Neuroscience Letters, 114 (1990) 339-344 339 Elsevier Scientific Publishers Ireland Ltd.

NSL 06968

Neuropeptide Y expression in rat brain: effects of adrenalectomy

R o g e r G. D e a n I a n d B. D o u g l a s W h i t e 2

1Department of Animal and Dairy Science and 2Department of Foods and Nutrition, University of Georgia, Athens, GA 30602 (U,S.A.)

(Received 15 November 1989; Revised version received 5 February 1990; Accepted 28 February 1990)

Key words: Neuropeptide Y; Glucocorticoid; Gene expression; Response element

Neuropeptide Y (NPY) messenger RNA was measured by hybridization of mRNA from the cortex, hippocampus, hypothalamus and striatum of rat brains. Adrenalectomized rats showed lowered level of NPY message in the striatum. A similar decline was found in the hypothalamus, while the cortex and hip- pocampus were unchanged. Levels of NPY message per unit total RNA were about the same for hypothal- amus, cortex and striatum and about 50% less for hippocampus. Adrenalectomized rats that received re- placement corticosterone had levels of NPY message that had returned to the levels found in rats receiving sham operation. Response elements consistent with our findings are reported in the NPY genomic se- quence,

Neuropept ide Y (NPY) is abundant ly expressed th roughout much o f the central

and peripheral nervous system. N P Y is thought to act as a neurotransmit ter and pro- duces a variety o f effects when administered exogenously. It is known to increase vasoconstr ict ion [9, 10], decrease glucose-stimulated insulin release [17], affect lutei-

nizing ho rmone secretion through gonadot rop in releasing ho rmone [15] and shift cir-

cadian rhythms [1]. One o f its most dramatic effects is to induce food intake in

satiated animals when administered intracerebroventricularly [6, 18]. N P Y ' s broad

expression, relative abundance, diverse effects and highly conserved sequence [5] leads to the conclusion that it may have impor tant functions. However, in spite o f intensive research, the mechanisms of expression, release and receptor binding are not well unders tood and the functions o f N P Y are not known.

A key to unders tanding the functions o f N P Y lies in finding mechanisms and fac-

tors that regulate N P Y expression. Very little is currently known about N P Y expres- sion. Tissue distribution studies indicate cell specificity generally restricted to neural

tissues [14, 16] and a study o f N P Y expression in cell culture [14] has indicated that

Correspondence." R. Dean, Dept. of Animal and Dairy Science, Livestock-Poultry Building, University of Georgia, Athens, GA 30602, U.S.A.

0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

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phorbol ester, cAMP, calcium ionophore and glucocorticoids may act as inducers in a cell specific way. We have focused our work on the expression of NPY in the brain and its regulation by adrenal factors by measuring NPY mRNA levels in hypothala- mus, striatum, hippocampus and cortex of adrenalectomized (ADX) rats. In addi- tion, we have examined sequences [16] in the 5' control area through the first intron of NPY in an effort to identify sequences that may serve to regulate NPY in conjunc- tion with adrenal glucocorticoids.

Sprague-Dawley rats were either bilaterally adrenalectomized (with or without corticosterone replacement) or given sham operations. For replacement, ADX rats received, immediately after adrenalectomy, osmotic pump implants (Alzet, 2001 Palo Alto, CA) working at a rate of 38.25 pg/h (corticosterone). ADX and sham rats were implanted with control pumps containing vehicle (peg) alone. All rats were given ac- cess to Purina lab chow and water (0.9% saline for adrenalectomized rats) and main- tained on a 12 hour light-dark cycle. Rats were decapitated (09.00-10.00 h) on the fourth day after adrenalectomy, trunk blood was collected for serum corticosterone determination and the cortex, hippocampus, hypothalamus and striatum were dis- sected [12] and homogenized in RNAzol (Cinna/Biotecx labs, Friendswood, TX). RNA was purified according to manufacturers instructions. Total RNA was electro- phoresed on formaldehyde gels and blotted [11] on Gene Screen Plus or dot blotted (Dupont Wilmington, DE) and dried at 80°C in a vacuum oven. For replacement studies, hypothalamus and striatum from groups of 10 rats representing ADX (with and without replacement) and sham were blotted on a single membrane. An addi- tional northern was done in parallel to insure hybridization and wash stringency. Membranes were prehybridized for 1 h and hybridized in phosphate buffer (0.25 M NaPO4, 0.25 M NaC1, I mM EDTA) and 50% formamide with 7% SDS at 62°C over- night [3] using a rat NPY mRNA sequence [2]. Membranes were then washed and exposed to X-ray film for 2-4 days. Image analysis was done on autoradiographs to determine the amount of bound probe. These values were corrected for the amount of RNA loaded on the gel or dot blot, which was also determined by image analysis or OD 260. For northern analysis, brain areas from paired rats (ADX-sham) were compared on each blot. The percent change in mRNA due to adrenalectomy was determined for each tissue on each membrane. This was calculated as the corrected mRNA for the adrenalectomized rat minus the corrected mRNA value for the sham rat divided by the smallest value. The mean percent change in NPY message levels for each tissue was tested against the hypothesis that the change was equal to zero [19}. Adrenalectomies were evaluated by measuring serum levels of corticosterone using an RIA kit (Research Systems Labs, Carson, CA). Analysis and searches for DNA sequences were done using Intelligenetics software programs.

