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HYPOTHYROIDISM INCREASES VASOACTIVE INTESTINAL POLY PEPTIDE (VIP) IMMUNOREACTIVITY AND GENE EXPRESSION IN THE RAT HYPOTHALAMIC PARAVENTRICULAR NUCLEUS

ROBERTOTONI*, ILKDIKO KAKUCSKA“, SALVATORE MOSCA*, PAOLOMARRAMA”, RONALDM. LECHAN” *Istituto di Anatomia Umana Normale, Universird di Bologna, Italy, Yattedra di Endocrinologia. Universitd di Modena, Italy and ‘Department of Medicine, Division of Endocrinology, Diabetes, Metabolism and Molecular Medicine, New England Medical Center Hospitals, Boston, Massachusetts 02111 ABSTRACT: Vasoactive intestinal polypeptide (VIP) is produced by neurons in the rat hypothalamic paraventricular nucleus (PVN) and may have an important role as a prolactin-releasing factor. Recent work from our laboratories has shown that thyroid hormone regulates the content of VIP and VIP mRNA in the rat anterior pituitary, but its effect on VIP in the PVN is not known. To determine whether thyroid hormone alters VIP biosynthesis in the PVN, we studied the effect of hypothyroidism on the content of immunoreactive (IR)-VIP and VIP mRNA in PVN neurons using histochemical techniques. By irnmunocytochemistry. only scattered IR-VIP fibers were present in the PVN of control animals whereas IR-VIP perikarya and fibers were present in hypothyroid rats. By in situ hybridization histochemistry. no labeled neurons were recognized in the PVN in control animals whereas PVN neurons were labeled in hypothyroid rats. These findings raise the possibility that hypothyroidism exerts negative feedback regulation on VIP-producing neurons in the PVN and suggest that this may be important to modulate the stimulatory effects of VIP on anterior and/or posterior pituitary function.

Vasoactiveintestinalpolypeptide(VIP) is presentin bothneurons of thehypothalamic paraventricular nucleus (PVN)(1) andin apopulationof anteriorpituitarycells(2). Thispeptidemayplay a rolein theregulationof prolactin (PRL)secretion, bothasa hypophysiotropic factor(3)or as anautocrine(4) or paracrine(5) regulatorin thepituitary. Inducedhypothyroidismin the rat wasreportedby Lam andcolleagues to leadto anincrease in pituitarycontentof VIP (6)aswellasanincrease in mRNAcodingfor VIP (7), unacommpanied by anychangeincontentof hypothalamic IR-VIP or VIP mRNA(6). Thisfindingwasinterpretedto meanthat thehypothyroidism-induced response wasspecific to theanteriorpituitary. However,thepossibilitythat only a subpopulation of VIPergicneuronsmightbeaffectedby thyroid deficiencywasnot excludedby measurementsof wholehypothalamicextracts(6). Indeed,previousstudiesfrom ourlaboratories haveshownthatchangesin tbyrotropin-releasing hormone(TRH)mRNAinducedby hypothyroidismoccuronly in thePVN, andnot in other TRH-synthesizingneuronalsystems(8). In this study,light microscopic immunocytochemistry andin situ hybridizationofhypothyroidrathypothalamus wascarried out usingspecificantibodyandmRNAprobesfor ratVIP. Hypothyroidismwasshownto inducemarkedchanges highly localizedto a subpopulation of PVN cells. Materials

and Methods

Animalsandtissueoreparation: 3 Sprague-Dawley malerats(200-250g) weremadehypothyroidby the additionof 0.02%methimazole (Sigma ChemCo, St Louis,MO) to their drinking waterfor 3 weeks;3normalratswereused ascontrols.After pentobarbitalanesthesia ([email protected].). theanimals wereperfused throughthe left ventricle with 0.9% salinecontaining

