Co-existence of CRF and vasopressin immunoreactivity in parvocellular paraventricular neurons of rat hypothalamus

Peptides. Vol. 7, pp. 891-898, 1986. ~ Ankho International Inc. Printed in the U.S.A.

0196-9781/86 $3.00 + .00

Co-Existence of CRF and Vasopressin Immunoreactivity in Parvocellular Paraventricular Neurons of Rat Hypothalamus DIANE

T. P I E K U T

I AND SHIRLEY

A. J O S E P H

N e u r o e n d o c r i n e Unit, Center f o r Brain R e s e a r c h University o f R o c h e s t e r S c h o o l o f Medicine and Dentisto,. R o c h e s t e r , N Y R e c e i v e d 7 A p r i l 1986 PIEKUT, D. T. AND S. A. JOSEPH. Co-existence of CRF and vasopressin immunoreactivity in parvocelhdar paraventricular neurons of rat hypodmlamus. PEPTIDES 7(5) 891-898, 1986.---New dual immunocytochemical staining procedures were used in the same tissue section to elucidate the distribution and co-existence of CRF and vasopressin in parvocellular neuronal perikarya in the paraventricular nucleus (PVN) of rat hypothalamus. CRF immunostained cells were for the most part concentrated in the medial parvocellular component of PVN. Few vasopressin-immunoreactive (it) neurons were seen in this area in the normal and colchicine-treated animals. Vasopressin-containing neurons predominated in the magnocellular component of PVN. In the adrenalectomized and adrenalectomized-colchicine-treated animals, a dense accumulation of vasopressin-ir cells were observed in the medial parvocellular area of PVN ; this region is normally vasopressin-ir poor and CRF-ir rich. The vasopressin immunostained cells appeared to have an anatomical distribution similar to that seen for CRF-containing cell bodies. Results of this study unequivocally establish the co-existence of vasopressin and CRF in the same parvocellular perikarya of PVN following pertubation of the pituitary-adrenal axis. Brain Paraventricular nucleus Dual staining immunocytochemistry

Corticotropin-releasing factor

FOLLOWING the isolation and characterization of corticotropin-releasing factor (CRF) by Vale and c o - w o r k e r s [23,28], considerable progress has been made in our understanding of the control by C R F of the brain-pituitary-adrenal axis. C R F - i m m u n o r e a c t i v e fir) neurons have been localized in brain with a remarkable density of cells seen in the parvocellular c o m p o n e n t of the paraventricular nucleus (PVN) of rat hypothalamus [2, 3, 7, 9, 13, 15, 22]. T h e s e C R F - i r cells project to the zona externa o f median e m i n e n c e [1, 14, 16, 22] where their influence on anterior pituitary function is manifested. N e u r o h y p o p h y s i a l h o r m o n e s have been implicated in the control of the pituitary-adrenal axis, h o w e v e r little is known about the precise relationship of C R F and neurohypophysial peptidergic systems. Recent studies [11, 19, 22, 27] have provided anatomical e v i d e n c e for the presence of vasopressin-ir cells in the parvocellular area of PVN in the a d r e n a l e c t o m i z e d animal; this area is normally C R F - i r rich and vasopressin-ir poor except in the a d r e n a l e c t o m i z e d model. Disparate studies have generated c o n t r o v e r s y concerning the co-existence of these two peptidergic systems in the same neuronal cell body [1, 4, 5, 11, 19, 22, 26, 27]. Co-localization implies that two substances are found in

Vasopressin

separate perikarya but in the same region of brain, w h e r e a s co-existence indicates that two antigens are localized and probably synthesized within the same cell. The present study was designed to establish with clarity the co-localization versus co-existence of C R F and vasopressin in paravocellular neuronal perikarya in PVN using a new dual imm u n o c y t o c h e m i c a l m e t h o d for the staining of two antigens in the same section. This method e m p l o y s antibodies and the peroxidase-antiperoxidase (PAP) technique as a first sequence in the protocol followed by antibodies and the glucose oxidase conjugated avidin as a second sequence. A rich brown versus vibrant blue reaction product respectively is obtained. The applicability of this i m m u n o c y t o c h e m i c a l protocol to determine the co-localization versus co-existence o f neuropeptides is discussed. METHOD F o u r groups of adult male Sprague-Dawley rats were used. One group ( n - 8 ) consisted of normal untreated animals. A second group (n=8) was infused with colchicine (100 p,g/20 p,1) stereotaxically into the lateral cerebral ventri-

