� Immunocytochemistry for LHRH Neurons in the � Arcuate Nucleus Area of the Rat

‘This work was supported in part by NIH grants HD-15040 and

AA-06014 and a NIH postdoctoral fellowship to W.L.D. from Train-

ing Grant HD-07062.

83

0022-1554/84/13.00

The Journal of Histochemistry and CytochemistryCopyright © 1984 by The Histochemical Society, Inc.

Vol. 32, No. 1, pp. 83-91, 1984Printed in U.S.A.

II

� Immunocytochemistry for LHRH Neurons in the

� Arcuate Nucleus Area of the Rat:

Fact or Artifact?1

GERALD P. KOZLOWSKI and W. LES DEES

Department of Physiology, University of Texas Health Science Center at Dallas, Southuestern Medical School, Dallas, Texas 75235

Received for publication March 29, 1983 and in revised formJuly 20, 1983; accepted August 12, 1983 (OA 83-136)

Antisera to luteinizing hormone-releasing hormone (LHRH)generated against hapten-conjugates in which BSA (bo-vine serum albumin) is used as a carrier can contain both

anti-LHRH and anti-BSA antibodies. Such antisera cancause the false-positive staining of neuronai cell bodies andother elements of the medial basal hypothalamus owing tothe presence of anti-BSA, which cross-reacts with albu-minoid substances within these cells. Differential blocking

experiments using either BSA or LHRH or both BSA and

LHRH as immunoabsorbents demonstrated that recent re-

Introduction

The presence of LHRH in neurons of the rat arcuatc nucleus

has been an important historical question for ncurocndocri-

nologists, and remains a source of debate. Since the rat is a

widely used animal model for studies of the hypothal-

amo-hypophysial-gonadal axis, it is of prime importance to

determine those brain areas having LHRH-containing neu-

rons. Although useful, other experimental approaches such as

Hal#{225}szknife-cuts (Hal#{225}sz, 1969) and radioimmunoassay for

LHRH in Palkovits-type punch specimens from various brain

areas (Palkovits et al., 1974) have provided only indirect in-

formation about the location of LHRH-containing neurons.

Even immunocytochemical localization studies for LHRH have

been controversial, despite their potential for providing the

most direct evidence of LHRH-containing neurons. Studies

by Naik (1974, 1975a, l975b, 1976) are yet to be confirmed,

and according to Kelly et al. (1982), there is concern about

the use oflow-titer anti-LHRH antisera. The claim of Hoffman

et al. ( 1978) of LHRH in arcuate neurons of the rat was later

retracted when it was discovered that the anti-LHRH anti-

ports of LHRH-containing neurons in the medial basal

hypothalamus of the rat have arisen from false-positive

results due to the presence of anti-BSA within the anti-LHRH antisera used. Immunocytochemical evidence of

LHRH-containing cell bodies in the medial basal hypo-thalamus (especially within the arcuate nucleus) of the rat

remains unproven.KEY WORDS: Luteinizing hormone-releasing hormone; Immu-

nocytochemistry; Arcuate nucleus; Median eminence; Rat.

serum these authors used contained anti-ACTH antibodies

(Clayton and Hoffman, 1979).

Perikarya containing LHRH have been demonstrated within

the human medial basal hypothalamus (MBH) (Barry, 1976;

Bugnon et al., 1977; Paulin et al., 1977) and that of several

species of laboratory animals including primates (Barry and

Carette, 1975; Marshall and Goldsmith, 1980; Silverman et

al., 1982), guinea pigs (Barry et al., 1974; Silverman, 1976),

dogs and cats (Barry and Dubois, 1975), hamsters (Pickard

and Silverman, 1976), and rabbits (S#{233}t#{225}lOet al., 1978). Several

large domestic animals have also been studied in this regard.

