Immunohistochemical Demonstration of Neurophysin in the Hypothalamoneurohypophysial System

Immunohistochemical Demonstration of Neurophysin in the Hypothalamoneurohypophysial Systern W . B . WATKINS Postgraduate School of Obstetrics and Cynaecology. University of Auckland. Auckland. New Zealand

1. Introduction

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. . . . . . . . . . . . . . . .. .

11 Relationship between Neurosecretory Material

and Neurophysin . . . . . . . . . . . . . . . I11. Methods of Extraction of Neurophysin . . . . . . . A . Extraction from Posterior Pituitary Glands . . . . B. Extraction from Whole Pituitary Glands . . . . . IV Purification of Neurophysin Antigens . . . . . . . A . Molecular Sieve and Ion-Exchange Chromatography B Preparative Polyacrylamide-Gel Electrophoresis . C . Preparative Isoelectric Focusing . . . . . . . . D . Extraction of Neurophysin from Electrophoretic Gels V. Production of Antibodies against Neurophysin A Cross-Species Reactive Antibodies . . . . . . . B Species-Specific Antibodies . . . . . . . . . VI . General Considerations of Antibody Production and Detection . . . . . . . . . . . . . . . . VII Immunohistochemical Techniques . . . . . . . . A . Light Microscope Level . . . . . . . . . . . B. Electron Microscope Level . . . . . . . C Photographic Procedures . . . . . . . D Preparation of Tissues . . . . . . . . . . . VIII . Demonstration of Neurophysin in the Hypothalamoneurohypophysial System Using Cross-Species Reactive Antineurophysin A Magnocellular Nuclei . . . . . . . . . . . . B. Other Areas of the Hypothalamus and Brain . . . C . Proximal Neurohypophysis . . . . . . . D . Pituitary Stalk . . . . . . . . . . . E . Posterior Pituitary Gland . . . . . . . . . . Ix. Use of Species-Specific Antisera for the Demonstration of Neurophysin . . . . . . . . . . A . Pig . . . . . . . . . . . . . . . B. Ox . . . . . . . . . . . . . . . x. Conclusions . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

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Introduction

Osborne and Vincent (1900)were the first to report on the association of a high-molecular-weight material with the hormones in the 24 I

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W. B. WATKINS

posterior pituitary gland. These workers demonstrated that treatment of a saline extract of the ox infundibulum, with either excess alcohol or by the addition of ammonium sulfate, resulted in precipitation of the pressor activity found in the tissue. In an extension of this work, Kamm et al. (1928)showed that both the oxytocic and pressor principals of the gland extract were salted out with 30% sodium chloride, but that the efficiency of the precipitation depended upon the dilution of the initial extract. By using relatively concentrated solutions, approximately 100% of the pressor activity was salted out and, furthermore, the ratio of pressor activity to oxytocic activity approximated that found in the fresh gland. Addition of sodium chloride to a dilute solution containing 10 IU of both oxytocic and pressor activities per milliliter gave incomplete precipitation of the biological activities. Rosenfeld (1940)subsequently found that the oxytocic and pressor activities sedimented in an ultracentrifuge with a sedimentation coefficient of about one-half to one-third that of egg albumin. Partial purification and biochemical characterization of the proteinaceous material isolated by Kamm et al. (1928)and Rosenfeld (1940)was begun by van Dyke et al. (1942).Employing the techniques available at that time, for example, constant solubility, electrophoresis, and differential centrifugation, it was concluded that the “van Dyke protein” was homogeneous and had a molecular weight of approximately 30,000. The protein is rich in residues of cystine, glutamic acid, and glycine as determined by amino acid analysis (Block and van Dyke, 1952).The finding that electrodialysis of the van Dyke protein can result in the dissociation of the oxytocic principal led Haselbach and Piquet (1952)to suggest that the polypeptide hormone is electrostatically bound to the proteinaceous component. Other procedures such as dialysis, ultrafiltration, electrolysis, treatment with trichloroacetic acid, and countercurrent distribution (Acher et al., 1955;Acher and Fromageot, 1957) further confirmed the noncovalent binding of hormone to protein. The protein component of the van Dyke protein was given the name neurophysin by Acher et al. (1955). When the van Dyke protein was initially isolated, it was considered a single protein, but more recent studies into the homogeneity of the protein by Frankland et al. (1966)established the presence of four discrete protein components. The presence of these multiple components has subsequently been attributed to the action of proteolytic enzymes during the extraction procedure (see Section I1I ,A,1).

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11. Relationship between Neurosecretory Material and Neurophysin

Histologically, the concept of neurosecretion is characterized by the presence of neurosecretory material (NSM) within the magnocellular hypothalamic nuclei and neurosecretory axons of the hypothalamoneurohypophysial system (HNS) (for reviews, see Bargmann, 1966; Bern and Knowles, 1966; Rinne, 1966; Sachs, 1969; Scharrer and Scharrer, 1954; Sloper, 1958). This material was first demonstrated by Bargmann (1949a,b, 1950, 1951) by the application of the acid permanganate chrome alum hematoxylin stain (CAH) which had been previously used by Gomori (1941) to stain the /3 cells of the endocrine pancreas. The basis of action of CAH and the more sensitive aldehyde-fuchsin (AF) (Gomori, 1950; Landing et al., 1956) is the oxidation of disulfide bonds in the tissue to sulfonic acid residues which then combine with hematoxylin and fuchsin residues, respectively. A more sensitive reagent reacting with sulfonic acid groups is pseudoisocyanin chloride which gives rise to an intense fluorescence (Sterba, 1964). NSM can also be demonstrated by using methods detecting protein-bound cystine such as performic acid-alcian blue (Adams and Sloper, 1956) and thioglycolate-ferric ferricyanide (Sloper, 1955). The thioglycolate-dihydroxy-dinaphthyl disulfide (Barnett, 1954; Sloper, 1955) and alkaline tetrazolium (Sloper, 1955; Howe and Pearse, 1956) techniques reveal NSM through reactions with protein-bound sulfhydryl groups formed by the reduction of disulfide bonds. Material containing arginine residues was detected in the infundibulum of the ox and rat by Howe (1959,1962), using a modification of the Sakaguchi reaction. An improved method for the detection of arginine in NSM was used by Bock and Schluter (1971). This method is based on the fact that arginine reacts with a derivative of ninhydrin to give a fluorescent compound which can be visualized in tissue slices (Rosselet, 1967). It was initially proposed (Schiebler, 1952) that NSM is a glycoprotein since it gives a positive reaction with periodic acid-Schiff, Millon, and sudanophilic lipid stains. However, later studies using the periodic acid-Schiff reaction gave inconclusive results (Gabe, 1960) which were attributed to species variation. It was implied that NSM is a lipid-protein because it was not present in sections fixed with lipid-dissolving solvents such as alcohol and acetone (Schiebler, 1951; Hild and Zetler, 1953). However, subsequent work

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showed that, when these tissue sections were postfixed with formalin or floated on Bouin’s fluid instead of water, NSM was retained in the tissue (Sloper, 1955). Further studies on the dog (Sloper, 1955) and rat (Howe and Pearse, 1956) confirmed that NSM does not contain lipid or carbohydrate residues but is a protein rich in cystine. It is now being recognized that NSM present in the mdmmalian HNS and neurophysin, may be one and the same species. This correlation is based on the following observations.