15,000U/L heparinsulfatefor 60 sec.followedby 4% paraformaldehyde (PFA) for 20min. For lightmicroscopic immunocytochemistry, 40 urn coronalsectionswerecut throughthePVN usingaLancerVibratome(TedPella,Inc., Tustin,CA), collectedinto0.1M phosphate bufferedsaline (PBS), pH 7.2 and clearedof fixative in 0.05 M Trisbufferedsaline(TBS), pH 7.6. For in sifu hybridization histochemistry, brainswerepostfixedovernightat 4” C in 4% PFA containing20%sucroseand 18urncoronalsectionswerecut throughthePVN on a cryostat.The 18urn sectionswereadheredto chrom-alum-coated glassslides anddessicated overnightat 42°C. Immunocvtochemistry: Immunocytochemical localizationof VIP in thePVN was performed onfree-floatingsections aspreviouslydescribed (9), basedontheavidin-biotin-peroxidase complex(ABC) techniqueof Hsuand Raine(10). IR-VIP neuronswere labeledusinga well characterized rabbitanti-VIP antiserum(3) dilutedI:750 in 0.2%Triton X- lOO/rBSfor 36-48 h at 4°C. The tissueswere then washedin PBS and incubatedwith biotinylatedgoatantirabbitimmunoglobulin G (IgG; Vector Laboratories, Inc., Burlingame,CA) at a titer of 1:lOOfor 1h, followedby theABC complex.The immunoreaction productwasdevelopedwith 0.025%3-3’diaminobenzidine tetmhydtochloride (DAB, Sigma)-0.045% hydrogenperoxidein TBSfor 6-10minat roomtemperature, followedby silver intensificationof the DAB-labeled structures (11).Thespecificityof theimmunocytochemical reactionwasestablished by preabsorption of the anti-VIP antiserumwith lo6 M syntheticVIP over 18h beforethe immunocytochemical procedure.Sectionswerecounterstainedwith methylgreenandvisualizedby lightmicroscoPY. In situ hvbridizationhistochemistry: In situ hybridizationwasperformed aspreviouslydescribed(8). Singlestranded‘?!iUTP-labeledantisense RNA

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Figure 1. Light microscopic immunocytochemical distribution of IR-VIP perikarya in the rat hypothalamic paraventricular (PVN) nucleus in control (A) and hypothyroid (B) animals. IR-VIP neurons were apparent only in the PVN of the hypothyroid animal (B). Round brackets denote PVN IR-VIP parvocellular neurons, magnified in(C), squaredbrackets denote PVNIR-VIPmagnocelhtlar neurons, magnified in (D). III = thiid ventricle; original magnification: A-B x125, C-D x1250.

probes were generated by transcription from a 380 bp fragment of the rat VIP/PI-II cDNA subcloned into the plasmid vector pSP64 (PromegaBiotec, Madison, Wise.), that contains the entire coding region and 69 bp of the 3’untranslated region (7). Hybridization was performed in buffer containing 50% formamide, 2 x SSC, 10%dextran sulfate, 0.25% BSA, 0.25% Ficoll400, 0.25% polyvinylpyrrolidine 360,250 nM Tris @H 7.5). 0.5% sodium pyrophosphate,0.5% sodiumdodecyl sulfate.250 @ml denaturated salmon sperm DNA and 6 x l@ cpm/slide radiolabeled VIP antisenseprobe for 16 h at 55” C (8). Slides were exposedfor 6-7 days and viewed by darkfield microscopy. Results No IR-VIP perikarya could be demonstratedin the periventicular and medial parvocellular subdivisions of the PVN in euthyroid control rats (Fig. 1A) although numerous IR-VIP axons extended towards the ventral aspectsof the nucleusappearingto arisefrom intenselylabeledneuronsin the suprachiasmaticnucleus(SCN). In contrast,the PVN of hypothyroid rats contained numerous IR-VIP neurons in both parvocellular and magnocellular subdivisions of the nucleus (Fig. lB-D). No apparent differences in IR-VIP werenoted in other peroxidasepositive regions in the brain. By in situ hybridization histochemistry,no neurons hybridizing VIP mRNA were observed in the PVN of euthyroid control rats (Fig. 2A), but adensecollection of hybridizedcells was seenin the ventrolateral nucleus of the thalamusin the sametissuesection(Fig. 2C). In contrast,asmall number of hybridized neurons in the PVN were noted in

