Proc. NatI. Acad. Sci. USAVol. 82, pp. 5555-5559, August 1985Neurobiology

Vasopressin mRNA in situ hybridization: Localization andregulation studied with oligonucleotide cDNA probes innormal and Brattleboro rat hypothalamus

(gene expression/dehydration/histohybridization/paraventricular nucleus)

GEORGE R. UHL*tt, HANS H. ZINGG§¶, AND JOEL F. HABENER*§*Howard Hughes Neuroscience Group at Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; tDepartment of Neurology,Massachusetts General Hospital, and Departments of Neurology and Neuroscience, Johns Hopkins Hospital, Baltimore, MD 21205; and §Laboratory ofMolecular Endocrinology, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114

Communicated by Francis 0. Schmitt, April 1, 1985

ABSTRACT Hybridizable vasopressin mRNA may bequantitatively localized in situ in sections from rat hypothala-mus. Radiolabeled oligonucleotide cDNA probes, synthesizedby chemical and enzymatic means, provide strong hybridiza-tion in zones known to contain vasopressin cell bodies, Multiplesingle-stranded 32p, 355-, or 3H-labeled oligonucleotides dem-onstrate localized hybridization that increases as probes arelengthened from 8 to 75 bases. Competition studies, RNaseexperiments, anatomic localization, and use of multiple probesall support hybridization specificity. An approximate doublingof hybridizable mRNA in both supraoptic and paraventricularnuclei can be detected with dehydration of the animals.Hybridizable mRNA densities are virtually normal in hypo-thalamic nuclei of Brattleboro rats given free access to water.These methods can provide insight into regional mRNA dynam-ics and may reflect functional activity of peptidergic neurons.

The anatomical topographies of many brain peptide neuro-transmitters and their receptors are now delineated by usingtechniques such as immunohistochemistry and receptorautoradiography (1-3). These anatomic methods, however,have limited ability to discern transmitter-specific functionalactivity. Regional reductions in level of peptide transmitters,for example, could occur as a consequence of either reducedgene expression and neuropeptide synthesis in a quiescentneuron or depletion of stores of peptide from an activelyfiring cell.Recent identification of nucleotide sequences that code for

several peptide neurotransmitters allows direct study of geneexpression (4-14). Hybridization techniques can providequantitative evaluation of identified mRNAs as indices of theextent ofgene expression (14-16). Variations in the secretoryactivity of certain endocrine cells are closely paralleled byalterations in the expression of the gene for the secretedproduct (14, 17-19). Assessment of cellular content of trans-mitter-specific mRNAs in neurons could also provide onemeasure of gene expression and reflect functional neuronalactivity. Known regional differences in brain neurotransmit-ters and their functions necessitate both anatomic accuracyand biochemical specificity in the measurements of theseneuronal mRNAs. Hybridization of mRNA within cells insitu can provide such accuracy and specificity (20-32).Hypothalamic neurons that synthesize vasopressin are

particularly amenable to study with in situ techniques be-cause: (i) They have been localized anatomically (33-35). (ii)The sequence of the vasopressin gene is known (14, 36, 37).Furthermore, although much of the vasopressin gene se-quence shares homology with the gene encoding the related

peptide oxytocin, a lengthy 3' sequence unique to vasopres-sin allows construction of vasopressin-specific oligonucleo-tide probes (37). (iii) Gene expression is regulated by knownstimuli such as dehydration (14, 38, 39). (iv) Vasopressin-deficient Brattleboro rat strains allow assessment of the rolesof transcriptional and post-transcriptional processes in aninteresting disorder of these hypothalamic systems (40, 41).We report in situ localization of vasopressin mRNA in normaland Brattleboro rat hypothalamus by using homogeneous3H-, 35S-, and 32P-labeled cDNAs prepared by chemical andenzymatic syntheses.