1. The positive histological reactions performed on the HNS with CAH, AF, and pseudoisocyanin chloride stains can be considered to be due to the interaction with the large number of cystine residues present in neurophysin. It is interesting to note that neurohypophysial hormones, like neurophysin, contain approximately 16%cystine and are also capable of giving a positive reaction with pseudoisocyanin chloride (Gutierrez and Sloper, 1969). 2. The performic acid-pseudoisocyanin chloride reagent reacts with the neurophysin-hormone complex as prepared by the method of Acher, Light, and du Vigneaud (1958; also see Sterba, 1964). 3. Neurophysins obtained from the species so far studied contain between four and six arginine molecules per molecule of protein, which would be capable of reacting with the ninhydrin fluorescent system described by Bock and Schliiter (1971). 4. Osmotic stimulation of an animal reduces the amount of NSM in the posterior pituitary lobe concomitantly with neurophysin (Watkins and Evans, 1972) (see Section VII1,E). 111. Methods of Extraction of Neurophysin

A.

EXTRACTION FROM POSTERIOR PITUITARY GLANDS

1. Use of Dilute Acids In their initial preparation of the protein-neurohypophysial hormone complex, van Dyke et al. (1942)extracted fresh-frozen ox posterior pituitary glands with 0.01 N sulfuric acid overnight at 4°C. Subsequent fractionation of the van Dyke protein by gel exclusion and ion-exchange chromatography (Hollenberg and Hope, 1967) produced six discrete proteins as revealed by starch gel electrophoresis at pH 8.1. Two of the proteins possessed the ability to bind oxytocin and vasopressin, confirming their identity as neurophysins. Proteolytic enzymes present in acetone-dried posterior lobe

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powder have optimum activity at pH 3-4 using hemoglobin as a substrate (Dean et al., 1967; Pickup and Hope, 1971,1972). If the extraction of ox tissue is carried out at a pH that irreversibly destroys the catheptic acitvity, the number of neurophysin components subsequently identified is reduced (Hollenberg and Hope, 1968). Despite the degradation of neurophysins by the action of proteolytic enzymes, many workers have persisted in using dilute acid for the initial extraction procedure. Breslow and Abrash (1966), Fawcett et al. (1968), and Legros et al. (1969) extracted bovine neural lobes with 0.01 N sulfuric acid according to the method of Acher et al. (1958), while 0.1 M acetic acid was used to extract neurophysins from ox (Ginsburg and Ireland, 1965; Ginsburg et al., 1966) and pig (Ginsburg et al., 1966)posterior pituitary glands. Pickering (1968) extracted neurophysins from cod pituitaries with 0.05 N acetic acid. 2. Use of Strong Mineral Acids The degradation of neurophysins by the action of proteolytic enzymes during extraction with dilute acids is avoided by using 0.1 N hydrochloric acid for the initial extraction procedure. Neurophysins from ox (Rauch et al., 1969; Pligka et al., 1972; Breslow et al., 1971; Robinson et al., 1971), pig (Uttenthal and Hope, 1970), sheep (Watkins, 1972a, 1973a), rat (Burford and Moens, 1971; Coy and Wuu, 1972; Watkins, 1972b), guinea pig (Watkins and Ellis, 1973), human (Watkins, 1971), and dog (Watkins, unpublished results) have been isolated using 0.1 N hydrochloric acid. It can be argued that use of hydrochloric acid at a pH of 1.5-2 may cause chemical disruption of the “native” neurophysin molecule. There is however, some evidence to suggest that this phenomenon does not occur to any marked degree: (1) Proteins extracted from either fresh or acetone-dried posterior pituitary glands give a similar electrophoretic pattern on starch. gel irrespective of whether hydrochloric acid (pH 1.5) or electrophoresis buffer (pH 8.1) is used for their extraction (Hope and Uttenthal, 1969; Watkins, 1972b, 1973a; Watkins and Ellis, 1973). (2) Furthermore, ox neurophysins extracted with 0.1 N hydrochloric acid have an electrophoretic mobility on starch gel similar to those of proteins obtained by lysis of neurosecretory granules from bovine neurohypophysis (Dean et al., 1967). It is believed that the proteins present in the neurosecretory granules represent native molecules. (3)Pig neurophysins extracted with hydrochloric acid (Uttenthal and Hope, 1970) give amino acid analyses similar to those of pig neurophysins obtained by Wuu and SaEran (1969) and Cheng and Friesen (1971b) using mild extraction

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procedures (see Section III,A,3). (4) All the neurophysins so far characterized possess alanine as the N-terminal residue. This is observed irrespective of the method used for extraction. 3. Extraction by the Percolation Method In their purification of porcine neurophysin, Wuu and SafFran (1969) extracted acetone-dried posterior pituitary lobe powder by percolation with a discontinuous gradient of water and acetic acid in ethanol. The fraction extracted with 70% aqueous ethanol containing 0.25 M acetic acid was then subjected to further purification by molecular sieve and ion-exchange chromatography (see Section IV). A similar extraction procedure was used by Martin et al. (1972) for the purification of bovine neurophysin. Neurophysins of the pig (Friesen and Astwood, 1967; Cheng and Friesen, 1971a,b) and human (Cheng and Friesen, 1972, 1973) were obtained by initially stirring acetone-dried neural lobes powders with 10 volumes of 40% acetone or ethanol followed by percolation with further acetone. Under these conditions neurophysin is extractable into the liquid phase (Martin et al., 1972). Addition of further organic solvent to a concentration of 90% (vlv) causes precipitation of the neurophysins.

B. EXTRACTION FROM WHOLEPITUITARYGLANDS

In his isolation of neurophysin from the cod, Pickering (1968)used whole pituitary glands as starting material. More recent work from this laboratory (W. B. Watkins and J. J. Evans, unpublished work) has shown that it is also possible to extract and purify sheep neurophysins from intact pituitary glands employing the standard methods developed for the isolation of neurophysins from neural lobes.

IV. Purification of Neurophysin Antigens CHROMATOGRAPHY A. MOLECULAR SIEVE AND ION-EXCHANGE 1. Method Used by Hope Neurophysin present in posterior pituitary lobe extracts (adjusted to pH 4) can be precipitated out of solution by the addition of sodium chloride to a final concentration of 10 gml100 ml (Acher et al., 1958). Dissociation of the neurohypophysial hormones, oxytocin and vaso-

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pressin, from the protein-hormone complex can be achieved by chromatography on a column of Sephadex G-25 using an acidic eluting solvent (Frankland et al., 1966). The chromatographic elution profile for such a column is shown in Fig. l a which represents the

voknrat.IRUII1M

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FIG. 1. Elution profiles obtained during column chromatographic purification of sheep neurophysin from the protein-neurohypophysial hormone complex. Circles, Ultraviolet absorption. (a) Chromatography on a column (2.2 x 146 cm) of Sephadex G-25 to separate oxytocin and vasopressin from high-molecular-weight material. The sample (103 mg) was applied to the column in 0.1 M formic acid and eluted with the same solvent at a rate of 25 ml per hour. Squares, Oxytocic activity; triangles, pressor activity. (b) Chromatography of the hormone-free protein (obtained from the void volume in Fig. la) (93 mg) on a column (2.2 x 146 cm) of Sephadex G-75. The column was eluted at a flow rate of 20 ml per hour to give albumin in peak A and a mixture of sheep neurophysin in peak B. (c) Purification of ovine neurophysin I11 from the material in peak B (b) by ion-exchange chromatography on a column (2.8 X 45 cm) of DEAE Sephadex A-50. The sample of neurophysins (93 mg) was applied to the column in tris-hydrochloric acid buffer (pH 8.1), and the column then eluted with tris-hydrochloric acid buffer (pH 8.1)with increasing concentrations of sodium chloride from 0 M to 0.3 M sodium chloride over a gradient of 700 ml. Open circles, sodium chloride concentration in milliequivalents per liter. The flow rate was 8 ml per hour. A large fraction of ovine neurophysin 111 (peak C) was separated from the neurophysin mixture, while peak D contained ovine neurophysins I and I1 and residual amounts of neurophysin 111. (d) Purification of neurophysins in peak D (Fig. lc) by chromatography on a column (2.2 x 19 cm) of C M Sephadex C-50. The neurophysins (33.9 mg) were applied to the column in 0.1 M acetic acid, and the column eluted with a gradient of sodium acetate buffer (pH 4.40 + 5.0; I = 0.1)over a total volume of 250 ml at a flow rate of 4 ml per hour. Open circles, pH. Peaks E, F, and G correspond to ovine neurophysins I, 11, and 111, respectively. (Adapted from Watkins, 1973a.)