Figure 2. Darkfieldphotomicrograph of coronal sections through the hypothalamic paraventricular (PVN) (A, B) and thalamic ventrolateral (VLN) (C, D) nuclei on the same tissue sections, Sections were hybridized with a 35S-labeled rat VIP mRNA antisense probe. Note presence of hybridized cells in the PVN only in the hypothyroid animal (B) but conspicuous absence in the euthyroid control (A). Arrow denotes the PVN; III = third ventricle.

hypothyroid animals, clustered primarily in more caudal regions in the PVN (Fig. 2B), without apparentalteration in hybridization signal in the thalamus (Figure 2D). Discussion In this study we have demonstratedthat hypothyroidism increasesVIP immunostaining and VIP mRNA in populations of neurons in the rat hypothalamic PVN, suggesting that as in the anterior pituitary (6,7), thyroid hormone also exertsnegative feedback regulation on the biosynthesisof VIP in the PVN. The selectivity of the thyroid hormone action for this nucleus is suggestedby the observation that other areas in the forebrain including the SCN and the thalamic ventrolateral nucleusshowednoqualitativealterations in either IR-VIP or VIP mRNA hybridization. The previously reported failure to detect changesin hypothalamic VIP content or its mRNA observed by studyof hypothalamic fragments during hypothyroidism (6,12), therefore, probably was due to the dilution by VIP and its mRNA in regions of the diencephalon other than the PVN. Recent studiesfrom our laboratories (6,7) and by Lam and Srivastava (12) have shown that hypothyroidism increasesthe content of IR-VIP and VIP mRNA in cellsof the rat anterior pituitary. As anti-VIP antiserum inhibits basal PRL releasefrom cultures of both normal (4) and hypothyroid (5) anterior pituitary cells, it is presumedthat VIP may have a stimulatory autocrine or paracrine action on lactotrophs and may contribute to the hypothyroid-induced hyperprolactinemia seenin man(13). The presentobservations raise the possibility that stimulation of VIP biosynthesisin the PVN by hypothyroidism comprisesan additional,

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neuroendocrine mechanism whereby VIP regulates PFU secretion from the anterior pituitary. In addtion to effects on anterior pituitary secretion, VIP may also act as a pituitary growth factor, recently proposed by Prysor-Jones et al (14) on the basis of the ability of VIP to stimulate cell growth in human growth hormone- and PFU-secreting pituitary adenomas. Since long-standing hypothyroidism can lead to pituitary thyrotroph hyperplasia and ultimately adenoma formation (15,16), enhanced VIP secretion may be an additional factor responsible for the hyperplastic/neoplastic process that occurs in the hypothyroid anterior pituitary. The presence of IR-VIP and VIP mRNA in vasopressinergic magnocellular neurons has recently been reported to occur following hypophysectomy (17). Although this increase may be partly secondary to hypoadrenalism (18), our studies would indicate that hypothyroidism may also be contributory. In addition to perikaryal immunostaining, I&VIP has also been identified in axon terminals of the magnocellular system in the posterior pituitary (19) where it may function to stimulate vasopressin secretion (20). Since increased levels of plasma (21) and hypothalamic (22) vasopressin have been observed during hypothyroidism in experimental animals and man (23), it is conceivable that the simultaneous increase in VIP in the magnocellular system may serve to potentiate vasopressin secretion and contribute to the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) often associated with myxedema (23). In conclusion, this study indicates that hypothyroidism stimulates VIP biosynthesis in distinct populations of PVN neurons and suggests that this might be a mechanism to modulate anterior and/or posterior pituitary function. Aknowledgements: ragement

and advice

assistance

with

We thank Dr. Seymour Reichlin for encouand AttreIio Valmori for excellent technical

the photography.

CNR9100335CTO4,MPI60%,

Studies

supported

by Grants

CassaRisparmioImola,NIHDK

37021.

