones.

then dissected freehand, using the top of the third ventricle as the dorsal boundary and the lateral hypothalamic sulci as the lateral boundaries. With the coronal face of the dissection facing up, the block was then dissected in half horizontally, to produce the mediobasal portion (MBH) and the mediodorsal portion (MDH). The MDH contains the PVN and dorsomedial nucleus (DMN), areas that are dense with GAL cell bodies as well as terminals. The MBH, in contrast, contains the suprachiasmatic nucleus (SCN) and median eminence (ME), which have a dense GAL innervation but few cell bodies, and also the arcuate (ARC) and supraoptic (SON) nuclei, which have a moderate concentration of cell bodies. For Experiment 2, the brains were quickly removed and frozen on dry ice. Serial sections of 300 #m were cut in a cryostat, and hypothalamic sites were microdissected according to the method of Palkovits (33) and following the atlas of Paxinos and Watson (34), as described elsewhere (17). A 300-t~m needle was used to punch the smaller areas [the medial (mPVN) and lateral (1PVN) portions of the PVN, ARC, SCN, and SON], while the other areas were punched using a 500-um needle [DMN, medial preoptic area (MPO), and ME].

METHOD

Animals

Galanin Radioimmunoassay

Adult, male Sprague-Dawley rats, obtained from the Charles River Laboratory (Kingston, NY), were used in these studies. The animals (275-300 g) were individually housed in stainless steel cages (43 X 22 X 19 era) in two temperature-controlled rooms (22 + 2°C) illuminated on a 12:12 h light:dark schedule, with lights on at 1130 h in one room and lights offat 1300 h in another room. They were maintained ad lib on Purina lab chow pellets and tap water. Animals were maintained in the laboratory for 4 weeks before sacrifice.

The MBH and MDH samples in Experiment 1 were homogenated in 0.5 N acetic acid and heated in a boiling water bath for 20 rain. Aliquots were taken for protein assay and then centrifuged at 12,000 rpm at 4°C for 15 min. The supernatant was lyophilized and stored frozen at -80°C. Micropunched tissue samples in Experiment 2 were expelled into 200 #1 of 2.0 N acetic acid and heat extracted for 20 min, then centrifuged at 12,000 rpm at 4°C for 15 min. Aliquots were taken from the supernatant, lyophilized, and stored frozen at -80°C, and the tissue precipitates were stored for protein assay. The extraction procedure and protein assay were modified for the micropunched tissue, because these small samples were not homogenated to minimize tissue loss. Because no comparisons are being made between the absolute values for the two experiments, these procedure differences should present little problem. Polyclonal antiserum generated in rabbits to synthetic rat GAL (11) was used. Radiolabeled [125I]GAL was purchased from Peninsula Lab. Inc. (Y7141, Belmont, CA). The assay procedure was similar to that previously used for hypothalamic GAL measurements (11), with protein content analyzed by the method of Lowry (29). This assay has a sensitivity of 4 pg, an EDs0 of 55 pg, and intra- and interassay coefficients of variation of 7% and 18%, respectively. The specific characteristics of the rat GAL radioimmunoassay and the extraction of peptide immunoreactivity have been described in previous reports (11-13). The properties of brain tissue subjected to heat extraction in dilute acetic acid followed by size exclusion chromatography have also been extensively described (11). Under such conditions, extracts from ME tissue eluted as a single peak that was not different from the synthetic rat GAL standard following Sephadex G-50 chromatography (Kay = 0.49). In hypothalamic tissue extracts, an additional, lower molecular weight peak, comprising approximately 20% total immunoreactivity, was seen with Sephadex G-50 separation (Kay = 0.67). In this report, additional characteristics of rat GAL-iike immunoreactivity in extracts following reverse-phase high pressure liquid chromatography (HPLC) are presented. Pools of acetic acid, heat-extracted hypothalamus, and ME tissues were applied to a Waters Model 6000 pump with solvent programmer (Milford, MA) with an analytical 5 ~z C-18 column (Microsorb, Rainin Inc., Woburn, MA). These extracts or synthetic standards