METHODS

Preparation of Oligonucleotides. Oligonucleotides of 8, 11,15, 19, 24, 45, and 75 bases, complementary to severalregions of the rat vasopressin mRNA (Table 1) were synthe-sized by using an automated oligonucleotide synthesizer(Applied Biosystems, Foster City, CA). Oligonucleotidesequences were selected so that the 3H- or 35S-labeledKlenow product (VP-J) (see below) and the 24-, 45-, and75-base 32P-labeled cDNAs (VP-E, VP-F, and VP-I) hybrid-ized to vasopressin mRNA sequences with virtually no ho-mology to the oxytocin mRNA.

Radiolabeling of Oligonucleotides. For 32P end-labeling, 25pmol of individual synthetic oligonucleotides reacted with 1mCi of [32P]ATP (Amersham, 7000 Ci/mol; 1 Ci = 37 GBq)and 20 units ofT4 polynucleotide kinase (Bethesda ResearchLaboratories) as described (42). The labeled oligonucleotidewas isolated by electrophoresis on a 20% polyacrylamide gel,detected by autoradiography, and eluted with boiling water.

3H- and 35S-labeled oligonucleotides were prepared byenzymatic synthesis. One picomole of the 45-base oligonu-cleotide corresponding to the mRNA encoding vasopressin(VP-G) and 5 pmol of the 15-base complementary oligonu-cleotide (VP-H) were hybridized overnight at 370C in 0.1 MNaCl/0.01 M MgCl2/0.1 mM dithiothreitol/50 mM Tris HCl,pH 7.5. For 3H-labeling, [3H]dATP (50 Ci/mM), [3H]dGTP(25 Ci/mM), [3H]dCTP (58 Ci/mM), and [3H]dTTP (96Ci/mM) (New England Nuclear), previously dried in thereaction tube, were added to final concentrations of 50-100,tM. For 35S-labeling, 30 ,tM [35S]dCTP and [35S]dATP (1300Ci/mM; Amersham) and 100 ,uM unlabeled dGTP and dTTPwere used. Five units of DNA polymerase I (Klenowfragment) (Bethesda Research Laboratories) was added tothe mixture after the hybridization step and allowed to reactat 22°C for 30 min. The reaction was terminated by boiling themixture, which was then separated by electrophoresis on a

VPresent address: Laboratory of Molecular Endocrinology, RoyalVictoria Hospital, Montreal, Quebec H3A1A1.tTo whom reprint requests should be addressed at: Wellman 405,Massachusetts General Hospital, Fruit Street, Boston, MA 02114.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Table 1. Oligonucleotide sequences and specific/nonspecific hybridization ratios

mRNA Position Oxytocin mRNA Specific/nonspecificDesignation sense Length numbers homology Radiolabel estimate

Chemical synthesisVP-A - 8 13-20 + 32p 1.04VP-B - 11 11-19 + 32p 1.22VP-C - 15 1-15 + 32p 1.42VP-D - 19 1-19 + 32p 1.65VP-E - 24 382-405 - 32p 2.03VP-F - 45 361-406 - 32p 3.47VP-G + 45 372-417 - 32pVP-H - 15 398-412 - 32pVP-I - 75 330-405 - 32p 3.30

Enzymatic synthesisVP-J - 40 372-412 - 3H 29.5VP-J - 40 372-412 - 35S 38.0

This table describes the locations, sense, length (number ofbases), and oxytocin mRNA homology ofeach oligonucleotidesequence used here. Sequence numbers are derived from ref. 36, although the rat sequence is used. VP-A, -B, -C, -D, and-F were end-labeled with 32p; VP-J was synthesized using Klenow-directed polymerization of [3H]- or [35S]dNTPs. Filmautoradiograms from in situ hybridization were examined for six regions of interest (each supraoptic nucleus, two zonesfrom cerebral cortex, and two areas of film with no tissue). Mean optical density was derived for each region from digitizedimages by using an image analysis system (Loats Associates, Westminster, MD) (42). Anatomically specific hybridizationwas estimated as the increase in density over the vasopressin-rich supraoptic nucleus (son) compared to film divided bythe density in vasopressin-poor cerebral cortex (cx) compared to film background. The specific/nonspecific estimate is,therefore, derived from [OD (son) - OD (film)]/[OD (cx) - OD (film)].