~Requests for reprints should be addressed to Dr. Diane T. Piekut, Neuroendocrine Unit, Box 609, University of Rochester, 601 Elmwood Avenue. Rochester, NY 14642.

891

892

PIEKUT

AND JOSEPH

I

V

t

,o

V~ ,0 I

2

V~

3 FIG. I. Frontal brain section of adrenalectomized-colchicine-treated rat d e m o n s t r a t i n g immunoreactive C R F n e u r o n s in the medial parvocellular c o m p o n e n t of the paraventricular nucleus (PVN). V = ventricle, pm posterior magnocellular area of PVN. × 160. FIG. 2. V a s o p r e s s i n - i m m u n o r e a c t i v e fir) n e u r o n s are observed in the magnocellular region of PVN in the colchicine-treated animal: few vasopressin-ir cells extend into the medial parvocellular (rap) c o m p o n e n t of PVN. V = t h i r d ventricle. × 160. FIG. 3. V a s o p r e s s i n - i m m u n o s t a i n e d n e u r o n s are seen in the medial pavvocellular and magnocellular c o m p o n e n t s of PVN in the adrenalectomized-colchicine-treated animal. The mp ~rea is normally CRF-rich (Fig. l) and vasopressin-poor lFig. 2) except in the adrenalectomized animal. × 160.

CRF AND VASOPRESSIN IN PVN cle in a saline vehicle over a 15 minute interval at a rate of approximately 1.5-2 tzl/minute. Survival time following colchicine treatment lasted 48 hi'. A third group ( n - 8 ) was bilaterally adrenalectomized using a dorsal approach and subsequently maintained on 0.9c/~ NaCI in place of the drinking water. Remowd of adrenal glands was confirmed at autopsy. Animals were allowed to survive for 14 days before pet-fusion. A fourth group of rats ( n - 8 ) was bilaterally adrenalectomized and 48 hr prior to sacrifice, this group received a single injection of colchicine. At the time of sacrifice, adult male Sprague-Dawley rats were anesthetized with sodium pentobarbital 10.4 ml/200 g intraperitoneally) and perfused through the ascending aorta. Perfusion was initiated with physiological saline rinse (200-300 ml) until the effluent remains clear followed by 300 ml of fixative containing 4% paraformaldehyde, 0.2c~ picric acid in 0.25 M sodium phosphate buffer with 0.9c/~ sodium chloride (PBS), pH 8.0. Brains were removed fiom the calvarium, immersed overnight in the fixation solution and blocked for sectioning the following day. Brains were cut serially on a Lancer Vibratome at 40 #m, tissue sections were collected into wells containing phosphate buffered saline (PBS, pH 7.6) and they were washed overnight to completely remove umeactive fixative prior to immunostaining. The immunocytochemical staining protocol that was used to demonstrate two antigens with contrasting colors in the same tissue section consisted of the following sequence. (I) Free floating tissue sections were incubated in antiserum to the first antigen, diluted in PBS containing I% BSA (Bovine serum albumin) and 0.4% Triton X-100, for 48 hours at 4°C. (2) Rinsed several times in PBS containing 0.04% Triton X-100 IPBS-TX). (3) Incubated in goat anti-rabbit immunoglobulin G (IgG, Antibodies Inc., diluted 1:75) or biotinylated goat anti-rabbit immunoglobulin G (lgG) of the Vectastain ABC kit (Vector Laboratories) in PBS-TX for 40 minutes at room temperature. {4) Rinsed in PBS-TX. 15) Incubated in rabbit anti-peroxidase-horseradish peroxidase complex !PAP, 24), diluted 1:100 or ABC reagents (Avidin DH and biotinylated horseradish peroxidase H, 8) of the Vectastain ABC kit in PBS-TX for 41t-60 minutes. 16) Rinsed in PBS-TX. 17) Incubated in 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma) in the presence of the co-substrate hydrogen peroxide (H_,O._,), 0.03%, which was added just prior to use. DAB concentration was 75 mg/100 ml in 0.05 M PBS, pH 7.6. The tissue was incubated at room temperature with the DAB mixture for approximately 3-10 minutes or until the brown immunoreaction product was seen. The progress of the reaction was monitored using a dissecting scope and terminated by placing the tissue sections in PBS before non-specific background staining developed. 18) Rinsed in PBS several hours or overnight. (9) Incubated in antiserum to the second antigen diluted the same as in step 1. (10) Rinsed in PBS-TX. (11) Incubated in biotin conjugated goat anti-rabbit IgG IJackson lmmunoResearch Laboratories, Inc.) diluted 1:2000 as in step 3 for 30 min. 112) Rinsed in PBS-TX. 113) Incubated in glucose oxidase conjugated avidin (Jackson ImmunoResearch Laboratories, Inc.) diluted 10 p.g/ml in PBS containing 0.04% Triton X-100 for 60 minutes at room temperature.

