Neuropeptides (1998) 32 ( 1 ), 63-66 @ Harcourt Brace and Company Ltd 1998

Neuropept ide Y in the magnocel lu lar hypotha lamic neurons of obese Zucker rats

S. Fetissov, S. Nicola'l'dis Groupe de Neurobiofogie des ReguJations, IESGCA de Dijon, CNRS UPR 9054, College de France, 11 place Marcelin Berthelot 75231, Paris Cedex 05, France

Summary The obesity syndrome in Zucker rats is associated with an elevated neuropeptide Y (NPY) content in the hypothalamus. It is recognized that the axons of NPY-ergic neurons arriving from the arcuate nucleus and the midbrain are the major source of NPY in this area. In magnocellular hypothalamic neurosecretory neurons (MCN) of normal rats NPY is expressed only in response to hyperosmotic stimulation. This study used immunohistochemistry with colchicine treatment aimed at MCN to compare NPY localization in obese (fa/fa) and in lean (Fa/Fa) Zucker rats. It was found that the obese (fa/fa), in contrast to the lean (Fa/Fa) Zucker rat, displays NPY immunoreactivity in numerous MCN of the paraventricular, supraoptic as well as accessory neurosecretory nuclei. This finding suggests local synthesis of NPY in the MCN of obese (fa/fa) rats and involvement of hydro-osmotic disorders in the Zucker syndrome of obesity.

I N T R O D U C T I O N

The hypothesis that elevated neuropeptide Y (NPY) expression in the hypothalamus is involved in the pathogenesis of hyperphagia and obesity is generally accepted.~ This hypothesis is partly based on data regard- ing elevated NPY production in the Zucker (fa/fa) rat, an animal model of genetic obesity. 2-~ Indeed, in obese (fa/fa) Zucker rats, when compared with their lean litter- mates, many hypothalamic regions - such as the supraoptic nucleus (SON), median and lateral preoptic area and also the regions directly involved in food intake regulation, such as the paraventricular (PVN) and the arcuate (ARC) nuclei - show a significantly increased NPY concentration? Moreover, an increased NPY secre- tion in the PVN 7 and an increased preproNPY mRNA content in whole hypothalamus and particularly in the ARC have been demonstrated. 8

In the hypothalamus of normal rats, the neurons of ARC are the major source of NPY. 9 These neurons project to the PVN, 1° the median eminence (ME) and the neuro-

Received 26 June 1997 Accepted 22 A ugust 1997

Correspondence to: Dr S. Fetissov, Department of Surgery, SUNY HSC Syracuse, 750 E. Adams St, Syracuse, NY 13210, USA. Tel: 315 464 62 77, Fax: 315 464 47 71.

intermediate lobe of the pituitary. ~j The PVN has a dense NPY-ergic innervation, not only originating from the ARC, but also from the midbrain. ~2 No NPY is found in the hypothalamic magnocellular neurons (MCN) of nor- real rats? Thus, the increased hypothalamic NPY level in obese (fa/fa) Zucker rats was generally considered to originate from the ARC.

In normal rats, NPY and tyrosine hydroxylase (TH), as well as their mRNA, are expressed in MCN during hyper- osmotic stimulation produced by a 2-week replacement of water with a 2% NaCI solution or by water depriva- tion. ~-~6 A recent study demonstrated that TH was expressed in MCN of obese (fa/fa) Zucker ratsY Following on from these data, this study tested the possi- ble localization of NPY in the MCN of the hypothalamus in obese Zucker rats by means of an immunohisto- chemical study.

MATERIALS AND M E T H O D S

To block the axonal transport, intracerebroventricular colchicine was administered (50 gg in 5 gl of 0.9% NaCI in each lateral ventricle) to 6-month-old male lean (Fa/Fa) and obese (fa/fa) Zucker rats (n = 6). Twenty-four hours later, the rats were aneasthetized and then per- fused intracardially (40/0 paraformaldehyde with 0.050/0 glutaraldehyde). Brains were immersion-fixed in the

63

64 Fetissov & Nicolafdis

Fig. 1 lmmunohistochemical NPY identification on frontal sections from the hypothalamus of obese (fa/fa) (A, C, E, F) and of lean (FaiFa) (B, D) Zucker rats. NC, nucleus circularis; OC, optic chiasm; PVN, paraventricular nucleus; SON, supraoptic nucleus; rSON, retrochiasmatic SON; Ill, third brain ventricle. Scale bar (A-F) = 50 pm.