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first stage in the purification of sheep neurophysins (Watkins, 1973a). Although oxytocin and 8-arginine-vasopressin have similar molecular weights, the latter peptide is retarded further on Sephadex G-25 because of the electrostatic interaction between the carbonyl groups on the Sephadex and the basic residues on the peptide (Gelotte, 1960). The protein eluted in the void volume from Sephadex G-25 is rechromatographed on a column of Sephadex G-75 (Fig. lb). Of the two peaks that are resolved, the first consists mainly of serum albumin. The profile of the second peak, with its slowly rising leading edge and rapidly falling tailing edge is characteristic of the presence of neurophysin. During the purification of pig neurophysins, however, pig neurophysin I1 is eluted prior to pig neurophysins I and I11 because its molecular weight is substantially greater than that of either neurophysin I or I11 (Uttenthal and Hope, 1970). Subsequent purification of individual neurophysins is achieved by ion-exchange chromatography. The choice of type of ion-exchange resin to be used is best determined empirically. The three ox neurophysins are successfully resolved on a column of DEAE Sephadex A-50 (Rauch et al., 1969), while a column of CM-Sephadex C-50 is employed in the separation of pig neurophysin I from neurophysin I11 (Uttenthal and Hope, 1970). Because the relative distribution of sheep neurophysins I, 11, and I11 is approximately 1:1:5, it has been found convenient to use both anion- and cation-exchange columns for their isolation (Watkins, 1973a). The major proportion of neurophysin I11 is isolated by the use of a DEAE-Sephadex A-50 column (peak A in Fig. lc). A CM-Sephadex C-50 column is then used to separate neurophysin I from neurophysin I1 (Fig. Id). 2. Method Used by Saffran The percolate fraction containing pressor activity was applied to a column of Sephadex G-25, and the material eluted in the void volume rechromatographed on a column of Sephadex G-50. Fractionation of the major protein peak from this column was carried out on DEAE-cellulose from which pig neurophysin was isolated (Wuu and SafFran, 1969).

3. Method Used by Friesen Neurophysins isolated in H. G. Friesen’s laboratory have been extracted by methods similar to that described above (Section IV,A,2).

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249

In the purification of pig neurophysins, the percolate material was first passed through a DEAE-cellulose column and protein fractions subsequently resolved by chromatography on a Sephadex G-75 column (Cheng and Friesen, 1971b). A side fraction obtained during the purification of gonadotrophins was used as starting material for the isolation of human neurophysins (Cheng and Friesen, 1972). An extract of this fraction was applied to a Sephadex G-100 column, and the peak containing material immunologically cross-reactive against antineurophysin serum was purified by pH gradient chromatography on DEAE-cellulose. Two proteins with hormone-binding properties were obtained and named human neurophysins I and 11. 4. Method Used by Ginsburg The proteins present in the supernatant of Ginsburg and Ireland’s (1965) acid extract of ox neurohypophysis were freed from oxytocin and 8-arginine-vasopressin by gel exclusion chromatography on Sephadex G-25. Separation of the neurophysins from other highmolecular-weight material was carried out by ion-exchange chromatography on CM-cellulose.

5. Method Used by Pickering The hormone-free protein mixture extracted from cod pituitaries was passed through a Sephadex G-75 column, and the peak containing proteins with hormone-binding properties collected. Further purification of the neurophysin was performed on DEAE Sephadex (Pickering, 1968). B. PREPARATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS Coy and Wuu (1971) demonstrated that a mixture of pig neurophysins could be purified by means of a preparative polyacrylamide gel electrophoresis system. This method was subsequently used for the isolation of three proteins from rat posterior pituitary glands (Coy and Wuu, 1972), which were later identified as neurophysins (Watkins, 1972b). C. PREPARATIVE ISOELECTFUC FOCUSING Bovine neurophysins I and I1 as obtained by isoelectric focusing, in a 0-40% sucrose gradient, were of purity comparable to that of

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ion-exchange chromatography preparations (PliBka et al., 1972). Although there are current problems associated with the quantitative removal of proteins from the column matrix, further advances in the development of ampholites will undoubtedly make the technique of preparative isoelectric focusing invaluable for the separation of molecules with similar isoelectric points such as the neurophysins.

D. EXTRACTION OF NEUROPHYSIN FROM ELECTROPHORETIC GELS Large-scale column chromatographic separation of neurophysins can be achieved conveniently only with species in which it is practical to collect sufficient quantities of posterior pituitary gland tissue. When small amounts of tissue are available, soluble proteins can be resolved by either starch or polyacrylamide gel electrophoresis and neurophysin components tentatively identified by immunological techniques (Cheng and Friesen, 1971a; Ellis et al., 1972). Microgram quantities of neurophysin extracted from these gels can then be used for the production of antibodies (Vaitukaitus et al., 1971). V. Production of Antibodies against Neurophysin A. CROSS-SPECIES REACTIVE ANTIBODIES

1. Ox Neurophysin a. Crude Preparation of Neurophysins as Antigen. The first report of the raising of cross-species reactive antibodies to neurophysins was by Fawcett et al. (1968). These workers gave a total of 42 mg of a crude mixture of ox neurophysin to each of a series of rabbits over a period of 6 months. Antibodies collected after this period crossreacted with beef neurophysin and dog posterior pituitary lobe homogenates as demonstrated by microimmunodihsion on agar gels. However, the antisera also reacted with a high-molecular-weight protein fraction (fraction A), indicative of the presence of serum albumin in the initial neurophysin preparation. It must also be pointed out that the protein-hormone complex used by these workers was prepared according to the method of Acher and co-workers (1958), and therefore the neurophysin was subjected to partial degradation by tissue enzymes. Legros et al. (1969), using a total bovine neurophysin mixture as

IMMUNOHISTOCHEMISTRY OF NEUROPHYSIN

25 1

prepared by the method of Fawcett et a2. (1968), raised antibodies in rabbits according to the protocol in Table I. Their antisera crossreacted with components in human serum and extracts of rat neurohypophysis (Legros et al., 1971a,b). b. Purified Ox Neurophysin Antigen. The purified ox neurophysin preparation used by Martin et al. (1972) as a source of antigen was extracted by the method suggested by Wuu and Saffran (1969). Antibodies raised against ox neurophysins cross-reacted immunologically with extracts from the posterior pituitaries of humans, sheep, guinea pigs, and rats. It is worthy of note that nasal insufflation of acetone-dried posterior pituitary tissue (pituitary snuE), used in the treatment of diabetes insipidus, can result in the production of antibodies to the various protein constituents of the preparation (Pepys et al., 1965; Mahon et al., 1967). Using an antiserum raised against a purified mixture of ox neurophysins, Martin (1971) determined the antibody titer of the peripheral serum of patients who had been treated, for periods up to 45 years, with a commercial preparation of a mixture of bovine and porcine posterior pituitary lobe powder (Pitressin, Parke Davis). In view of the finding that aqueous Pitressin contains neurophysin at a level of approximately 10 pg/ml, it is therefore not unexpected that prolonged administration of the preparation elicits antibodies to neurophysins in humans. 2. Pig Neurophysin a. Purified Pig Neurophysin Antigens. The high degree of antigenicity of porcine neurophysin I1 is reflected in the relative ease with which this protein elicits the antibody response in rabbits. Four weeks after a single injection of this antigen, precipitating antibodies can be detected (see Section V,B,2). Multiple injections of porcine neurophysin I1 give rise to an antiserum which cross-reacts with neurophysins extracted from the posterior pituitary glands of rats (Norstrom et al., 1971; Watkins and Evans, 1972) and sheep (Livett and Parry, 1971; Watkins, 1973b). The cross-species reactivity of the antineurophysin serum was further demonstrated by its ability to form insoluble antibody-antigen complexes with neural lobe proteins obtained from a selection of domestic and exotic mammalian species (Ellis et al., 1972). In a similar study antibodies raised against peptide I1 (porcine neurophysin I) cross-reacted with homogenates from the neural lobes of several mammals (Cheng and Friesen, 1971a).