893 (14) Rinsed in PBS-TX. (15) Development of the blue reaction product was accomplished by incubation of sections in the appropriate substrate for approximately 30 minutes at 37°C or until the appropriate contrast was achieved. The enzymatic disclosing step as originally described by Clark et al. [6] was modified as follows: 3.3 mg/ml (final concentration) /3-D-glucose (Calbiochem-Behring Corp.) and 0.33 mg/ml (final concentration) p-nitro blue tetrazolium chloride (Research Organics, Inc., Cleveland, OH) in PBS, pH 8.3, were mixed and preheated in oven at 37°C for I hour: 0.0167 mg/ml phenazine methosulfate (Sigma) was added to this mixture just prior to incubation with the sections. Reaction was terminated once cells and fibers were detected under a dissecting scope and minimal background was obtained. (16) Rinsed sections in PBS. (17) Mounted from distilled water and coverslipped. Controls for the immunocytochemical staining procedure included the omission of the primary antiserum or goat antirabbit antiserum, the substitution of normal or pre-immune rabbit serum for the primary antiserum, and preincubation of primary antiserum with the synthetic antigen (2-50 p~g/ml of diluted antiserum) for 48 hours prior to its application to tissue sections. Controls for the dual staining immunocytochemical procedure consisted of (a) replacing the primary antiserum of the first sequence with buffer or preabsorbed antiserum in the complete staining protocol, (b) replacing the primary antiserum of the second sequence with buffer or preabsorbed antiserum. (c) utilizing the same primary antiserum in both the first and the second sequence of the immunostaining procedure, and (d) reversing the primary antiserum utilized in the first and second sequence of the immunostaining protocol. Antisera used in these studies consisted of anti-ovine CRF [9], anti-wlsopressin [10,19] and anti-oxytocin 110,19]. All antisera were generated in rabbits. RESULTS We have previously described in detail the distribution of CRF-immunoreactive fir) cells [19] and oxytocin- and vasopressin-containing magnocellular neurons [ 17,19] in the paraventricular nucleus (PVN) of rat hypothalamus. CRFimmunostained perikarya were localized predominantly in the medial parvocellular component of PVN (Fig. I) in the normal, colchicine-treated, adrenalectomized and adrenalectomized-colchicine-treated animals. In the former, the perikarya were scarce and weakly stained. In the latter three groups, a remarkable density of CRF immunostained neurons were seen. lmmunoreactive material was observed in the perikarya, dendrites and axonal processes of parvocellular neurons. Vasopressin and oxytocin immunoreactive cells were distributed primarily in the magnocellular component of PVN [17,19]. Few immunostained cells were seen in the parvocellular area of PVN (Fig. 2) in the normal and colchicinetreated rat. In the adrenalectomized and adrenalectomizedcolchicine-treated animals, a dense accumulation of immunostained vasopressin cells was observed in the magnocellular and medial parvocellular components of PVN (Fig. 3): the latter appeared to have an anatomical distribution similar to that seen for CRF-containing cell bodies (Fig. 1). The extension of vasopressin-ir cells in this CRF rich region suggests a potential co-existence of these neuropeptidergic systems. The distribution and number of oxytocin-