Correspondence: Umana

Normale,

Roberto Via Imetio

Toni

MD,

48.40126,

Istituto Bologna,

di Anatomia Italy

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5. Lam KSL, Reichlin S 1989 Pituitary vasoactive intestinal peptide regulates prolactin secretion in the hypothyroid rat. Neuroendocrinology 50: 524-528 6. Lam KSL, Lechan RM. Minamitani N, Segerson TP. Reichlin S 1989 Vasoactive intestinal peptide in the anterior pituitary is increased in hypothyroidism. Endocrinology 124: 1077-1084 7. Segerson TP, Lam KSL. Cacicedo L, Minamitami N, Fink JS, Lecban RM, Reichlin S 1989 Thyroid hormones regulates vasoactive intestinal peptide (VIP) mRNA levels in the rat anterior pituitary gland. Endocrinology 125: 2221-2223 8. Segenon TP, Kauer J, Wolfe HC, Mobtaker H. Wu P. Jackson IMD, Lechan RM 1987 Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus. Science 238: 78-80 9. Toni R, Jackson IMD, Lechan RM 1990 Neuropeptide-Y-immunoreactive innervation of thyrotropin-releasing hormone-synthesizing neurons in the rat hypothalamic paraventricular nucleus. Endocrinology 26: 24442453 lO.HsuS-M,RaineL 1981 ProteinA,avidinandbiotininimmunocytochemistry. J Histochem Cytochem 29: 1349-1353 11. Gallyas F, Gorks T, Merchenthaler I 1982 High-grade intensification of the end-product of diaminobenxidine reaction for peroxidase histochemistry. J Histochem Cytochem 30: 183- 184 12. Lam KSL, Srivastava G 1990 Sex-related differences and thyroid hormone regulation of vasoactive intestinal gene expression in the rat brain and pituitaty. Brain Res 526: 135-137 13. Reichlin S 1988 Neuroendocrine significance of Vasoactive Intestinal Polypeptide. In: Said SI, Mutt V (eds) Vasoactive Intestinal Peptide and Related Peptides, AM NY Acad Sci ~01527: 431-449 14. Prysor-Jones RA,SilverlightJJ.Jenkins JS 1989Oestradiol.vasoactive intestinal peptide and fibroblast growth factor in the growth of human pituitary tumor cells in vitro. J Endocrinol 120: 171-177 15. Furth J 1955 Experimental pituitary tumors Ret Prog Horm Res 11: 221-249 16. Smallridge RC 1987 ‘Ibyrotropin-secreting pituitary tumors. In: Molitch ME, (ed) Pituitary Nmors: diagnosis and management, Endocrin Metab Clinic North Am. vol 16: 765-792 17. Ceccatelli S, Fahrenkrug J, Villar M. Hokfelt T 1991 Vasoactive intestinal polypcptide/bistidine isoleucine immunorcactive neuron systems in the basal hypothalamus of the rat with special reference to the portal vasadature: and immunohistochemical and in situ hybridization study. Neuroscience 45: 483-502 18. Mexcy E. Kiss JZ 1985 Vasoactive intestinal peptide-containing neurons in the paraventricular nucleus may participate in regulating prolactht secretion. Proc Natl Acad Sci USA 82: 245-247 19. Mikkelsen JD 1989 Immunohistochemical localization of vasoactive intestinal peptide (VIP) in the circumventricular organs of the rat. Cell Tissue Res 255: 307-313 20. Bardmm B, Gttensen B, Fahrenkmg J. Fuchs A-R 1988 Release of oxytocin and vasopressin by intracerebroventricular vasoactive intestinal polypeptide. Endocrinology 123: 2249-2254 21. Seif SM. Robinson AG, Zenser TV, Davis BB, Huallmantal AB, Halusxczak C 1979 Neurohypophyseal peptides in hypothyroid rats: plasma levels and kidney response. Metabolism 28: 137-143 22. Ali M. Clos J 1987 Histological, biochemical and immunocytochemical data on the postnatal development of the hypothalamic magnocellular nuclei in the congenital hypothyroid rat. J Physiol (Paris) 82: 25-35 23. Goldberg M, Reivich M 1962 Studies on the mechanism of hyponatremia and impaired water excretion in myxedema. Ann Intern Med 56: 120130

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