METHODS USED Neurophysin antigen

TABLE I FOR THE RAISING OF ANTIBODIES IN RABBITS AGAINST

Protocol'

MAMMALIAN NEUROPHYSINS Cross-reactivity

Reference

Bovine mixture

6 mg in Freund's complete adjuvant into toepads, 6 mg i.v. 1month later, 9 mg i.m. at second and third month, and 6 mg i.v. at fourth and sixth month; bled 5 days after each injection

Ox, dog

Fawcett et al. (1968)

Bovine mixture

Antigen in Freund's complete adjuvant injected twice a week for 3 weeks into toepads, followed by monthly i.p. injections

Man, ox, dog, rat, pig

Legros et al. (1969, 1971a,b)

Bovine mixture

1 mg antigen in Freund's complete adjuvant injected into neck every 2 weeks for 6 months; bled 10-14 days after each injection

Ox, man, pig, sheep, rat, guinea pig

Martin et al. (1972)

Bovine I

1 mg antigen in Freund's complete adjuvant injected into toepads and back weekly for six weeks and then monthly for six times; bled 1 week after each monthly injection

Bovine I, monkey

Robinson et al. (1971); Zimmerman et al. (197313)

Bovine I1

As for bovine I

Bovine I1

Robinson et al. (1971)

Porcine mixture

20 mg antigen in Freund's complete adjuvant injected into back each fortnight for three times and boosters given at intervals of 3-4 months

Pig

Ginsburg and Jayasena (1968)

Rat, rabbit, guinea pig, dog, sheep, ox, monkey, man

Cheng and Friesen (1971a)

Porcine I 2 mg of either antigen in Freund's adjuvant injected weekly (peptide 11) on four occasions into dorsal surface, followed by an i.v. and 11 injection of 2 mg; blood collected 10 days later (peptide 111)

E;; N

9 m 4

v)

Porcine I1

1.5 mg of neurophysin in Freund's complete adjuvant injected S.C. twice at fortnight intervals; blood collected 1 month after the final injection

Porcine I

Ovine 111

"

Ellis et al. (1972)

Porcine I, 1% against porcine I1

As for porcine I

Porcine 11, 0.1% against porcine N-I

Pickup et al.

Warthog

Uttenthaland Hope (1972)

1.5 mg antigen in Freund's complete adjuvant injected S.C. twice at fortnightly intervals; blood collected 1 month

Sheep, rat

Watkins and Evans (1972)

2 mg of neurophysin suspended in Freund's adjuvant injected weekly for four times into back, followed by i.v. booster of 2 mg of antigen; blood collected 10 days later

Human I and I1

after final injection

Human I

Watkins and Evans (1972)

0.5 mg neurophysin in Freund's adjuvant injected S.C. into a single site; 5 weeks later 0.5 mg antigen bound to polymethylmethacrylate particles injected i.v.; blood collected 1 week later

Porcine I1

Giraffe, muntjac deer, coatimundi, pig, hippopotamus, ox, sheep, fallow deer, dog, cat, mouse, rat, guinea pig, dama wallaby, pig-tailed macaque, woolly monkey, chacma baboon, man, rabbit, hedgehog

i.v., Intravenously; i.m., intramuscularly; i.p., intraperitoneally; s.c., subcutaneously.

(1973);

Livett et al.

(1971)

z 3c z

zce

U

E

Cheng and Friesen (1973) Q .t

u(

w

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W. B. WATKINS

3. Sheep Neurophysins a. Purified Sheep Neurophysin Antigen. Antisera raised in rabbits by using multiple doses of the major sheep neurophysin, neuro-

physin 111, as antigen cross-reacted with neurophysins from the rat (Watkins and Evans, 1972) and guinea pig (Evans and Watkins,

1973).

4. Human Neurophysins a. Purified Human Neurophysin Antigen. In the development of a homologous radioimmunoassay for human neurophysin, Cheng and Friesen (1973) used purified human neurophysins I and I1 as antigens for antibody production. Both antisera cross-reacted with each antigen. ANTIBODIES B. SPECIES-SPECIFIC

1. Ox Neurophysin A radioimmunoassay specific for bovine neurophysins I and I1 has recently been developed by Robinson et al. (1971).The specific an-

tisera were raised by giving six weekly injections of the antigen into the back and footpads of rabbits. Although there was minimal crossreactivity of antineurophysin I serum with neurophysin 11, the antiserum cross-reacted with human and monkey neurophysin (Robinson and Zimmerman, 1973). Earlier work by Livett et al. (1971) failed to produce precipitating antibodies against either bovine neurophysin I or I1 after the use of various injection regimes.

2. Pig Neurophysin Antibodies produced against the pig neurophysin isolated by Ginsburg and Jayasena (1968) were found to be species-specific and did not cross-react with proteins extracted from the rat, ox, or guinea pig neurohypophysis. In view of the massive doses required for antibody production, and the method used for preparation of the antigen, it is questionable whether the antibodies raised were in fact against pig neurophysin. Furthermore, these workers also obtained cross-reactivity against extracts of kidney, uterus, and mammary gland. The presence of neurophysin in these tissues has not been confirmed by other workers (Livett et al., 1971; Robinson et al., 1971). If 0.5 mg of purified pig neurophysin I1 is injected into a single site in rabbits, the antisera collected after 1 month cross-react specifically with the antigen as determined by microimmunodihsion on

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agarose (W. B. Watkins, unpublished results). A week later the titer of the antiserum can be increased by an intravenous injection of the neurophysin bound to polymethylmethacrylate particles (Livett et al., 1971). The warthog, which has the same phylogeny (suborder Suiformes) as the domestic pig, is the only other species so far studied that has a neurophysin possessing immunological determinants sufficiently similar to those in porcine neurophysin I1 to form a precipitin line with this antiserum (Uttenthal and Hope, 1972). Using the injection regime developed for the production of specific antibodies against porcine neurophysin 11, Pickup and colleagues (1973) obtained an antiserum against porcine neurophysin I with only 1% cross-reactivity against porcine neurophysin 11.

VI. General Considerations of Antibody Production and Detection The success of any protocol employed in the raising of antibodies is only as good as the techniques available for detection of the antibodies. The most convenient method for determining the presence of many antibodies is by microimmunodihsion or microimmunoelectrophoresis on a supporting matrix such as agar, agarose, or cellulose acetate (Ouchterlony, 1968).This method, however, is suitable only for those antibodies that form insoluble complexes with the antigens. During an extensive study carried out in our laboratory on the raising of antibodies to neurophysins, it became apparent that not all the antibodies produced give immunoprecipitates with the antigen. In fact, we showed (H. K. Ellis and W. B. Watkins, unpublished work) that an antiserum that did not give a precipitin line possessed a higher titer (as determined by radioimmunoassay) than another antiserum that gives a strong precipitin line in the Ouchterlony system. The most sensitive method for antibody detection is undoubtedly the displacement of iodinated neurophysin by the addition of “cold” antigen. The failure of Livett et al. (1971) to raise antibodies against ox neurophysins probably lay in their inability to detect such species. This is supported by the finding that the antibodies to bovine neurophysin I and I1 (Robinson et al., 1971) did not readily give precipitin lines with the corresponding antigen (A. G. Robinson, personal communication). In order to raise the titer of circulating antibodies, Cheng and Friesen (1971a)gave a booster intravenous injection a week prior to the bleeding of the animal. Since intravenously administered neuro-

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physin is rapidly cleared by the kidney and has a half-life of only approximately 3 minutes (Forsling et al., 1973), the effect of such a booster may be expected to be minimal.