894

P1EKUT AND JOSEPH TABLE 1 COLOR MIXING UPON INCOMPLETE OCCUPANCY OF ANTIGEN SITES Staining Lof PV neurons Dilution of 1st Sequence A.

B.

anti-CRF no antiserum anti-CRF

anti-CRF

Dilution of 2nd Sequence l: 1000

anti-AVP

1:8000 1:8000 1:8000 1:8000 1:8000 1:8000 1:8000

Br Br Br Br Br Br --

BI BI BI B1 BI BI BI

anti-CRF

1:400(I 1:4000 1:4000 1:4000 1:4000 1:4000 1:4000

Br Br B~>>B1 Br>BI BI>Br BI > > B r B]

--------

anti-OXY

1:8000 l :8000 1:8000 1:8000 1:8000 1:8000 1:8000

Br Br Br Br Br Br --

l :1000 1:4000 1:20000 1:40000 1:80000

anti-CRF

-Bl BI BI BI BI BI BI BI

1: 1 0 0 0

1: 120000 1:160000 D.

Br BI Br Br Br>>BI Br>BI Bl>Br BI> >Br BI

1: 1 0 0 0 1:4000 1:20000 1:40000 1:80000 1: 120000 1:160000

1: 120000 l : 160000 anti-CRF

Magnocellular

no antiserum anti-AVP 1:8000 anti-AVP 1:8000 1:8000 1:8000 1:8000 1:8000 1:8000 1:8000

1:4000 1:20000 1 : 40000 I : 80000

C.

Parvocellular

l: 1 0 0 0 1:4000 1:20000 1 :40000 1:80000

1:120000 I: 160000

Bl B1 B] B1 BI BI BI

~Staining of PV (paraventricular) neurons refers to the color observed for the majority of cells in the parvocellular or magnocellular component of this nucleus. Br=brown; Bl-blue; >=stronger than: > > = m u c h stronger than: - - = n o staining. c o n t a i n i n g cells w e r e not a f f e c t e d by a d r e n a l e c t o m y . A n e w dual staining i m m u n o c y t o c h e m i c a l p r o t o c o l for the s i m u l t a n e o u s localization o f t w o a n t i g e n s in the s a m e tissue s e c t i o n has b e e n d e v e l o p e d and applied to the p r e s e n t study. Biotin-goat anti-rabbit i m m u n o g l o b u l i n G (IgG), aviding l u c o s e o x i d a s e and a p p r o p r i a t e s u b s t r a t e s w e r e u s e d to obtain a blue r e a c t i o n p r o d u c t . A n t i - C R F and a n t i - v a s o p r e s s i n , at their optimal dilution o f 1:1000 and 1:8000 r e s p e c t i v e l y , b o t h i m m u n o s t a i n e d cells intensely in the medial p a r v o c e l l u lar c o m p o n e n t o f P V N in the a d r e n a l e c t o m i z e d and a d r e n a l e c t o m i z e d - c o l c h i c i n e - t r e a t e d animals. W h e n antiC R F was u s e d in the first reaction s e q u e n c e o f the dual i m m u n o s t a i n i n g p r o t o c o l and a n t i - v a s o p r e s s i n in the s e c o n d s e q u e n c e , b r o w n and blue i m m u n o r e a c t i v e cells w e r e obs e r v e d in the p a r v o c e l l u l a r c o m p o n e n t o f P V N (Fig. 4). N o c o l o r mixing o c c u r r e d . H o w e v e r , the n u m b e r o f blue parvocellular n e u r o n s o b s e r v e d w a s few and it did not equal that o f an a d j a c e n t tissue s e c t i o n in w h i c h only v a s o p r e s s i n - i r cells w e r e stained. In all c a s e s , a d e n s e a c c u m u l a t i o n o f blue v a s o p r e s s i n - c o n t a i n i n g n e u r o n s w a s seen in the m a g n o c e l l u lar c o m p o n e n t o f P V N . C o l o r mixing due to i n c o m p l e t e oc-