Neuropep tides (1998) 32(i), 63-66 0 Harcourt Brace and Company Ltd 1998

NPY in hypothalamic neurons of obese Zucker rats 65

same fixative, rinsed in 0.05 M Tris-buffered saline (TBS), pH 7.4, and kept in 20% sucrose in TBS for at least 24 h. Brains were cut into 30-gm thick frontal sections on a cryostat. The floating sections were processed for immunohis tochemis t ry with NPY anfisera (Sigma; 1:1000) according to the routine avidin-biotin-peroxi- dase complex (Vector) procedure. TM They were reacted with 3.3 '-diaminobenzidine and examined under a Leitz photomicroscope.

RESULTS

It was found that major parts of the populat ion of MCN in obese (fa/fa) rats displayed NPY immunoreact iv i ty (NPYi). NPYi was observed in MCN of the SON (Fig. 1A), of the PVN (Fig. 1C), of the accessory neurosecre tory hypothalamic nuclei such as the nucleus circularis (Fig. IF) and of the retrochiasmatic parts of the SON (Fig. 1E) as well as in the individual MCN (Fig. 1F). NPYi was also found in typical hypothalamic areas for NPY cell b o d y localization such as the ARC. The MCN NPYi had a typical cytoplasmatic character of distribution and was observed t h rough all parts of SON and PVN wi thout pref- erence for oxytocinergic or vasopressinergic cell localiza- tion. A marked increase of NP¥i fibres in these nuclei and in the whole hypotha lamus was also observed in obese (fa/fa) rats compared with lean (Fa/Fa) rats. In contrast to obese (fa/fa) rats, no NPYi reaction was observed i n M C N of lean (Fa/Fa) rats (Fig. 1B, D).

DISCUSSION

These findings clearly demonstra te tha t in obese (fa/fa) rats, NPY is localized no t only in the typical hypothala- mic areas, bu t also in the entire magnocel lular system which can undergo neurochemical modification during osmotic stimulation. 13-j6 In obese (fa/fa) Zucker rats this st imulation may be related to hydro-minera l abnormali- ties such as increased plasma osmolality and diuresis. 3,19 With regard to the physiological funct ion of NPY in MCN, it is unclear whe ther it has the same action on food and water intake regulation - which is observed at the postsynaptic level in the PVN - because NPY in MCN m a y attain target cells in the neurohypophysis . However, NPY in MCN can have a synergistic action with the NPY- ergic neurons which are localized in the ARC and project to the ME and the neurohypophysis . NPY synthesis in the MCN has also been shown in Brattleboro rats, 2o that also display increased plasma osmolality. The comparison of the peculiarity of NPY expression with nutri t ional and hydro-minera l balance in the obese (fa/fa) Zucker and Brattleboro rats can help to explain the physiological or pathological role of NPY expression in the MCN of the hypothalamus.

Overexpression of NPY in the ARC together with what seems to be a neo-expression in whole MCN of obese (fa/fa) Zucker rats could consti tute the pr imary patho- genesis of the Zucker syndrome that includes hydro- osmotic abnormalit ies in addit ion to hyperphagia and obesity.

REFERENCES

1. Frankish H M, Dryden S, Hopkins D, Wang Oo Williams G. Neuropeptide Y, the hypothalamus, and diabetes: insights into the central control of metabolism. Peptides 1995; 16: 757-771.

2. Beck B, Buffet A, Nicolas J-P, Buffet C. Hypothalamic neuro- peptide Y (NPY) in obese Zucker rats: implications in feeding and sexual behaviors. Physiol Behav 1990; 47: 449-453.