VII. Immunohistochemical Techniques A. LIGHT MICROSCOPELEVEL

1. lmmunofluorescence Histochemisty The technique of immunofluorescence histology is a powerful tool in the detection of trace amounts of tissue antigen (for a review, see Nairn, 1969). Two approaches are available for fluorescent protein tracing. (1) The direct method involves the conjugation of antineurophysin serum with a fluorochrome such as l-dimethylaminonapthalene-5-sulfonic acid or fluorescein isothiocyanate. This conjugate is then applied to the tissue section, and the fluorescence observed using a fluorescence microscope. (2) The difficulties often encountered in conjugation of the antisera to the fluorochrome can be overcome by using the indirect or “sandwich technique.” The tissue containing neurophysin antigens is first treated with antineurophysin serum which acts as the middle layer of the “sandwich.” After 30 minutes excess antiserum is washed from the tissue, and then commercially available sheep antirabbit yglobulin coupled to fluorescein isothiocyanate is applied (at a dilution of one-fifth to one-tenth) to the section. Excess stain is removed after 30 minutes, and after the sections are mounted in a glycerolsaline mixture (Kawamura, 1969) they are ready for observation. Areas containing neurophysin appear yellowish green against a paleblue background. Serum components, which often give rise to nonspecific absorption onto the tissue, can often be eliminated by treatment of the antiserum with acetone-dried liver powders. The specificity of the antiserum reaction is confirmed using preimmune serum and fluorochrome alone. 2. lmmunoperoxidase Histochemistry The immunoperoxidase technique described by Nakane (1968)has been used, with several modifications by Zimmerman et al. (1973b), for the cellular localization of neurophysin. In the two-layer method

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the tissue is first treated with antineurophysin serum followed by horseradish peroxidase-labeled sheep antirabbit yglobulin. Treatment of the peroxidase conjugate with 3,3’-diaminobenzidine in the presence of hydrogen peroxide gives a brown deposit representing the position of neurophysin. With the three-layer technique the tissue is treated sequentially with the following reactants: antineurophysin serum, sheep antirabbit y-globulin, rabbit antiperoxidase, peroxidase, and finally 3,3’-diaminobenzidine in hydrogen peroxide. In contrast to immunofluorescence histochemistry, immunoperoxidase sections are permanent and can be counterstained.

B. ELECTRONMICROSCOPE LEVEL Although the immunoperoxidase method has been successfully applied to the detection of tissue antigens in ultrathin sections (Kawarai and Nakane, 1970; Nakane, 1971), the localization of neurophysin in the HNS has not been reported. C. PHOTOGRAPHICPROCEDURES The fluorescent micrographs reported from this laboratory were all taken through a Leitz Laborlux fluorescent microscope fitted with UG-1 and BG-38 primary filters and a K430 secondary filter. Photographs were taken with either Kodak High Speed Ektachrome at exposures of approximately 2-3 minutes and developed at 160 ASA, or Kodak Tri-X film exposed at 10-15 seconds. A major disadvantage of the immunofluorescence technique is the difficulty in recording, on photographic emulsion, areas of weak fluorescence. This problem is not encountered when the immunoperoxidase procedure is used.

D. PREPARATION

OF

TISSUES

1. Cyostat Sections Neurophysin was first demonstrated by immunofluorescence histochemistry on fresh-frozen tissue of pig hypothalamus and posterior pituitary gland (Livett et al., 1971). Subsequent work from this laboratory on the distribution of neurophysin in the rat (Watkins and Evans, 1972) and guinea pig (Evans and Watkins, 1973) was carried out on cryostat sections from fresh-frozen tissues. The freezing of tissues by immersion in a slurry of dry ice-acetone often results in

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FIG.2. Immunofluorescence localization of neurophysin-containing cells close to the third ventricle of the guinea pig hypothalamus. (a) Transverse sections were cut from snap-frozen tissues. (b) Hypothalamic tissue was fixed in saline-formalin prior to paraffin embedding. The tissues were stained with rabbit antiovine neurophysin 111 and fluorescein-labeled sheep antirabbit yglobulin. x 170. (From Watkins and Evans, 1974.)

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damage due to ice crystal formation. These artifacts are more likely to occur in the freezing of large blocks of tissue such as hypothalamus from large animals. It is our experience that even faster freezing in liquid nitrogen causes the tissue to become brittle and fragment. Immunofluorescence staining of neurophysin in cryostat sections of hypothalamus often appears diffuse and lacks discrete morphological integrity (Fig. 2a). Postfixation of cryostat sections with 86-92% ethanol does not destroy the antigenicity of neurophysin antigens (Livett et al., 1971; Watkins and Evans, 1972; Evans and Watkins, 1973). Livett et al. (1971), however, found that fixation in formalin-saline, Bouin’s fluid, chloroform-methanol (2:l), and 70 and 100% ethanol gave unsatisfactory results. 2. Paraffin-Embedded Tissues Livett and Parry (1971) reported the successful use of 95% ethanol-fixed tissues embedded in paraffin for immunofluorescent localization of sheep neurophysin. Fixation of tissue in either salineformalin (pH 7.2) or Bouin’s reagent (pH 1.8) prior to embedding in paraffin dramatically increases the resolution and histological detail of the neurophysin-containing structures as compared with that of fresh-frozen tissues (Fig. 2b) (Watkins and Evans, 1974). Immunoperoxidase techniques are equally successful when applied to sections fixed in saline-formalin or Bouin’s fluid (Zimmerman et al., 1973b).There is now a growing realization that many proteins retain their immunoreactivity after treatment with formalin. Beck et al. (196913) demonstrated the presence of human placental lactogen by immunofluorescence in formalin-fixed placentas, and Pasteels et al, (1972) reported excellent results when studying growth hormone and prolactin by immunofluorescence in formalin-treated pituitary tissues that had been stored for extended periods in paraffin blocks. These findings are in contrast to Pearse’s (1961) statement: “Some antigens can withstand prolonged formalin fixation and remain antigenic enough to be demonstrated in routine paraffin embedded sections. There are exceptions, however, and for most work short precipitation of the protein in the section with ethanol is all that is required.” One advantage of the fluorescence technique over the immunoperoxidase system is that staining for NSM with A F can be performed on the same slide after the immunohistochemical reaction has been carried out.