c u p a n c y o f antigen sites (Table 11 as d e s c r i b e d by S t e r n b e r g e r and J o s e p h [25] was t e s t e d to d e t e r m i n e the potential c o - e x i s t e n c e o f C R F and v a s o p r e s s i n in p a r v o c e l l u l a r neurons. Adrenalectomized and adrenalectomizedc o l c h i c i n e - t r e a t e d animals (Table 1A) w e r e used. W h e n a dilution o f 1: 1000 o f a n t i - C R F was used in the first s e q u e n c e and primary a n t i s e r u m o m i t t e d in the s e c o n d s e q u e n c e , only b r o w n parvocellular n e u r o n s w e r e seen. This s e r v e d as a " b r o w n " control. W h e n the primary a n t i s e r u m was o m i t t e d in the first s e q u e n c e and a 1:8000 dilution o f a n t i - v a s o p r e s s i n was used in the s e c o n d s e q u e n c e , blue p a r v o c e l l u l a r and blue m a g n o c e l l u l a r n e u r o n s w e r e p r e s e n t . This s e r v e d as a "'blue'" control. At I:1000 or 1:4000 dilutions o f a n t i - C R F , b r o w n parvocellular n e u r o n s w e r e seen (Fig. 4). W h e n prog r e s s i v e dilutions o f a n t i - C R F w e r e used in the first seq u e n c e and a c o n s t a n t dilution o f 1:8000 o f a n t i - v a s o p r e s s i n in the s e c o n d s e q u e n c e , c o l o r s b e c o m e m i x e d in parvocellular n e u r o n s . A l t h o u g h m i x e d , a b r o w n c o l o r i m m u n o r e a c t i o n p r o d u c t p r e d o m i n a t e d at 1:20,000 and 1:40,000 (Fig. 5) dilutions o f a n t i - C R F , w h e r e a s at dilutions o f 1:80,000 and 1:120,000 (Fig. 6) o f a n t i - C R F , a blue i m m u n o r e a c t i o n prod-

CRF AND VASOPRESSIN

IN PVN

895

D

8

FIGS. 4-6. Frontal brain sections o f adrenalectomized-colchicine-treated animals d e m o n s t r a t i n g CRF-ir and v a s o p r e s s i n (AVP)-ir n e u r o n s in PVN. At a 1: 1000 and 1:8000 dilution of anti-CRF and anti-AVP respectively, brown CRF-ir cells are seen in the parvocellular c o m p o n e n t and blue AVP-ir n e u r o n s in the magnocellular region of PVN (Fig. 4). As progressive dilutions of anti-CRF were used in the first s e q u e n c e of these dual stained preparations, e.g., 1:40,000, the cells in the parvocellular area appear brown-blue (or mixed) in color (Fig. 5) and at a 1:120,000 dilution, they are predominantly blue (Fig. 6). At all dilutions o f anti-CRF, cells in the magnocellular region of PVN are a vibrant blue. Figs. 4-6. x 122. FIGS. 7-9. Frontal brain sections o f colchicine-treated animal at level and magnification comparable to that seen in Figs. 4-6. W h e n anti-CRF was used at a dilution of 1:4000 followed by anti-AVP at a 1:8000 dilution, b r o w n C R F i m m u n o s t a i n e d cells are seen in the parvocellular region of PVN and blue v a s o p r e s s i n n e u r o n s in the posterior magnocellular area (Fig. 7). The n u m b e r and staining intensity o f brown CRF-ir n e u r o n s is reduced at a 1:40,000 dilution (Fig. 8) o f anti-CRF and at a 1:120,000 dilution of this a n t i s e r u m brown cells are barely detectable (Fig. 9). Blue magnocellular n e u r o n s are o b s e r v e d in all preparations with a 1:8000 dilution o f anti-AVP. N o color mixing occurred in parvocellular or magnocellular PV neurons.