Procedures Two experiments were conducted. In Experiment 1, 48 rats were sacrificed at eight different time points in = 6/time point; hours 6, 12, 16, 17, 18 (dark onset), 19, 20, and 24] to examine any change in GAL levels within dissections of the medial hypothalamus. In Experiment 2, 60 animals were sacrificed at eight different time points [3-h intervals, n = 7-8/time point), hours 3, 6, 9, 12, 15, 18 (dark onset), 21, and 24] to examine levels of the GAL peptide in micropunched samples. (In Experiment 1, rats sacrificed during hours 16-24 were housed in one room, and the remaining rats were maintained in the other room. In Experiment 2, the rats were equally distributed such that those sacrificed during hours 3-12 were in one room and during hours 15-24 were in the other.) All animals at a single time point were sacrificed within a 5-min period. They were quickly taken from their cages and decapitated within the same room, and their trunk blood was collected in heparinized tubes. Brains were rapidly removed, microdissected as described below, and frozen at -80°C. Plasma was separated and frozen at - 8 0 ° C until assayed.

Microdissection Technique For Experiment 1, a 2-ram coronal section of the brain was made using a brain matrix (Activational Systems, Inc.; Warren, MI) prechilled on wet ice. With the ventral surface of the brain facing up in the matrix, a razor blade was inserted into a slot in the matrix around the middle of the caudal optic chiasm, and another razor blade was inserted into a slot 2 mm caudal to the first blade. The coronal section was removed from the matrix and laid onto a glass plate, prechilled on wet ice, with the rostral surface of the section facing up. The medial hypothalamus was

DIURNAL RHYTHM OF GALANIN

1439

were eluted with a triethylamine buffer pH 3.0 (50) in a linear acetylnitrile gradient (10-90%) over 60 min at a flow rate of 1 ml/min. Representative HPLC profiles for hypothalamic and ME extracts, with corresponding positions of the void volume and the retention time for the synthetic rat GAL standard, are illustrated in Fig. 1. Immunoreactivity eluted in a single peak with a retention time that was similar to the synthetic standard (ME = 38.8 + 1.1 min, n = 3; hypothalamus = 39.5 + 1.4 min, n = 4; standard = 39.7 _+ 0.5 min, n = 7). Recovery for applied sample for these 14 runs was 88 + 6%. The presence of one peak after HPLC chromatography vs. two after size exclusion chromatography perhaps represents the hydrophobic similarities of the two different molecular weight moeities.

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Circulating Hormone Determination Plasma CORT and insulin levels were assayed by radioimmunoassay similar to the method of Krey et al. (21) and the methods of Herbert et al. (14), respectively. Plasma aldosterone (ALDO) levels were assayed using the commercially available kits (TKAL2, Diagnostic Products Corporation, Los Angeles, CA), whereas plasma glucose levels were analyzed with a Beckman Glucose Analyzer No. 2.

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RESULTS

Experiment 1: Light/Dark Rhythm of GAL in Medial Hypothalamic Dissections Analyses of peptide levels in the hypothalamic dissections showed a clear light/dark rhythm in the MDH, F(7, 40) = 3.11, p < 0.05, but not the MBH, F(7, 40) = 1.62, p > 0.10 (Fig. 2). The MDH rhythm was characterized by relatively low basal levels throughout the light period and a rise in GAL concentrations,

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approximately 50% above baseline, that peaked during the sixth hour of the dark period (hour 24, p < 0.05). In the MBH, basal GAL levels were relatively stable across the light/dark cycle, ranging from 3.4 to 4.2 ng/mg protein. Measurements of circulating hormones showed clear rhythms for the adrenal steroids CORT, F(7, 40) = 6.83, p < 0.001, and ALDO, F(7, 40) = 3.36, p < 0.01 (Fig. 3, upper portion). These steroids increased dramatically between hours 12 and 18, 2-6 h before dark onset; they peaked around dark onset and then declined gradually to reach basal levels at light onset. A very different rhythm was detected for circulating insulin, F(7, 40) = 2.38, p < 0.05 (Fig. 3, lower portion), which remained at low levels, between 50-60 ttU/ml, throughout the light period. Dur-

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FIG. 2. Light/dark rhythm ofgalanin (GAL) levelsin medial basal (MBH) and medial dorsal (MDH) hypothalamus. Two points for hour 6 are the same data. *p < 0.05 relative to hours 16-19 by Duncan's New Multiple Range test.