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VIII. Demonstration of Neurophysin in the Hypothalamoneurohypophysial System Using Cross-Species Reactive Antineurophysin

A. MAGNOCELLULAR NUCLEI

1. Normal Animals Paraventricular (PVN) and supraoptic (SON) nuclei have generally been recognized to be associated with the synthesis of oxytocin and vasopressin, respectively (Olivecrona, 1957; Aulsebrook and Holland, 1969; Sokol, 1970; Bisset et al., 1971). Sokol and Valtin (1967) presented evidence for synthesis of neurohypophysial hormones within separate neurons of the rat. Later it was shown that NSM was still present in the posterior pituitary lobes of Brattleboro rats after destruction of the PVN, indicating a limited synthesis of oxytocin in the SON (Sokol, 1970). Data available on the vasopressinloxytocin ratio in the PVN and SON (Lederis, 1962; Bisset et al., 1973) indicate that the SON are more specific for the production of vasopressin than the PVN are for the production of oxytocin. This has been demonstrated in the guinea pig (Tindal et al., 1968) and cat (Bisset et al., 1967, 1970, 1971), in which electrical stimulation of the hypothalamic nuclei caused the release of both oxytocin and vasopressin from the PVN and only vasopressin from the SON. Since neurophysins are closely associated with the synthesis, storage, and release of neurohypophysial hormones (for reviews, see Ginsburg, 1968; Sachs, 1969),it is expected that neurons in the brain containing the hormones also represent sites of neurophysin. Vice versa, the detection of neurophysinlike antigens, by sensitive immunohistochemical techniques, offers a method for the identification of the neuronal elements that should contain oxytocin and/or vasopressin. The apparent specificity of the SON for the production of vasopressin might be reflected in their association with a specific neurophysin (see Section IX,A,B). The concept of one neurophysin being specifically associated with one hormone was proposed when it was found that the ox posterior pituitary gland contains two major neurophysins present in ratios approximating the ratios of the hormones (Hollenberg and Hope, 1968). Furthermore, evidence has been presented for the storage of oxytocin with neurophysin I and of vasopressin with neurophysin I1 in separate ox neurosecretory granules (Dean et al., 1968a). The pig also has two major neurophysins (Uttenthal and Hope, 1970), each of which may be as-

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FIG.3. Immunofluorescence in the cell bodies and processes of sheep PVN (a) and SON (b). The sections were taken from saline-formalin-fixed tissues, and the immunofluorescence carried out by the “sandwich” technique using antiporcine neurophysin I1 serum. x 170. (From Watkins, 1973b.)

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sociated with one or the other of the neurohypophysial hormones (Pickup et al., 1973).Both of the two major neurophysins in the rat also appear to have functional relationships with the hormones (Burford et al., 1971). More recent studies on the relative proportion of neurophysins present in other species (Ellis et al., 1972) suggest that the simple stoichiometric relationship between molar ratios of oxytocin and vasopressin and of the two major neurophysins found in the ox (Dean et al., 196813)and rat (Burford et al., 1971)may not be common among all mammals. Magnocellular nuclei can readily be identified by the intense fluorescent reaction obtained with acridine orange (Bertalanffy, 1960; Evans and Watkins, 1973) as a result of its interaction with cellular DNA and RNA. Intense immunofluorescence in the magnocellular cells of the PVN and SON of the rat (Watkins and Evans, 1972), dog (Alvarez-Buylla et al., 1973), guinea pig (Evans and Watkins, 1973), and sheep (Watkins, 1973b)(Fig. 3a and b), indicates the presence of neurophysinlike proteins. Occasionally, sections of nuclei were obtained that possessed a distinct speckled appearance (Fig. 4). Resolution of the cell nuclei and processes emanating from the perikaryon of the sheep SON (Fig. 3b) are clearly seen after formalin fixation of

FIG.4. Speckled nature of the structures containing neurophysin in guinea pig PVN as revealed by immunofluorescence histology. Cryostat section from a freshfrozen tissue block. x 170.

FIG.5. Transverse section of a rhesus monkey hypothalamus treated by the threelayer immunoperoxidase bridge method for the demonstration of neurophysin using antibody to bovine neurophysin I. Neurophysin is present in the PVN and SON concentrated near blood vessels (arrows) and in neurons of the paraventricular tract (PVT) optic tract (OT), and third ventricle (111). X42. (From Zimmerman et al., 1973b.)

FIG.6, Higher magnification of the PVN in Fig. 5. Note the varying amounts of immunoreactive neurophysin present in the magnocellular nuclei. x575. (From Zimmerman et al., 197313).

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the tissue. The antisera used for these studies cross-reacted with each of neurophysins present in the various animals, Neurophysin was also demonstrated in the cells of monkey PVN and SON by Zimmerman et al. (1973b) using the three-layer immunoperoxidase bridge method (Figs. 5 and 6) in association with an antiserum raised against bovine neurophysin I. Beaded segments of nerve fibers can be seen leaving the PVN and SON (Fig. 6). Areas around several blood vessels also stain for neurophysin. Certain magnocellular cells in both PVN and SON of ox and monkey were found to contain only small amounts of neurophysin, while a few others were almost completely devoid of immunoreactive protein (Zimmerman et al., 1973b). The significance of these cells, which have also been observed in the guinea pig (Watkins and Evans, 1974), is not known. Do they represent cells with an inherent reduced level of protein synthesis? Or are they associated with highly active secretion of their contents? Could they be cells in a “resting phase”?

2. Dehydrated Animals In our studies on the distribution of neurophysin in rats (Watkins and Evans, 1972) and guinea pigs (Evans and Watkins, 1973), the intensity of neuophysin immunofluorescence in the PVN and SON of normal animals did not differ markedly from that observed in an animal deprived of water for extended periods. The effect of osmotic stimulation on the content of NSM and neurohypophysial hormones in the hypothalamus appears to be in conflict, Dehydration of dogs (Ortmann, 1951; Hild and Zetler, 1953) causes depletion of NSM in the magnocellular nuclei. These findings are in contrast with those reported by Andersson and Jewel1 (1957), who showed that in excessively hydrated dogs a high proportion of the cell bodies in the PVN and SON are also depleted of NSM. Fendler et al. (1968) failed to observe a depletion of NSM in the rat magnocellular system after water deprivation, nor was Diamond (1956) able to demonstrate a loss of antidiuretic hormone activity in dehydrated rat hypothalamus. In rats dehydrated for 10 days (Vilhardt, 1970), there was approximately an 80% reduction in vasopressin content of the hypothalamus. However, Cheng et al. (1972) measured a fivefold increase in neurophysin content of the PVN after dehydration. There was no significant increase in the neurophysin content of the PVN. Dehydration of rats has been shown to have the effect of increasing the in vivo uptake of cystineand cysteine-=S into the PVN and SON (Wells, 1963; Talanti,

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1971).There is also an increase in the amount of c ~ s t e i n e - ~incor~S porated into vasopressin during the in uitro incubation of hypothalamic slices obtained from dehydrated guinea pigs 4 days (Takabatake and Sachs, 1964). After an 8-day dehydration, however, the incorporation of radioactive label was less than normal. The inability to observe a marked change in the intensity of neurophysin immunofluorescence in the main body of the PVN and SON on dehydration might be due in part to the nature of the immunologically active components present in the cytoplasm of the perikaryon. Since the chemical nature of neurophysin antigens in the cytoplasm is unknown, a direct correlation with the neurophysin ex-

b

C

d

FIG. 7. Diagrammatic representation of transverse sections of the guinea pig hypothalamus showing areas of cells (represented by hatching) which contain neurophysin demonstrated by immunofluorescence histochemistry using antiserum raised against porcine neurophysin 11. OC, Optic chiasma; SOC, supraoptic complex; ASO, accessory supraoptic nucleus; SO, supraoptic nucleus; PV, paraventricular nucleus; APV, accessory paraventricular nucleus; OT, optic tract; 111, third ventricle; F, fomix. (a) and (e) represent the most rostra1 and caudal positions, respectively. Tracts of neurosecretory fibers are also represented in (c) and (d). (From Evans and Watkins, 1973.)

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tracted from the neural lobe may not be valid. It has been our experience in guinea pigs, however, that immunoflurorescent cells situated away from the main areas of the PVN and SON (see Section VIII,B,l) are more easily visualized in the dehydrated animal. Beaded fibers also become more apparent in the stimulated animal. These findings are suggestive of increased protein biosynthesis as a result of osmotic stress. B. OTHER AREAS OF

THE

HYPOTHALAMUS AND BRAIN

1. Normal Animals In a recent study we cut transverse sections of guinea pig hypothalamus from extreme rostral to caudal positions and observed those regions outside the main area of the PVN and SON that contained neurophysin (Evans and Watkins, 1973). Neurophysinlike material was seen at the extreme rostral part of the hypothalamus and in a few cells positioned ventral and closely dorsal to the third ventricle (Fig. 7a). Advancing caudally, the cells of the SON became apparent. The anterior portion of the SON observed in several animals had a fi-

FIG. 8. Beaded fibers in the anterior SON of the dehydrated guinea pig. Neurophysin is demonstrated using antiovine neurophysin I11 serum in association with fluorescein-labeled sheep antirabbit y-globulin. Section from fresh-frozen tissue. X 170. (From Evans and Watkins, 1973.)