896

P I E K U T AND J O S E P H

act predominated in parvocellular neurons. In all cases, few immunostained cells were only brown or only blue. At a 160,000 dilution of anti-CRF, only blue immunoreactive cells were seen. It is noteworthy that at all dilutions of CRF, distinct blue vasopressin immunostained cells were evident in the magnocellular component of PVN in the same tissue section: color mixing did not occur in this region. In the colchicine-treated animals, CRF-immunostained neurons were observed in the parvocellular component of PVN and vasopressin-ir cells in the magnocellular region: the latter did not extend into the parvocellular component in this group of animals. At 1:1000 and 1:4000 (Fig. 7) dilutions of anti-CRF in the first sequence and a 1:8000 dilution of anti-vasopressin in the second sequence, brown immunostained CRF cells were present in the parvocellular area and blue immunostained vasopressin cells were seen in the magnocellular region of PVN (Table 1B and Fig. 7). As progressive dilutions of anti-CRF (see Table 1B) were used in the first sequence and a constant 1:8000 dilution of antivasopressin in the second sequence, a decrease in the number and staining intensity of CRF-ir cells was observed (Figs. 8 and 9). CRF-ir cells were lightly stained at a 1:80,000 dilution and barely delectable at 1:120,000 (Fig. 9): at a I: 160,000 dilution of anti-CRF, no brown cells were seen. In all cases, vibrant blue magnocellular neurons were observed. No color mixing was detected in the parvocellular or magnocellular neurons of PVN in these animals. To further test the validity of color mixing, two additional studies were conducted in the colchicine-treated, adrenalectomized and adrenalectomized-colchicine-treated animals. In the first study (Table 1C), anti-CRF was used in the first sequence at varied dilutions and a 1:4000 dilution of the same antiserum was used in the second sequence. As anticipated due to incomplete occupancy of antigen sites, color mixing in parvocellular neurons became evident as progressive dilulions of anti-CRF were used in the first sequence as outlined in Table IC. At a 1:160,000 dilution in the first sequence, only blue parvocellular CRF-ir neurons were seen in PVN. A second study employed anti-CRF at varied dilution in the first sequence and a 1:8000 dilution of anti-oxytocin in the second sequence (Table 1D). At a 1:1000 dilution of anti-CRF and a 1:8000 dilution of anti-oxytocin, brown immunostained cells were present in the parvocellular area and blue immunoreactive neurons in the magnocellular component of PVN. Blue parvocellular neurons were not seen as oxytocin-containing cells were localized in the magnocellular, not parvocellular, component of PVN. A decrease in the number and intensity of brown CRF-containing cells occurred in parvocellular neurons as progressive dilutions of anti-CRF were used. CRF-ir cells were weakly stained at dilutions of 1:80,000 and 1:120,000: no brown immunostained cells were seen at a 1: 160,000 dilution of anti-CRF. In all cases, a dense accumulation of blue magnocellular oxytocin-ir neurons was present. No cokn- mixing was detected in the parvocellular or magnocellular neurons of PVN. DISCUSSION

A dense aggregation of CRF-immunoreactive cells has been localized in the parvocellular component of the paraventricular nucleus (PVN) of rat hypothalamus. Recent anatomical studies [I 1, 19, 22,271 have revealed a remarkable density of vasopressin-containing neurons in this CRFrich area of brain in the adrenalectomized animal; the anatomical distribution of vasopressin-ir cells was similar to