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FIG. 1. Representative immunoreactivity profile for hypothalamic (left panel) and median eminence (fight panel) tissue extract following HPLC purification. The arrow denotes retention time for the synthetic rat galanin (GAL) standard. (Iio denotes void volume).

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FIG. 4. Light/dark rhythm ofgalanin (GAL) levels in the lateral portion of paraventricular nucleus (IPVN). Two points for hour 6 are the same data. *p < 0.05 relative to hours 3, 6, 15, and 18 by Duncan's New Multiple Range test.

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FIG. 3. Top: light/dark rhythm of circulating adrenal steroids, corticosterone (CORT) and aldosterone (ALDO); *p < 0.05 relative to hours 6 and 12 by Duncan's New Multiple Range test. Bottom: light/dark rhythm of circulating insulin (INS) and glucose (GLUC); *p < 0.05 relative to hours 6, 12, 16-18, and 20. Two points for hour 6 are the same data.

only two sites, namely, the IPVN (Fig. 4) and the SCN (Fig. 5). In the IPVN, F(7, 52) = 3.77, p < 0.01, similar to the M D H , G A L levels were relatively low during the 3 h before and also around dark onset, ranging from 32.9 to 38.5 ng/mg protein. However, after the first 3 h of the nocturnal period, G A L rose sharply to peak levels of 83.3 _+ 20.4 ng/mg protein by hour 21, twofold higher than the average baseline of the light period (p < 0.05), and then it returned to basal levels by the end of the

120-

ing the first hour of the nocturnal feeding cycle, insulin increased dramatically to an average peak level of 113 + 18 # U / m l (p < 0.05); it then significantly declined over the next 5 h to around 80 u U / m l (p < 0.05), and dropped further to reach basal levels by the end of the dark period. Although relatively stable throughout the cycle, F(7, 40) = 1.80, p > 0.10, circulating glucose tended to drop between hour 19 and 20, from 137 _+ 6 to 124 _+ 7 mg/dl, immediately after the peak in insulin. Correlational analyses relating these hormones to hypothalamic GAL levels failed to reveal any relation between the adrenal steroids and GAL, in either the M D H or MBH, or at any time of the diurnal cycle, However, analyses relating insulin and G A L in the M D H across all time periods revealed a significant inverse relationship (r = -0.32, p < 0.05). When examined separately at each of the eight time points, this association between G A L and insulin was strongest during the sixth hour of the natural feeding cycle (r = -0.84, p < 0.05). This relation was not detected for G A L in the MBH. Nor was any association seen between hypothalamic G A L and circulating glucose levels.

Experiment 2: Light~Dark Rhythm of GAL in Discrete Hypothalamic Nuclei Analyses of peptide levels in eight different hypothalamic areas across the light/dark cycle revealed significant rhythms in

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FIG. 5. Light/dark rhythm of galanin (GAL) levels in the suprachiasmatic nucleus (SCN). Two points for hour 6 are the same data. *p < 0.05 relative to hour 3, 12, 15, and 24 by Duncan's New Multiple Range test.

DIURNAL RHYTHM

OF GALANIN

1441

TABLE 1 DIURNAL RHYTHM OF GAL LEVELS (ng/mg PROTEIN) IN DISCRETE HYPOTHALAMIC AREAS Hypothalamic Areas Time of Day (h) 0300 0600 0900 1200 1500 1800 2100 2400

ME

[8] [8] Light onset [7] [7] [7] [8] Dark onset [7] [8]