20% polyacrylamide sequencing gel. Labeled cDNA (VP-J)was thereby isolated from the unlabeled oligonucleotidetemplate.

Experimental Animals. Sprague-Dawley, Long-Evans, orBrattleboro (Blue Spruce Farms, Altamont, NY) rats (225 g)were maintained on a 12-hr on, 12-hr off, day-night cycle.Three groups of three Sprague-Dawley rats each weresubjected to water deprivation for 96 hr, during which timethey lost 15-20% of their body weight. We have shownpreviously that this period of dehydration results in bothincreased blood levels of vasopressin and increased hypo-thalamic levels of mRNAs encoding pre-propressophysin(14). Brattleboro and Long-Evans rats were examined with-out fluid deprivation.

Tissue Preparation. Animals were anesthetized withpentobarbital between 11 a.m. and 3 p.m. and perfusedthrough the left ventricle for 20 sec with phosphate-bufferedsaline, followed by perfusion with 0.5% depolymerizedparaformaldehyde/1% glutaraldehyde/75 mM lysine/37.5mM NaPO4, pH 7.4/10 mM sodium periodate to -1 ml perg of body weight (31). The brains were removed, post-fixedin the same fixative for -2 hr at 4°C, soaked for an additional2 hr in 7% sucrose in phosphate buffer, cut into slabs, andrapidly frozen on cryostat chucks using powdered dry ice.Sections (10 ,um) of appropriate areas were cut with a Harriscryostat, thaw-mounted onto slides pretreated by acetyla-tion, and coated with Denhardt's solution (0.02% bovineserum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone)and stored at -20°C (20, 43).

Sections were pretreated for 20 min at 22°C with 0.2 MHCl, 30 min at 70°C in 0.3 M sodium chloride/0.03 M sodiumcitrate, pH 7.4, and for 15 min at 37°C in proteinase K (1,g/ml)/10 mM Tris-HCl, pH 7.4/2 mM calcium chloride.Some slides were also incubated with RNase A (25 ,g/ml)(Sigma) at 42°C for 1 hr. All slides were dehydrated in ethanoland dried at room temperature.

Hybridization and Washing. Twenty-five microliters ofsolution containing the labeled oligonucleotide was applied toeach coronal section through the rat brain. The hybridizationsolution contained 1-2 x 104 cpm of 32p- or 35S-labeled cDNAper ,ul or 1-5 x 103 cpm of 3H-labeled oligonucleotide per ,ulin 0.8 M NaCl/7 mM Tris HCl, pH 7.4/1.3 mg of bovineserum albumin per ml/26 mg of polyvinylpyrrolidone per

dl/26 mg of Ficoll per dl/0.13 g of dextran sulfate per ml/0.3mg of salmon sperm DNA per ml/0.6 mM dithiothreitol/0.6mM EDTA/60% (vol/vol) deionized formamide. The hybrid-ization reaction was sealed under siliconized coverslips usingrubber cement, and sections were incubated for 18-24 hr at370C.

After the primary incubation, the coverslips were gentlyremoved and sections were washed overnight in 0.3 MNaCl/0.03 M sodium citrate/0.001 M EDTA, pH 7.4, at 22°C.Sections were next washed for 1 hr in the same solution at500C, dehydrated in 0.3 M sodium acetate with increasingethanol concentrations, and dried at room temperature.Autoradiograms of tissue were generated by apposition of

32P-labeled sections to X-Omat film (Kodak), or to emulsion-coated coverslips (Kodak NTB3) (44). 3H- and 35S-labeledsections were exposed to Ultrofilm (LKB) or emulsion-coated coverslips. After appropriate incubation, films andemulsions were developed and tissue underlying the emul-sion was stained with cresyl violet as described (44).

Competition Experiments. In some experiments, sectionsadjacent to the study sections were treated identically, exceptthat 20-50 pmol of unlabeled cDNAs was added to theprimary incubation to establish a control. This was performedboth with unlabeled homologous cDNA and with unlabeledheterologous cDNAs directed against glucagon (23 bases),somatostatin (25 bases), dynorphin (45 bases), and oxytocin(19 bases). Hybridization competition with 3H- and 35S-labeled probes (VP-J) was performed with the complemen-tary 45-base oligonucleotide (10 pmol).