The hippocampus, striatum, hypothalamus and cortex are areas of the brain known to have high levels of NPY message [ 14, 16]. Our results showed levels of NPY message per unit of total RNA to be about the same in striatum, cortex and hypo- thalamus and about 50% less in the hippocampus (data not shown). These results agree with the previous reports except that we found relatively higher levels of NPY message in the hypothalamus. Adrenalectomy resulted in lower levels of NPY

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Fig. I. NPY expression levels in adrenalectomized and sham operated rats. A: autoradiograph showing cortex (COR, lane 1, 2); hippocampus (HIP, lane 3,4); hypothalamus (HYP, lane 5, 6) and striatum (STR, lane 7, 8) as paired rats. Adrenalectomized rat tissues are found in lanes 1, 3, 5, and 7 with sham-operated rat tissues in lanes 2, 4, 6 and 8. B: image analysis was performed on both the ethidium bromide gel nega- tives and nine autoradiographs to determine differences. NPY levels are given in dpm. Serum levels of glucocorticoid in the adrenalectomized rats was 2.8 ng/ml and in the shams was 105.5 ng/ml.

mRNA in the hypothalamus and striatum. In comparisons based on northern analysis, levels of NPY message in the striatum averaged 3-fold lower in ADX rats than in shams. This difference was statistically significant (P<0.05) (Fig. l). The decrease in the hypothalamus was greater than 3-fold but the data did not reach statistical significance (P=0.08). Cortex and hippocampus showed only negligible changes. This data suggest that NPY expression in the striatum and hypothalamus is depen- dent on adrenal factors, such as corticosterone, while expression in the cortex and hippocampus is relativley unaffected by adrenal factors.

To demonstrate the involvement of corticosterone, a second experiment was done, using hypothalamus and striatum, in which half of the ADX rats received replace- ment corticosterone. Fig. 2 compares the levels of NPY mRNA found in hypothala- mus and striatum for sham, ADX and corticosterone replacement (ADX+ Cort). In the striatum a significant difference is observed (P<0.05) between ADX and sham rats as was found in the northern analysis. Replacement with corticosterone increases NPY levels to the range found in sham rats. In the hypothalamus a decline in NPY mRNA is observed in ADX rats, however, as with the northern analysis, this data did not reach significance. Corticosterone replacement for the hypothalamus gives NPY levels indistinguishable from the sham control group.

Our data indicate that a loss of corticosterone results in lowered levels of NPY message in striatum and a similar trend in hypothalamus. The effects of adrenalec- tomy seen in hypothalamus may be more pronounced if specific nuclei were examined rather than the whole tissue. The magnitude of the change in NPY is probably most accurately reflected in our dot blot experiments where the averages of control and

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Fig. 2. NPY expression in ADX rats receiving replacement corticosterone. A: results of image analysis of dot blot densities for hypothalamus and striatum. Ten rats were used in each group (ADX, ADX- + Cort, Sham). Within each figure, different letters represent significant differences. B: levels of serum cor- ticosterone in ADX, sham control and corticosterone-replaced rats.

ADX groups are compared. In the northern analysis comparisons can only be made within each membrane which may magnify differences and variation. Our finding agrees with previous cell culture studies that indicate NPY expression is controlled in part by glucocorticoids in a cell specific manner [14]. This finding also parallels work indicating an abundance of glucocorticoid receptors (GR) in the hypothala- mus, hippocampus, and cortex [7] and colocalization of NPY and glucocorticoid receptors in neurons of the hypothalamus [13]. The decline in the striatum is some- what surprising since relatively little GR is found in this tissue and GR has been reported not to be colocalized with NPY neurons [13].

Our data also indicates a pronounced difference in the regulation of NPY in different brain areas. Hippocampus and cortex both of which have high levels of NPY message are unaffected by adrenalectomy while both hypothalamus and stria- turn are affected. The molecular mechanisms controlling tissue specificity are complex and therefore glucocorticoids do not completely control NPY expression. However, the action of glucocorticoids on the expression of NP¥ is likely to be direct since gene responsiveness to glucocorticoids is mediated by glucocorticoid response elements (GRE) [4]. Inspection of the published sequence for NPY [16] reveals two putative GREs (5'-CCCTGAGCTTGTTCT-3' at +154 to +168) and (5'-

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CCCTCCACCTGTCCT-3' at + 229 to + 243) located in the first intron of NPY. The 3' ends of these GREs are completely homologous with the highly conserved GRE 3' consensus heximer 5'-TGTYCT-3'. GREs having the 3' consensus heximer were not found 5' of the promoter; however, a 9 base pair triple repeat at -501 to -475 containing the sequence (5'-GGTCCAGTAGGTCCA-3') was found whose 5' end closely corresponds to the 5' heximer of the GRE consensus sequence (5'- GGTACA-3') and whose 3' end corresponds to the sequence (5'-CGTCCA-3') recently found by Drouin [8] to bind glucocorticoid receptors. While the sequence found by Drouin acts as a negative regulator it is not due to the sequence but rather due to overlap and interference with the promoter. Since the GRE found in NPY is well upstream of the promoter it may act as a positive element.