110 170 161 164 132 135 242 143

mPVN

± 13 ± 38 ± 26 ___23 _+ 42 ± 39 ± 40 ± 14

35.1 31.5 19.9 19.8 30.0 23.0 18.1 18.3

DMN

+ 13.2 ___ 2.5 + 6.3 + 5.0 _+ 13.2 ± 10.8 _+ 3.2 ___ 1.5

77.9 51.0 81.8 52.5 45.4 58.3 46.6 55.1

+ 25.0 ± 26.5 _+ 7.9 _+ 17.9 _+ 18.9 _+ 18.3 + 12.7 ± 15.1

ARC 16.8 15.6 15.8 13.8 14.6 17.2 14.8 14.5

SON

_+ 3.7 _+ 5.1 +_ 2.4 _+ 5.0 + 2.7 ± 5.0 + 3.4 ± 2.7

9.89 12.7 10.5 18.9 13.2 13.0 14.7 20.3

+ 3.0 _+ 2.9 _+ 1.1 ± 8.4 _+ 1.4 _+ 1.4 + 3.8 _+ 8.2

MPO 88.1 79.4 65.6 77.2 92.2 60.1 70.9 72.2

+ 10.2 _+ 7.1 ± 4.8 ± 10.5 + 14.1 + 6.8 _+ 3.6 + 9.9

Values given are means + SEM, with the number of rats given in brackets. Abbreviations: ME, median eminence; mPVN, medial paraventricular nucleus; DMN, dorsomedial nucleus; ARC, arcuate nucleus; SON, supraoptic nucleus; MPO, medial preoptic area.

feeding cycle. A smaller increase d u r i n g the mid-light period between hours 6 a n d 9, to 55 __ 9.3 n g / m g protein, failed to reach statistical significance. In the M E (Table 1), there was a t e n d e n c y for a peak (242 +_ 40 n g / m g protein) at 3 h after dark onset, F(7, 52) = 1.95, p < 0.10, similar to that seen in the 1PVN. This pattern is in contrast to t h a t observed in the SCN (Fig. 5). In this nucleus, a b i m o d a l r h y t h m of G A L was detected, F(7, 52) = 6.43, p < 0.01. Peak levels, o f approximately 70 n g / m g protein, were reached at the transitions from light to dark ( h o u r 18, p < 0.05) a n d from dark to light ( h o u r 6, p < 0.05). No other h y p o t h a l a m i c areas exhibited a significant t e m p o r a l r h y t h m of G A L (Table 1). Analyses of circulating h o r m o n e s yielded results similar to those o b t a i n e d in E x p e r i m e n t 1 (Table 2). Circulating C O R T , F(7, 52) = 3.15, p < 0.01, a n d ALDO, F(7, 52) = 4.39, p < 0.01, levels rose d u r i n g the several h o u r s before dark onset, peaking at lights out, a n d gradually declined over the next 6 h of the dark. In contrast, insulin increased after the onset of feeding a n d declined after the m i d - d a r k period, F(7, 52) = 1.85, p < 0.10. Once again, circulating glucose levels were relatively stable, F(7, 52) = 1.99, p < 0.10, although a decline after the third hour, from 151 +_+_9 to 138 _+ 3 mg/dl, was evident following the peak in insulin. As in Experiment 1, correlational analyses revealed a n inverse relationship between G A L levels a n d insulin across the light/

dark cycle. In this experiment, a similar pattern was detected in the 1PVN a n d ME, where a small b u t significant inverse relationship across all t i m e periods was obtained (r = - 0 . 3 2 a n d r = - 0 . 3 1 , p < 0.05, respectively). Analyses at specific time points showed this association to be significant during the feeding cycle, at the m i d - d a r k h o u r for l P V N (r = - 0 . 7 5 , p < 0.05) a n d M E (r = - 0 . 8 3 , p < 0.05) a n d the n i n t h h o u r for the IPVN (r = - 0 . 8 0 , p < 0.05). However, it was not detected in the SCN or in any other h y p o t h a l a m i c area examined. T h e r e was n o apparent relation between G A L levels in any h y p o t h a l a m i c area a n d either the adrenal steroids or glucose.

DISCUSSION This report suggests the existence o f two distinct r h y t h m s for h y p o t h a l a m i c G A L levels across the light/dark cycle. O n e r h y t h m , detected exclusively in the area of the SCN, is characterized by b i m o d a l peaks of G A L levels a r o u n d the onset o f the dark a n d light periods. T h e second r h y t h m shows a single peak o f G A L towards the middle o f the n o c t u r n a l feeding cycle, specifically between the third a n d sixth hour. This r h y t h m is evident in the dorsal region of the medial h y p o t h a l a m u s a n d is localized specifically to the lateral portion o f the PVN. W h e t h e r these peaks in G A L levels reflect changes in peptide synthesis, release, or degradation c a n n o t be d e t e r m i n e d at this time.