Analysis of Autoradiograms. Film autoradiograms wereanalyzed by semiquantitative visual rating and by a comput-erized image analysis system, allowing assessment of region-al digitized optical density measurements and comparisonswith standards of known radioactivity (45). Autoradiogramswith coverslips were subjected to confirmatory observationsin each case. 3H and 35S standards were prepared by usingbrain paste as described (45). Reported values represent themeans from two digitizations of each nucleus. An estimate ofanatomically specific hybridization was derived from theratio of the optical density in areas of film corresponding tothe supraoptic nucleus to optical density in film regionsexposed to the cerebral cortex. Uneven film background

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required subtraction of this density from supraoptic andcerebral cortical values.

RESULTS

Hybridization Signal and Background. Our standard 32p_,35S_, and 3H-labeled oligonucleotide probes (VP-F and VP-J)yield good hybridization in discrete vasopressin-containingareas of rat hypothalamus (Fig. 1). Little background hybrid-ization to other areas poor in vasopressin-immunoreactiveperikarya (33-35) is observed. Typically, optical densityratios [OD (supraoptic nucleus) - OD (film)]/[OD (cerebralcortex) - OD (film)] are 3.5 for normal brains hybridized withthe 45-base 32P-labeled probe VP-F and 30-40 for normalbrains hybridized with the 3H- or 35S-labeled probe VP-J(Table 1).Anatomic Distribution. Strong labeling occurs over

supraoptic and paraventricular nuclei, with lower graindensities over the suprachiasmatic nuclei (Fig. 1). Theseresults have been noted in >50 animals. Examination ofautoradiograms with coverslips reveals dense labeling overthe entire supraoptic nucleus. Within the paraventricularnucleus, uniform labeling is restricted to the more lateralmagnocellular portion. Within the parvocellular portion, highgrain densities are found only over scattered large cells (Fig.2). The medial aspects of the suprachiasmatic nucleus showthe highest grain densities in this nucleus.

Competition Studies. Addition of unlabeled homologouscDNA to hybridization reactions using 32P-labeled VP-Fvirtually eliminated specific supraoptic nucleus hybridization(Fig. 1). Addition ofunlabeled probe against other portions ofthe vasopressin gene, somatostatin, glucagon, dynorphin, oroxytocin failed to alter the hybridization (data not shown).Addition of unlabeled complementary cDNA to 3H- or

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FIG. 1. (A) Autoradiogram (film) of 32P-labeled VP-F hybridiza-tion to coronal rat brain section at the level of the supraoptic (so) andsuprachiasmatic (sc) nuclei. Increased whiteness corresponds toincreased grain density in these prints. cx, Cerebral cortex. (x7.4.)(B) Autoradiogram of competition experiment using section adjacentto A, treated identically except that 25 pmol of unlabeled VP-F was

added to the hybridization mixture to establish a control. (Onlybottom portion of section shown.)

FIG. 2. (A) Autoradiogram (emulsion) of 3H-labeled VP-J hybrid-ization to paraventricular nucleus magnocellular (pmc) andparvocellular (ppc) regions of a dehydrated rat. vIII, third ventricle.With plane of focus between cells and grains, grain densitiesassociated with specific parvocellular cells can be appreciated(arrowheads). (x30.5.) (B and C) Autoradiograms (emulsion) of3H-labeled VP-J hybridization to supraoptic nucleus (son) of a

normal rat. (B) Normal incubation. (C) Competition experiment,using adjacent section, with 10 pmol of unlabeled mRNA-codingsense strand complementary to VP-J also added to incubations. oc,Optic chiasm. Plane of focus between cells and grains allowsappreciation of both. (x 39.)

35S-labeled VP-J hybridization also dramatically decreasedgrain densities (Fig. 3).