The infundibular nucleus ofthe horse (Dees et al., 1981a) has

been shown to contain immunopositivc LHRH neurons, whereas

immunoreactive cells in the sheep (Dees et al., 1981b) and

cow (Dees and McArthur, 1981) have been reported in both

the infundibular and ventromedial nuclei. In contrast, the at-

tempted immunocytochemical localization of LHRH-contain-

ing perikarya in the rat has produced inconsistent and con-

flicting results. Kelly and Ronnekleiv (1981), Ronncklciv et

al. (1981), Fraser et al. (1982), Kelly et al. (1982), and Ron-

nekleiv and Kelly ( 1982) have reported numerous perikarya

in the rat median eminence (ME) and arcuate nucleus, and in

the area just lateral to the arcuate nucleus. Kawano and Dai-

koku (1981) localized LHRH-positive cell bodies adjacent to

the lateral border of the ME, but denied the existence of

LHRH-containing perikarya in the arcuate nucleus. The pres-

by guest on May 30, 2015jhc.sagepub.comDownloaded from

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Figure 1 . Section of the rostral median eminence of the rat using the

Kelch #12 anti-LHRH antiserum. With unabsorbed antiserum thereare stained cell bodies (arrows) in the region ofthe median eminence,

and bilateral staining for LHRH fibers in the zona externa (ZE). III-

third ventricle. Original magnification x 180. Bar = 55 p.m.

84 KOZLOWSKI, DEES

ent study was designed to continue the investigation and char-

acterization of presumed LHRH-containing perikarya in the

MBH of the rat.

Materials and Methods

Male Sprague-Dawley rats weighing 200-250 g were anesthetized

with sodium pentobarbital, flushed via cardiac perfusion with physi-

ologic saline, and fixed with 10% phosphate-buffered formalin. A

block containing the hypothalamus and median eminence was isolated

from each brain and postfixed in Bouin’s solution for 24 hr. Transverse

10-p.m paraffin sections were cut from the block, mounted on glass

slides, and dried overnight at 40#{176}C.Rehydrated sections were stained

for LHRH with the indirect immunoperoxidase technique of Stern-

berger et al. ( 1970), using the peroxidase-antiperoxidase complex

(PAP). Additionally, the brains of animals fixed by perfusion withZamboni’s fixative (see Kozlowski and Nilaver, 1983) were sectionedwith a Vibratome (Oxford Labs, CA) and immunostained for LHRH

as described by Kozlowski et al. (1980). The staining procedure in-

volved the sequential application of the following reagents prepared

in phosphate-buffered saline (PBS), pH 7.4, containing 0. 1% gelatin:

primary rabbit antiserum to LHRH (Arnel #104, Kelch #12, Koz-

lowski #705, Nett-Niswender #42, Nilaver #3, White-Porter #1)

used at dilutions of 1:700 for 24 hr at 4#{176}C;sheep anti-rabbit IgG(Miles Laboratories, Elkhart, IN) used at a dilution of 1:200 for 30

mm at room temperature; rabbit PAP complex (Miles) used at a 1:200

dilution for 30 mm at room temperature; and 7.5 mg 3,3’-diamino-

benzidinc tetrahydrochloride (Sigma, St Louis, MO) per 50 ml gel-PBS

with 50 p.1 of 3% hydrogen peroxide, used for 5-10 mm at room

temperature on an automatic shaker. The slides containing the sectionswere thoroughly washed with PBS, pH 7.4, between the steps of the

procedure.

The specificity of staining was verified through the absorption of

1.0 ml of each of the primary antisera, diluted 1:100 with 120 gg

synthetic LHRH (Beckman), 24 hr before use. The anti-BSA activity

present in the antiseragenerated against BSA conjugates (Kelch #12,

Arnel #104, and WP-1) was removed by the addition of 500 mg ofBSA to 1.0 ml of a 1: 100 dilution of each antiserum 24 hr before

use. Adding as much as 1.0 mg of BSA per milliliter of antiserum

eliminated albuminoid staining without affecting specific LHRH stain-

ing. The LHRH antisera were tested for cross-reactivity with other

peptides; 1 ml ofeach antiserum (diluted 1:100) was preabsorbed for

24 hr at 4#{176}Cwith 120 mg of oxytocin, arginine-vasopressin, thyro-

tropin-releasing hormone, and adrenocorticotrophic hormone (ACTH).