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FIG.9. Specific neurophysin immunofluorescence in cells of the guinea pig. (a) Supraoptic complex. (b) Posterior SON in a position medial to the optic tract. (c) Anterioventral portion of PVN. Sections were from snap-frozen tissue. X130. (From Evans and Watkins, 1973.)

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FIG.10. Immunofhorescent staining of cells extending from the third ventricle to an area dorsal to the fomix (a), of accessory PVN (b) in a position ventral to the accessory PVN and between the fornix and optic tract of the guinea pig (c).The tissue was snapfrozen prior to sectioning. x 130. (From Evans and Watkins, 1973.)

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brous appearance due to the presence of bundles of nerve fibers (Fig. 8). Other cells lying dorsal to the optic chiasma between the SON and the third ventricle (Figs. 7b and 9a) often gave rise to immunofluorescence and corresponded to the “supraoptic complex” described in the rabbit by Ford and Kantounis (1957). The posterior SON medial to the optic track also gave rise to fluorescence (Figs. 7e and 9b). In the region of the accessory SON (Bandaranayake, 1971) (Fig. 7b), a few fluorescent cells were seen. Neurophysin-containing cells of the anterioventral portion of the PVN (Fig. 7c) often stained in a diffuse manner, as shown in Fig. 9c. Fibers spread laterally from the PVN and around the fornix in a similar manner, as demonstrated in the vole (Clarke and Kennedy, 1967) and guinea pig (Knaggs et al., 1971) by using A F stain. Proceeding further posteriorly into the hypothalamus, immunofluorescent cells were seen extending from an area close to the third ventricle to a position just dorsal to the fornix (Figs. 7d and 10a); the group of cells regarded as the accessory paraventricular nucleus (Fig. 7d) were also immunoreactive against antineurophysin (Fig. lob). A few cells ventral to the accessory paraventricular nucleus between the fornix and the optic tract were sometimes seen to contain neurophysin (Fig. 1Oc). 2. Neurophysin in the Brain of the Scrapie Sheep In sheep affected with natural scrapie there is degeneration of the mossy terminals within the granular layer of the cerebellar cortex (Beck et al., 1969a). Livett and Parry (1971) observed neurophysinspecific immunofluorescence in this part of the cerebellum, and also in an area of the third cranial nerve. It is uncertain whether this material is identical to the neurophysin present in the magnocellular nuclei or even how the neurophysinlike protein reaches the cerebellum. Does the protein result from local synthesis in the hindbrain by neurons with synthetic capabilities similar to those of the PVN or SON? Or does it accumulate as a result of axonal transport from the hypothalamic nuclei? The loss of neurons in the PVN and SON of scrapie sheep (Beck et al., 1964) is associated with reduced levels of neurophysin as demonstrated by immunofluorescence histology (Livett and Parry, 1971). C. PROXIMALNEUROHYPOPHYSIS

1. Median Eminence and Internal Infundibulum The hypothalamo-distal neurohypophysial system originates in the magnocellular cells of the hypothalamus. This neurosecretory path-

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FIG.11. Coronal sections of sheep median eminence. Neurosecretory fibers containing neurophysin emanating from cells of the supraoptic nucleus (a) and proceeding down the median eminence (b). Herring bodies are marked with arrows. ~ 1 7 0 . (From Watkins, 1973b.)

way, which is also referred to as the supraopticoneurohypophysial system, consists of nerve fibers which descend from the hypothalamus via the internal zone of the infundibulum and terminate in the infundibular process (posterior pituitary gland). It is by this main neurosecretory pathway that the neurohypophysial hormones, together with neurophysin, enter the posterior pituitary lobe. Nerve fibers in the hypothalamus leaving the SON readily stain for neurophysin (Fig. lla). Herring bodies associated with these axons become more apparent as the fibers enter the median eminence (Fig. llb).

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FIG.12. (a) Neurophysin present on the ventricular surface of the monkey third ventricle and in the cytoplasm of tanyacte perikarya as demonstrated by the immunoperoxidase technique. x504. (From Robinson and Zimmerman, 1973.) (b) Immunofluorescence localization of neurophysin in tanyacte processes running laterally between the third ventricle (111) and the supraopticohypophysial tract (soh) of the sheep. X 170. (From Watkins, 1973b.)

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Kobayashi and his colleagues (1970) demonstrated the uptake of horseradish peroxidase into ependymal cells of the third ventricle when the tracer was injected into the ventricular spaces of the rat and quail. Since neurophysin has a molecular weight of about onequarter that of horseradish peroxidase, it too would be expected to be absorbed from the cerebrospinal fluid. Using the Gomori stain, Talanti and Kivalo (1961) showed the presence of NSM in the ependymal cellular layer of the infundibular recess and extending to the third ventricle of the camel. This work was extended by Robinson and Zimmerman (1973) in the monkey (Fig 12a) and by Watkins (197313) in the sheep (Fig. 12b), and confirmed the presence of neurophysin in the cytoplasm of tanyacte perikarya and in tanyacte processes running perpendicular to the ventricle surface and lateral to the supraopticoneurohypophysial secretory system. The role of the tanyactes is still in question, but several workers (Anand Kumar and Knowles, 1967; Knowles and Anand Kumar, 1969; Porter et al., 1970) have suggested that they may play an endocrine role in providing a pathway from the cerebrospinal fluid to the portal system of the hypothalamus. In fact, Knowles and Anand Kumar (1969) traced tanyacte processes from the third ventricle to positions abutting onto the pericapillary spaces of the pituitary portal plexus blood vessels. These terminal processes are part of the external palisade layer of the median eminence-a region directly involved in the release of hypothalamic releasing factors (see Section VIII,C,2). 2. External Znfundibulum Nerve fibers of the hypothalamo-proximal neurohypophysial system originate mainly in the infundibular (acuate) nucleus of the hypothalamus. The tuberoinfundibular tract initially proceeds along the internal zone of the infundibulum, together with the supraopticoneurohypophysial tract, and then turns into the external zone of the median eminence where the fibers terminate close to the mantle capillary plexus. Under normal conditions the external infundibulum gives a negative reaction for NSM using histochemical stains. However, there is an increase in the NSM following bilateral adrenalectomy and hypophysectomy (Bock and Muhlen, 1968; Bock et al., 1969; Bock and Forstner, 1969; Brinkmann and Bock, 1970). This accumulation of Gomori-positive granules can be reduced in the hypophysectomized animal by administration of adrenocorticotrophin hormone, and in the adrenalectomized animal by the application of corticosteroids (Bock et al., 1969), which implies that the amount of NSM in the external infundibulum is directly

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FIG. 13. Sagittal sections of sheep infundibulum stained for neurosecretory material and neurophysin. (a) AF stain for neurosecretory material in the infundibulum. ~250.(b) Immunofluorescence of neurophysin present in the external zone (ze) and internal zone (zi). pt, Pars tuberalis; ir, infundibular recess. X170. (From Watkins, 1973b.)