that seen for CRF-ir neurons. For decades, vasopressin (a posterior lobe hormone) has been implicated in the CRFpituitary-adrenal axis and substantiated by very convincing anatomical, physiological and pharmacological studies (for review, see [311). The precise relationship or interaction of vasopressin and CRF neuropeptidergic systems is yet to be elucidated. Several methodological approaches have been used to resolve the issue pertaining to the co-localization versus co-existence of these two neuropeptidergic systems in PV neurons and they include adjacent section immunocytochemistry and single section dual immunostaining. Dual staining procedures which demonstrate contrasting color staining of two antigens in the same tissue section are ideally suited to establish an anatomical relationship of two neuropeptide and/or neurotransmitter substances as reviewed by Joseph and Piekut [10]. The present study demonstrates the applicability of a new dual immunostaining procedure to elucidate the presence of two neuronal systems within the same cell body. Tramu e/ a/. 1271 have demonstrated synthesis of w~sopressin in perikarya localized in the parvocellular component of PVN in the adrenalectomized animal. This area of brain normally does not contain many w~sopressin-ir cells but is an area in which a remarkable density of CRF-containing neurons was seen. The co-existence of vasopressin and CRF in the same cell body has been suggested by Tramu el a/. [27] using immunocytochemistry and elutkm techniques l\w double immunostaining in the same tissue section. These anatomical studies have been confirmed by Kiss el a/. [I It and Sawchenko el al. [22] using similar methodology on alternate or the same tissue sections. A main disadvantage to the use of the elating solution (i.e., potassium dichromate) is that some antigens, for example those whose antigenic sites contain tryplophan, methionine, or disulfide groups, may be damaged in the process ]12]. The elation reagents used may not remove all antigen/antibody complexes and thus would allow the second series of immunoreagents to be reactive which would result in fidse positive data concerning the coexistence of two neuronal systems. Additionally, in a neuropeptide-rich area such as the PVN, analysis of in> munostained serial tissue sections or the same section in which the first antigen is stained, elated, and the second antigen then stained is often difficult to establish with certainty. Evidence fi)r the co-existence of vasopressin and CRF in the same perikarya has not been confirmed by Antoni el a/. [1] and Taniguchi ]26]. Sternberger and Joseph [25] have described a dual staining technique which demonstrates contrasting color staining of two antigens in the same section without antibody removal. They have shown that color mixing in dual staining immunocytochemislry does not occur even though they used the same anti-immunoglobulin and PAP in both sequences of the protocol and hydrogen peroxide was applied as an enzyme substrate twice. Apparently the DAB reaction product masked the antigen and catalytic sites of the first sequence of immunoreagents and thus prevented interaction with reagents of the second sequence. Using dual staining procedures with glucose oxidase-anti-glucose oxidase (GAG) complex as a substitute lkw PAP in the second sequence of the immunocytochcmical protocol, our studies [19] have provkled no evidence tbr the co-existence of CRF and wtsopressin in parvocellular neurons. We did however suggest that it was possible thai the first antibody/antigen complex masked other antigenic sites in the same neuron at the antisera dilutions and immunocytochemical protocol used and this did not allow vis-