Standardization. 3H and 35S standards in brain mashyielded linear standard curves for plots of optical densityagainst radioactivity. By contrast, 32P-labeled standard spotsvaried in both size and optical density with saturation ofX-Omat film over a narrow range of radioactivity (data notshown). The [OD (supraoptic nucleus) - OD (film)]/[OD(cerebral cortex) - OD (film)] ratios reported thus representgood estimates of binding specificity using 3H- and 35S-labeled oligonucleotides, but they must be regarded as

approximations for 32P-labeled oligonucleotides.Hybridization of Oligonucleotides. The supraoptic nucleus

was examined for anatomically discrete hybridization of 8-,11-, 15-, 19-, 24-, 45-, and 75-base 32P-labeled oligonucleo-tides complementary to vasopressin mRNA. Localizedsupraoptic hybridization was seen with the 11-, 15-, 19-, 24-,45-, and 75-base probes but not with the 8-base vasopressinoligonucleotide. Nonspecific background radioactivity was

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FIG. 3. Autoradiograms (film) of 3H-labeled VP-J hybridizationat the level of the paraventricular (pv) and caudal supraoptic (so)nuclei. (A) Normal rat. (B) Dehydrated rat (4-day water deprivation).cx, Cerebral cortex. (x4.8.) Autoradiograms (film) of 35S-labeledVP-J hybridization at the level of the supraoptic nuclei (son) andsuprachiasmatic nuclei (scn). (C) Normal Long-Evans rat. (D)Brattleboro rat. (x 6.5.)

minimal using the 24-, 45-, and 75-base oligonucleotides andwas greater using the shorter 32P-labeled cDNAs (Table 1).RNase Treatment. RNase pretreatment decreased supra-

optic hybridization. For the 3H probe VP-J, the value of [OD(supraoptic nucleus) - OD (film)]/[OD (cerebral cortex) -

OD (film)] was decreased from 46 to 19 in a typical experi-ment.

Effect of Dehydration. We used 3H-labeled probe VP-J toassess the influence of dehydration on hybridizable vaso-

pressin mRNA levels in supraoptic and paraventricularnuclei (Table 2). Water deprivation of rats substantiallyincreased hybridization strength over both of the magno-cellular hypothalamic nuclei. We quantitated this differenceby measuring optical densities in film zones exposed to these

nuclei and by comparing these numbers to a standard curvegenerated by coexposing sections of brain paste standards ofknown 3H radioactivity to the same sheets of film. Meansupraoptic values increased from 2.47 x 104 dpm per mg oftissue to 5.08 x 104 dpm/mg with dehydration (three to fouranimals in each group; two nuclei measured in each rat).Paraventricular hybridization also doubled, from 2.6 x 104dpm/mg to 5.62 x 104 dpm/mg. Each of these differences isstatistically significant (P < 0.005 by t test) and has beenreplicated in three independent groups of rats.

Brattleboro Rats. We used the 35S-labeled probe VP-J toassess the influences of dehydration on hybridizable vaso-pressin mRNA levels in supraoptic and paraventricularnuclei of Brattleboro rats and normal rats from their parentLong-Evans strain. Hybridization densities in the Brat-tleboro rats were not different from those found in normalLong-Evans rats. Brattleboro supraoptic values were 9 x 104dpm per mg of tissue (control = 8.5 x 104 dpm/mg) andparaventricular values were 9.4 x 10" dpm/mg (control = 9.1x 104 dpm/mg) (P >> 0.1).

DISCUSSION

These studies demonstrate the potential for elucidation ofbiochemically and regionally specific mRNA dynamics invasopressin neurons. Furthermore, they may represent ageneral strategy applicable to analogous explorations in thegrowing number of neuropeptidergic neuronal systems withdefined gene sequences.