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related to the presence of corticotrophin-releasing factor (CRF). While the external zone of the sheep infundibulum is devoid of NSM as demonstrated by AF (Fig. 13a), specific neurophysin immunofluorescence extends from an area near the infundibular recess to the external infundibulum (Fig. 13b) (Watkins, 1973b). In sheep affected with scrapie, there is an estimated twofold increase in the intensity of the immunofluorescence in the external infundibulum as compared with that in the normal animal (Livett and Parry, 1973; Parry and Livett, 1973). The presence of neurophysin in both regions of the infundibulum has been confirmed in the normal monkey by Zimmerman et al. (1973a) using the immunoperoxidase method (Fig. 14). These workers also found that the high concentration of immunoreactive neurophysin present around the monkey portal capillaries of the median eminence was reflected in the portal vessel levels of neurophysin being approximately 25 times greater than that in the systemic circulation. With the now confirmed presence of neurophysinlike molecules in the external infundibulum of normal animals, it is tempting at this stage to speculate upon a functional significance for the proteins.

FIG.14. Neurophysin concentrated around the portal capillaries in the external zone of the median eminence (ze) of the monkey as revealed by immunoperoxidase techniques. X504. (From Zimmerman et al., 1973b.)

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Although CRF has so far eluded isolation and biochemical characterization, it is believed to possess a structure similar to that of vasopressin (Schally et al., 1962; Schally and Bowers, 1964). It is therefore conceivable that the synthesis of CRF may also be associated with the elaboration of higher-molecular-weight protein. This protein may be sufficiently similar to neurophysin to be able to cross-react immunologically with antineurophysin serum.

D. PITUITARYSTALK The neurosecretory fibers from the PVN and SON converge as they enter the region of the pituitary stalk and appear as a mass of immunofluorescent material (Fig. 15).

The concept of the hypothalamic neurosecretory cells providing the posterior pituitary lobe with neurophysin by somatofugal transport has recently been verified by Alvarez-Buylla et al. (1973). Immunofluorescence histology was applied to parasagittal sections cut from a block of tissue containing the hypothalamus attached to the posterior pituitary gland obtained from a dog 20 hours after the pituitary stalk had been constricted. There was an accumulation of neurophysin proximal to the constriction, in contrast to the region of the stalk immediately distal to the crush, which was devoid of im-

FIG.15. Convergence of the fibers of the supraopticoneurohypophysialtract in the pituitary stalk of the sheep. Saline-formalin-fixed tissue. x 170. (From Watkins, 1973b.)

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FIG.16. Montage showing the distribution of neurophysin immunofluorescence in the pituitary stalk of a dog 20 hours after the stalk had been constricted (arrows). Snapfrozen tissue. (From Alvarez-Buylla et al., 1973.)

munofluorescent material (Fig. 16). Six days after stalk constriction, the amount of neurophysin in the hypothalamus and stalk proximal to the crush was twofold greater than that in the sham-operated animal, clearly indicating a proximal-distal flow of neurophysin.

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E. POSTERIORPITUITARYGLAND The distribution of neurophysin within the posterior pituitary gland does not appear to be the same for all species studied. In the guinea pig neurophysin distribution had a distinct lobular appearance (Fig. 17a) similar to the pattern of NSM (Fig. 17b) revealed with AF. In this respect the guinea pig resembles the opposum (Bodian, 1951; Roth and Luse, 1964), whose NSM also has a lobular appearance. This is in contrast to the closely packed neurophysin found in the rat (Watkins and Evans, 1972) (Fig. 18a) and NSM present in the dog (Ortmann, 1951) posterior pituitary gland. Osmotic stimulation of an animal by either removing the drinking water or by salt loading, results in a progressive decrease in the amount of neurophysin concomitant with loss of the neurohypophy-

FIG. 17. Distribution of immunofluorescent neurophysin (X130) (a) and NSM (b) 150) in the posterior pituitary lobe of the guinea pig. The immunofluorescent study was carried out on fresh-frozen tissue, while the NSM was demonstrated on salineformalin-fixed tissue. (X

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FIG.18. Effect of dehydration on the distribution of neurophysin [(a) and (b)] and neurosecretory material [(c)and (d)] in the posterior pituitary lobe of the rat. Normal animal [(a) and (c)] and animal dehydrated for 7 days [(b) and (d)], immunofluorescence [(a) and (c)]. (X238). AF stain [(b) and (d)] (X224). (Adapted from Watkins and Evans, 1972.)

sial hormones and AF material (Watkins and Evans, 1972; Evans and Watkins, 1973) (Fig. 18a-d).

IX. Use of Species-Specific Antisera for the Demonstration of Neurophysin A. PIG Using an antiserum that specifically cross-reacted against porcine neurophysin 11, Livett et al. (1971) showed that immunofluorescence

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was localized principally in the neurosecretory neurons arising from magnocellular SON. A few scattered cells close to the third ventricle in the region of the PVN also reacted. Since the bulk of the neurosecretory cells of the PVN did not cross-react with the antiserum, it was suggested by these workers that porcine neurophysin I1 may be associated with 8-lysine-vasopressin which was thought at the time to be the main hormone of the SON. However, more recent findings by Johnston et al. (1972) have confused this simple association of neurophysin I1 with vasopressin. Dissection of SON and PVN from cryostat sections of pig hypothalamus was performed, and the levels of oxytocin, 8-lysine-vasopressin, neurophysin I, and neurophysin I1 determined. It was found that both neurophysins and the neurohypophysial hormones were present only in the SON, and it is therefore difficult in the case of the pig to associate specifically one neurophysin with one hormone solely based on immunofluorescent histochemical studies.

B. Ox Antisera raised against bovine neurophysin I has been used to investigate the cellular distribution of neurophysin in ox brains using the immunoperoxidase technique. Neurophysin I was localized in both the SON and PVN (Fig. 19a and b), although results from radioimmunoassay suggest that there is more neurophysin I1 than neurophysin I in the SON (Zimmerman et al., 1973b). The latter results, together with those obtained from the subcellular distribu-

FIG. 19. Immunoperoxidase localization of bovine neurophysin I in the ox. (a) SON ~ 3 4 4(b) . PVN ~ 3 4 5 (From Zimmerman et al., 1973b.)

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tion work of Dean et al. (1968a), partially confirm that the SON are more specialized in the elaboration of vasopressin than oxytocin, in accord with other species (Bisset et al., 1973).

X. Conclusions In this article the techniques of immunohistochemistry have been applied to study tissue distribution of neurophysin throughout the HNS. The sensitivity of the method enables one to visualize immunoreactive material not only in the perikarya of hypothalamic nuclei but also in the fine axons leading from the hypothalamus to the posterior pituitary lobe. This method has provided positive evidence for Bargmann’s initial concept of neurosecretion, in which protein material synthesized in the hypothalamic nuclei is conveyed by axonal transport to nerve terminals in the posterior pituitary gland. The presence of immunoreactive neuroph ysin in areas that are apparently devoid of NSM, as revealed by AF, clearly demonstrates the insensitivity of the traditional histochemical stains as compared with immunofluorescent techniques. Although much information has been obtained during the short 3-year history of the localization of neurophysin by immunofluorescence methods, several avenues are still open for investigation:

1. Application of immunoperoxidase techniques to thin sections using antisera specific to one neurophysin or another within a particular species will establish whether or not individual nerve fibers are specialized in the transport of specific neurophysins. 2. The results obtained from studying the ontogeny of neurophysins may be correlated with the presence of arginine vasotocin in certain fetal mammals (Vizsolyi and Perks, 1969). 3. The significance of neurophysinlike material in the hindbrain of neurologically disturbed animals is as yet unclear. 4. Perhaps the most intriguing aspect of all the neurophysin work published is the presence of neurophysin or molecules structurally similar to neurophysin in the external zone of the infundibulum. The confirmation of the presence of a carrier molecule for CRF will revitalize the study of the elusive releasing factor. ACKNOWLEDGMENTS

I thank the Medical Research Council of New Zealand and the Auckland Medical Research Foundation for financing this work. Miss V. Bailey is thanked for assistance

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