CRF AND VASOPRESSIN

IN PVN

897

ualization o f t w o different p e p t i d e s in the s a m e n e u r o n . Subs e q u e n t l y , a m o d i f i c a t i o n o f this dual staining imm u n o c y t o c h e m i c a l p r o c e d u r e h a s b e e n d e v e l o p e d and we h a v e applied this new m e t h o d o l o g y to t h e p r e s e n t study. In this p r o c e d u r e , a p p l i c a t i o n o f p r i m a r y a n t i s e r u m , b i o t i n - g o a t a n t i - r a b b i t i m m u n o g l o b u l i n G (lgG), a v i d i n - g l u c o s e o x i d a s e and a p p r o p r i a t e s u b s t r a t e s were used to o b t a i n a blue reaction p r o d u c t as the s e c o n d s e q u e n c e o f the imm u n o c y t o c h e m i c a l p r o t o c o l . This i m m u n o c y t o c h e m i c a l m e t h o d has o f t e n p r o v e n to be m o r e s e n s i t i v e and disc r i m i n a t i n g [18l as c o m p a r e d to p r i o r dual staining proced u r e s used in this l a b o r a t o r y {17, 19, 20] e m p l o y i n g the G A G c o m p l e x or glucose o x i d a s e c o n j u g a t e d to lgG. T h e aviding l u c o s e o x i d a s e p r o t o c o l yielded a m o r e i n t e n s e imm u n o r e a c t i o n p r o d u c t a n d s o m e cell b o d i e s and f i n e - b e a d e d a x o n a l p r o c e s s e s w e r e visualized distinctly w h i c h w e r e o f t e n not seen with the latter t w o p r o c e d u r e s . T h e i n c r e a s e d sensitivity o f the p r o t o c o l used in the p r e s e n t s t u d y is a t t r i b u t e d to b e t t e r p e n e t r a t i o n and h i g h e r specific activity o f glucose o x i d a s e labeled avidin a n d b i o t i n y l a t e d a n t i b o d y ( S t e g e m a n , personal communication). V a s o p r e s s i n i n f l u e n c e s on the c o r t i c o t r o p i n - A C T H axis has b e e n d o c u m e n t e d (for r e v i e w , see [31]). A d r e n a l e c t o m y p o t e n t i a t e s v a s o p r e s s i n s e c r e t i o n and its i n f l u e n c e on the a d r e n a l axis [1, I1, 14, 16, 22, 271; t h e s e effects subside following d e x a m e t h a s o n e t r e a t m e n t [16] implicating a steroidal f e e d b a c k m e c h a n i s m o f action. W h e t h e r the a f f e c t e d site is at the pituitary o r b r a i n level is not p r e s e n t l y k n o w n alt h o u g h b o t h loci h a v e b e e n suggested. T h e a n a t o m i c a l data p r e s e n t e d here favors an effect at the cellular level suggest-

ing t h a t the C R F p e r i k a r y o n h a s the n e c e s s a r y m a c h i n e r y to s y n t h e s i z e v a s o p r e s s i n u n d e r stressful situations. R e c e n t d a t a by W o l f s o n et al. [30] using a n t i - C R F a n d v a s o p r e s s i n m R N A add c r e d e n c e to this h y p o t h e s i s . In m o n k e y a n d o t h e r species including n o r m a l rat, vasopressin terminals h a v e been s h o w n to abut on portal vessels in the e x t e r n a l layer o f m e d i a n e m i n e n c e . W e a n d o t h e r s h a v e o b s e r v e d i n t e n s e staining in this area o f m e d i a n e m i n e n c e following a d r e n a l e c t o m y . H o w e v e r , in this p a r t i c u l a r s t u d y it w a s difficult to d i s c r i m i n a t e b e t w e e n distinct blue a n d b r o w n colors in t e r m i n a l s using the dilutions o f a n t i s e r a w h i c h gave s e p a r a t e blue v e r s u s b r o w n colors at the level of the cells. A q u e s t i o n r e m a i n i n g t h e r e f o r e is w h e t h e r the vaso p r e s s i n a n d C R F are c o - t r a n s p o r t e d to m e d i a n e m i n e n c e as well as c o - s y n t h e s i z e d . E v i d e n c e for the c o - e x i s t e n c e o f C R F and v a s o p r e s s i n in the s a m e axonal t e r m i n a l s o f m e d i a n e m i n e n c e as well as in the same n e u r o s e c r e t o r y vesicles has b e e n r e p o r t e d [29]. F u r t h e r studies w h i c h p e r t a i n to vesicle packaging and transport, a coordinate secretion of these n e u r o p e p t i d e s into the pituitary portal vessels and the p h y s iological significance of t h e s e findings are n e e d e d . ACKNOWLEDGEMENTS The authors are grateful to ]'racy Sheppard for her excellent technical assistance. The authors thank Dr. Ludwig Sternberger 1University of Rochester) and Dr. William Stegeman IJackson lmmunoResearch Laboratories, Inc.) for their many helpful suggestions. This research was supported by NSF Grant BNS-831094 and NIH Grants NS18626 and NS/HL21323. Dr. Piekut is the recipient of an RCDA NS00869.

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