3. Bray G A. The Zucker-fatty rat: a review. Fed Proc 1977; 36: 148-153.

4. Brif D J, Sipols A J, Woods S C. Intaventricular neuropeptide Y injections stimulate food intake in lean, but not obese Zucker rats. Physiol Behav 1992; 51:1105-1110.

5. McKibbin P E, Cotton P E, McMillan Set al. Altered neuropeptide Y concentration in specific hypothalamic regions of obese (fa/fa) Zucker rats - possible relationship to obesity and neuroendocrine disturbances. Diabetes 1991; 40: I423-1429

6. Stricker-Krongrad A, Max J P, Musse N, Nicolas J-P, Buffet C, Beck B. Increased threshold concentrations of neuropeptide Y for a stimulatory effect on food intake in obese Zucker rats - changes in the microstructure of the feeding behavior, Brain Res 1994; 660: 162-166.

Z Dryden S, Pickavance L, Frankish HM, Williams G. Increased neuropeptide Y secretion in the hypothalamic paraventricular nucleus of obese (fa/fa) Zucker rats. Brain Res 1995; 690: 185-188.

8. Sanacora G, Kershaw M, Finkelstein J A, White J D. Increased hypothalarnic content of preproneuropeptide Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation. Endocrinology 1990; 127: 730-73Z

9. Chronwall B M, DiMaggio D A, Massari V J, Pickels V M, Ruggiero D .4, O'Donohue T L. The anatomy of neuropeptide Y- containing neurons in the rat brain. Neuroscience 1985; 15: 1159-1181.

10. Bai F L, Yamano IV[, Shiotani Yet al. An arcuato-paraventricular and dorsomedial hypothalamic neuropeptide Y-containing system which lacks noradrenaline in the rat. Brain Res 1985; 331: 172-175.

11. McDonald J K, Koening J I, Gibbs D M, Collins P, Noe B D. High concentration of neuropeptide Y in pituitary portal blood of rats. Neuroendocrinology 1987; 46:538-541. Sawchenko P E, Swanson L W, Grzanna R, Howe P R C, Polak J M, Bloom S R. Co-localization of neuropeptide Y immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus. J Comp Neurol 1985; 241: 138-153. Kiss J Z, Mezey E. Tyrosine hydroxylase in magnocellular nenrosecretory neurons: response to physiological manipulation. Neuroendocrinology 1986; 43:519-525. Larsen P J, Mi!d(elsen J D, Jessop D S, Lightman S L, Chowdrey H S. Neuropeptide Y mRNA and immunoreactivity in hypothalamic neuroendocrine neurons: effects of adrenalectomy and chronic osmotic stimulation. J Neurosci 1993; I3: I138-114Z

12.

13.

14.

© Harcourt Brace and Company Ltd 1998 Neuropepfides (1998) 32(1), 63-66

66 Fetissov & Nicola'{dis

15. Meister B, Cort6s R, Villar M J, Schalling M, Hokfelt T. Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels. Cell Tissue Res 1990; 260: 279-29Z

16. Young W SIII, Warden M, Mezey E. Tyrosine hydroxylase mRNA is increased by hyperosmotic stimuli in the paraventricular and supraoptic nuclei. Neuroendocrinology 1987; 46: 439-444.

1Z Fetissov S, Marsais F, Nicola'idis S, Calas A. Expression of tyrosine hydroxylase in magnocellular hypothalamic neurons of obese (fa/fa) and lean heterozygous (Fa/fa) Zucker rats. Mol Brain Res 1997; 5 O: 314-318.

18. Hsu S-M, Raine L, Fager H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedure. J Histochem Cytochem 1991; 29: 577-580.

19. York D A, Bray G A. Regulation of water balance in genetically obese rats. Proc Soc Exp Biol Med 1971; 136: 798-801.

20. Kagotani Y, Hisano S, Tsuruo Y, Daikoku S, Chihara K. Vasopressin-deficient paraventricular magnocellular neurons of homozygous Brattleboro rats synthesize neuropeptide Y. Neurosci Lett 1990; 112: 37-42.

Neuropeptides (1998) 32(1), 63-66 © Harcourt Brace and Company L td 1998