TABLE 2 DIURNAL RHYTHMS OF CIRCULATING HORMONES AND GLUCOSE Time of Day (h) 0300 0600 0900 1200 1500 1800 2100 2400

[8] [8] Light onset [7] [7] [7] [8] Dark onset [7] [8]

CORT (t~g%)

ALDO (pg/ml)

Insulin (t~U/ml)

Glucose (mg/dl)

5.3 _+ 2.8 0.3 _ 0.0 0.7+0.0 0.7 + 0.5 8.2 ___3.5 18.5 +_ 2.9* 11.6 + 4.6 9.2 +_ 4.7

121 _+ 28 86 ± 27 29_ 8 97 + 17 163 + 21 228 _+ 18" 144 ± 43 146 _+ 28

64.9 + 10.7 61.9 + 7.3 48.7+_ 5.2 57.4 +__ 7.4 45.3 + 4.4 56.2 _+ 13.8 81.7 + 13.1 78.8 +_ 6.2

141 _+ 3 147 + 2 151_+3 155 +_ 4 155 _ 4 153 _-!-4 151 + 9 138 _+ 3

Values given are mean _+ SEM, with the number of rats given in brackets. * p < 0.05 by relative to hours 6, 9, and 12 by Duncan's New Multiple Range test.

1442

Bimodal Rhythm of GAL in the SCN This bimodal pattern for GAL levels in the SCN, exhibiting peaks around the transitions between dark and light, has to our knowledge been detected with only one other peptide, namely, NPY (6,16,37). The change in light stimulation is critical in producing this NPY rhythm within the SCN, as evidenced by its loss during constant lightness or darkness (37). Moreover, the SCN is generally unique in exhibiting this pattern, reflecting the function of this nucleus as a circadian pacemaker and its anatomical connections with the lateral geniculate nucleus and retino-hypothalamic projection (35). In the case of GAL, the SCN has a rich peptide innervation but few GAL cell bodies (31). Although the origin of these GAL terminals is unknown, the bimodal pattern detected in the SCN indicates that GAL, like NPY, may also be involved in conveying visual information to the hypothalamus during the light/dark transition (37). This rhythm, which exhibited no relation to circulating hormones, may additionally originate from an endogenous pacemaker. These bimodal peaks were not detected in the MBH dissection, which contained the SCN, presumably because they were diluted by the high GAL levels contributed to the MBH by the ARC and ME.

Mid-Dark Rise in GAL in the lPVN The second rhythm detected in this study, a unimodal peak in GAL levels towards the middle of the dark period, is distinctive. It is apparent in the MDH, which contains the PVN as well as the DMN, and through analyses in micropunched tissue, it is found to be localized to the lateral portion of the PVN, where the rhythm is further amplified. Because the third hour was not sampled in the analysis of MDH GAL, the exact timing of this unimodal peak and the possibility that the MDH and IPVN peaks actually reflect the same rhythm require further investigation. The conclusion to be drawn at this time is that a unimodal peak occurs in these areas during the first half of the nocturnal feeding cycle, between the third and sixth hours. The relationship between this unimodal rhythm within the 1PVN and the bimodal rhythms of the SCN also needs to be examined. Considering the neuroanatomical connections between the SCN and PVN (52), it is likely that these two rhythms are closely related. Lesion studies also support an association between the SCN and diurnal rhythms of food-related processes that may, in part, be controlled through the PVN (4).

Dissociation of lPVN GAL and CORT From the measurements of CORT and ALDO, there appears to be little relation between this peptide in the 1PVN and these circulating steroids. These steroids and IPVN GAL exhibit very different diurnal rhythms (Fig. 4, Table 2). Moreover, they have been further dissociated through studies in ADX rats, which fail to reveal any impact of CORT on GAL peptide or mRNA levels in the PVN (3). Thus, GAL in the PVN, whether in its cell bodies or terminals, appears to function independently of circulating CORT and to have little relation to this steroid's nocturnal peak. As to the impact of GAL on circulating CORT, there is evidence to indicate that PVN GAL administration has an inhibitory effect on the hypothalamo-pituitary-adrenal axis in intact animals (15,19,45). Thus, the rise in 1PVN GAL a few hours into the feeding cycle may actually reflect its possible role in reversing the peak in CORT at dark onset.