Five features of the observed hybridization argue for itsspecificity. (i) Anatomic localization of hybridization signalto hypothalamic nuclei rich in vasopressin immunoreactivityis consistent with hybridization specificity. (ii) Diminution ofhybridization with RNase treatment also argues for a specificinteraction with RNA. (iii) Physiological regulation ofmRNAlevels with dehydration fits well with the known function ofthese neurons. (iv) Recognition of hybridized mRNA inidentical patterns by several different vasopressin-relatedcDNAs but not by unrelated probes of similar length andspecific activity provides good evidence for hybridizationspecificity. (v) Successful competition for hybridization usingexcess homologous but not heterologous unlabeledoligonucleotides also furnishes confirmation ofthe specificityof our observations. Competition experiments using heter-ogeneous nick-translated mixtures of probes have beenreported to be only partially successful (28). The homoge-neous nature of our labeled probes and competing unlabeledoligonucleotides may have resulted in the virtual eliminationof specific hybridization in our competition experiments.Comparisons between oligonucleotides of 8-75 bases sug-

gest that the longer probes in this series provide better

Table 2. 3H-labeled VP-J in situ hybridization regional mean optical density (x) determination innormal and dehydrated rats

Supraoptic Paraventricular Supraoptic Paraventricularnucleus nucleus nucleus nucleus

Normal rats Dehydrated rats1 0.4299 0.4823 1 0.6464 0.57932 0.4740 0.4280 2 0.6105 0.64123 0.4652 0.4930 3 0.5602 0.6122

R = 0.4561 0.4627 4 0.6076 0.6645x = 0.6061 x = 0.6243

Derived radioactivity calculations: supraoptic nucleus, 2.47 x 104 dpm/mg (normal); 5.08 x 104dpm/mg (dehydrated). Paraventricular nucleus, 2.60 x 104 dpm/mg (normal); 5.62 x 104 dpm/mg(dehydrated). Film autoradiograms from in situ hybridization and brain paste standards of knownradioactivity were examined. Values presented for each nucleus for each rat represent the means oftwoquantitations of optical density in film regions corresponding to the right and to the left nucleus. Derivedvalues for dpm per mg of tissue are obtained by comparing optical densities overlying appropriate ratbrain regions to the linear standard curve generated from the brain paste standard data.

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hybridization. Oligonucleotides of these lengths were select-ed because they can be readily synthesized with reasonableyield. We have not excluded the possibility that improvedsynthetic techniques allowing further lengthening of oligo-nucleotide probes, or even use of longer cloned cDNAprobes, might provide even better hybridization in situ.Preparation of homogeneous 3H- and 35S-labeled

oligonucleotides by Klenow-directed enzymatic synthesishas resulted in greatly improved anatomic resolution. Withemulsion autoradiograms, 3H probes allow detection ofhybridization associated with individual dispersed cells in theparvocellular portion of the paraventricular nucleus. 3H and35S probes have also aided in the quantification of in situresults, allowing better estimates of hybridized probe per unitof tissue than are possible with 32p label.

Magnocellular vasopressin neurons in the supraoptic andparaventricular nuclei send processes to the posterior pitu-itary, where vasopressin is released into the circulation inresponse to alterations in osmotic pressure and extracellularfluid volume (38). Our finding that specific mRNA levels inboth supraoptic and paraventricular nuclei appear to increasewith dehydration fits well with this anatomic distribution andconfirms roles for both nuclei in response to dehydration.The observed change is also in accord with previous reportsof dehydration-induced increases in vasopressin mRNAextracted from whole hypothalamus (14, 39).

Relative quantitation of vasopressin mRNA in hypo-thalami of vasopressin-deficient Brattleboro rats has beencontroversial, with similar or subnormal levels found indifferent studies conducted under differing dehydrating con-ditions (40, 41). In our studies of Brattleboro rats given ad libaccess to water, normal supraoptic and paraventricularvasopressin mRNA hybridization supports the likelihoodthat the reduced peptide levels found in these animals are notsolely a consequence of reduced levels of mRNA.

We gratefully acknowledge helpful discussion with G. Heinrich, T.Moench, H. Gendleman, and R. Grzanna; technical assistance of G.Hackney and V. Stranov; assistance with the manuscript by S.Cronin and J. Canniff; and support from the McKnight Foundation,Sloan Foundation, American Parkinson's Disease Association, andNational Institutes of Health Grant AM 34219.

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