In summary our data indicates that NPY expression in the striatum and possibly the hypothalamus is regulated in part by corticosterone, and that the mechanism of this control may be related to glucocorticoid response elements found in the NPY gene. The regulation shows tissue specificity in that after adrenalectomy NPY expres- sion is not affected in the hippocampus or cortex.

This work was supported by Public Health Service Grant HD-22226 from the Na- tional Institutes of Health and the Agricultural Experiment Station of the University of Georgia. We would like to thank Janet Allen for providing the NPY-5 mRNA probe.

1 Albers, H.E. and Ferris, C.F., Neuropeptide Y: role in light-dark cycle entrainment of hamster circa- dian rhythms, Neurosci. Lett., 50 (1984) 163-168.

2 Allen, J , Novotny, J., Martin, J. and Heinrich, G., Molecular structure of mammalian neuropeptide Y: analysis by molecular cloning and computer-aided comparison with crystal structure of avian homologue, Proc. Natl. Acad. Sci., U.S.A., 84 (1987) 2532-2536,

3 Amasino, R.M., Acceleration of nucleic acid hybridization rate by polyethylene glycol, Anal. Bio- chem., 152 (1986) 304-307.

4 Beato, M., Gene regulation by steroid hormones, Cell, 56 (1989) 335-344. 5 Blomqvist, A. and Larhammar, D., Evolutionary conservation of CCK and neuropeptide Y: cDNA

clones for chick CCK and gold-fish NPY, Soc. Neurosci. Abstr., 15 (1989) 839. 6 Clark, J.J., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Neuropeptide Y and human pancreatic poly-

peptide stimulate feeding behavior in rats, Endocrinology, 115 (1984) 427-429. 7 De Kloet, E.R. and Reul, J.M., Feedback action and tonic influence of corticosteroids on brain func-

tion: a concept arising from the heterogeneity of brain receptor systems, Psycboneuroendocrinology, 12 (1987) 83-105.

8 Drouin, J., Charron, J., Gagner, J., Jeannott, L., Nemer, M., Plante, R. and Wrange, O., Pro-Opiome- lanocortin gene: a model for negative regulation of transcription by glucocorticoids, J. Cellul. Bio- chem., 35 (1987) 293-304.

9 Emson, P.C. and Dequidt, M., NPY - A new member of the pancreatic polypeptide family, Trends Neurosci., 7 (1984) 31-35.

10 Edvinsson, L., Emson, P., McCulloch, J., Tatemoto, K. and Uddman, R., Neuropeptide Y: immuno- cytochemical localization to and effect upon feline pial arteries and veins in vitro and in situ, Acta Physiol. Scand., 122 (1984) 155-163.

11 Fourney, R., Miyakoshi, J., Day, R. and Patterson, M., Northern blotting: efficient RNA staining and transfer, Focus, 10 (1988) 5-7.

12 Glowinski, J. and Iversen, L.L., Regional studies ofcatecholamines in the rat brain I, J. Neurochem., 13 (1966) 655-669.

344

13 Harfstrand, A., Cintra, A., Fuxe, K., Aronsson, M., Wikstrom, A., Okret, S., Gustafsson, J. and Agnati, L., Regional differences in glucocorticoid receptor immunoreactivity among neuropeptide Y immunoreactive neurons of the rat brain, Acta Physiol. Scand., 135 (1989) 3-9.

14 Higuchi, H., Yang, H. and Sabol, S., Rat neuropeptide Y precursor gene expression, J. Biol. Chem., 263 (1988) 6288-6295.

15 Kalra, S.P. and Crowley, W.R., Norepinephrine like effects of neuropeptide Y on LH release in the rat, Life Sci., 35 (1984) 1173 1176.

16 Larhammar, D., Ericsson, A. and Persson, H., Structure and expression of the rat neuropeptide Y gene, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 2068-2072.

17 Moltz, JH. and McDonald, J., Neuropeptide Y: Direct and indirect action on insulin secretion in the rat, Peptides, 6 (1985) 1155 1159.

18 Stanley, G., Kyrkouli, S., Lampert, S. and Leibowitz, S., Neuropeptide Y chronically injected into hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity, Peptides, 7 (1986) 1189 1192.

19 Zar, J.H., Biostatistical analysis. Prentice Hall, New Jersey, 1974, 86 pp.