Relation Between lPVN GAL and Circulating Insulin In contrast to the adrenal steroids, there appears to be some relation between circulating insulin and endogenous GAL in the

AKABAYASH1 ET AL. PVN. This is revealed by a significant inverse correlation between peptide and hormone levels across the diurnal rhythm, detected in both Experiments 1 and 2, and by their distinctive, sequential rhythms after the onset of feeding. Shortly after the sharp rise in insulin during the first meal of the feeding cycle, there is a significant decline in hormone levels that occurs simultaneously to the peak in IPVN GAL around the mid-dark period. Although any causal relationship between this brain peptide and hormone remains to be demonstrated, other evidence suggests that high levels of GAL may reflect greater functional activity. Both hypothalamic (45) and pancreatic (9) administration of GAL has been shown to inhibit the secretion of insulin. Moreover, GAL levels in the pancreas are reduced under conditions of hyperinsulinemia (46), whereas an enhancement of GAL immunoreactivity in a subpopulation of PVN neurons has been described in diabetic rats (53).

Relation Between lPVN GAL, Insulin, and Fat Intake The significance of this relationship between 1PVN GAL and insulin may be further understood through studies of GAL's stimulatory effect on feeding behavior (24,43). In addition to inhibiting insulin secretion (45), hypothalamic or ventricular administration of this peptide has been shown to stimulate feeding and to produce a preferential increase in the ingestion of fat (22,23,44). Moreover, measurements of endogenous GAL or its gene expression in the PVN show a strong positive relation between this peptide and the animal's preference for fat, along with an inverse association between circulating insulin and both PVN GAL and spontaneous fat intake (26,48). This association between GAL and fat ingestion is supported by studies of the diurnal rhythms of eating patterns. These behavioral analyses show fat intake to remain generally low during the initial 2-3 h of the feeding cycle and then to rise sharply between hours 3-6 of the dark (38,39). This mid-dark peak coincides with the peak in GAL levels and the decline in insulin observed in the present report, and it is consistent with other evidence showing GAL to be more effective and selective in stimulating fat intake during the later hours of the dark period (43). Once again, whether these simultaneous events, changes in peptide and hormone in association with a behavioral response, are causally related remains to be established.

Diurnal Rhythm of Other Peptides Perhaps most remarkable are the dramatic differences detected between the two peptides, GAL and NPY, and their rhythms within the PVN where they are believed to act to stimulate feeding (25). Whereas GAL is linked to patterns of fat ingestion and to increased activity in the mid-dark period, endogenous NPY in the PVN, specifically its medial portion, has been associated with the rise in carbohydrate ingestion that is characteristic of the beginning of the dark period (25,27,38,39). This peptide preferentially stimulates the ingestion of carbohydrate (40), and its levels in the medial portion of the PVN, as well as ARC, are positively correlated with natural appetite for carbohydrate (17). Further, levels of this peptide or its gene expression rise just before or around the time of dark onset, in association with CORT as well as the onset of carbohydrate feeding (2,16). Thus, with NPY as well as GAL, it is suggested that the diurnal rhythms of these peptides evident in hypothalamic nuclei, such as the PVN, reflect their close relation to the natural feeding process and to associated changes in circulating hormones. Another neuropeptide, vasopressin (AVP), has also been shown to exhibit a diurnal rhythm within the hypothalamus,

DIURNAL RHYTHM OF GALANIN

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which differs from those of G A L and NPY. In the SCN, levels of AVP are highest during the middle hours of the light period and lowest during the mid-dark period (10,47,49). This pattern clearly distinguishes this peptide from G A L in the SCN, which peaks at the light/dark transitions. Although limited evidence exists in the PVN, several studies have failed to reveal any distinct light/dark rhythm for AVP in this nucleus, as well as in the SON (49), in contrast to the rhythm detected for GAL. Whereas AVP and G A L are known to coexist in neurons of the hypothalamus (31), including those within the PVN and SON, this evidence appears to distinguish these two peptides and suggests that they

may function differently and be differentially regulated in relation to the diurnal cycle. ACKNOWLEDGEMENTS This research was supported by USPHS Grant MH43422 (S.F.L.), by a fellowship from The Naito Foundation (A.A.), and by a fellowship from Conselho Nacional de Pesquisa (CNPq) and P.I.C.D., Brazil (C.T.B.V.Z.). The authors thank Ms. Yim Dam of the Obesity Core Center at St. Luke's-Roosevelt Hospital for assistance with the insulin and glucose determinations, and Ms. Jesline Alexander and Mr. HeeJin Chae of The Rockefeller University for their excellent technical assistance.

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