Finally, staining was eliminated when PBS was substituted for either

the primary antiserum, sheep anti-rabbit IgG, or the PAP complex.

Results

Using adjacent sections through the rostral, middle, and caudal

median eminence ofthc rat, we were able to demonstrate false-

positive staining of neurons in the median eminence and me-

dial basal hypothalamus, along with true-positive staining for

immunoreactive LHRH fibers in the median eminence. The

false-positive staining was caused by the presence of anti-BSA

antibodies in rabbit antisera to LHRH, generated against hap-

ten-conjugates when BSA was used as a carrier to enhance

their immunogenicity. Those antisera to LHRH that contained

anti-BSA antibodies included the Arnel #104, Kelch #12,

Nett-Niswender #42, and White-Porter #1 (WP-1 antisera).

Not only did these antisera demonstrate LHRH-positive

fibers in the median eminence, but also cell bodies of the

medial basal hypothalamus (Figures 1, 2a, 3a, 4a, and 5a). This

false-positive result was eliminated with either liquid- or solid-

phase immunoabsorption of the antisera with BSA (Figures

2c, 3c, 4b, and Sc), without affecting the staining of fibers.

Immunoabsorption with LHRH alone did not affect the stain-

ing of medial basal cells of the hypothalamus, but effectively

abolished the staining of LHRH-containing fibers in the tub-

eroinfundibular sulcus region of the median eminence (Figures

2b and Sb), and likewise abolished the staining of LHRH-

containing cell bodies and fibers in other brain areas. Liquid-

phase immunoabsorption with both BSA and LHRH elimi-

nated all immunopositive staining (Figures 2d, 3d, and Sd).

With the presence of anti-BSA in anti-LHRH antisera, there

is artifactual staining of fibers and cells of the magnocellular

system (Figures 3a, 3b, and 4a) as well as of ependymal sur-

faces (Figures 2a and b, 3a and b, and 4a), connective tissue,

and blood vessels. These structures and cells of the medial

basal hypothalamus are presumed to contain albuminoid sub-

stances able to cross-react with anti-BSA antibodies, causing

a false-positive staining reaction. When anti-BSA is not present

in anti-LHRH antisera, as is the case both for antisera gen-

crated against thyroglobulin as a conjugate (Kozlowski #705

or Nilaver #3) and antisera generated against BSA as a con-

jugate but absorbed with BSA (WP-I, Nett-Niswcnder #42,

Arnel #104, Kelch #12), the observed cytoarchitectural char-

acteristics of LHRH-containing cell bodies (Figures 7 and 8)

are similar, and differ in appearance from those containing

albuminoid substances (Figure 6). When anti-BSA was re-

moved from the antisera to LHRH, cells in the region of the

arcuate nucleus never showed immunopositivity (Figures 2c

and d, 3c and d, 4b and Sc and d). Although not demonstratedhere, Arnel #104 and Nett-Niswender #42 antisera to LHRH



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IMMUNOCYTOCHEMISTRY FOR LHRH NEURONS 85

:.

had reactivitics to albuminoids that were similar to those shown

for the Kelch #12 and WP-1 antisera. By contrast, with anti-

LHRH antisera in which thyroglobulin was used as a carrier

(Kozlowski #705 and Nilaver #3), there was no false immuno-

staining of cell bodies in the median eminence or medial basal

hypothalamus of the rat. However, in the case of antiserum

#705 there was one instance of a single positively LHRH-

staining cell body in the arcuate nucleus (Figure 8).

Discussion

A recent review by Flerk#{243}et al. ( 1980) summarized his and

his colleagues’ work throughout the years, and stated, as con-

clusions, that the MBH does not contain LHRH-synthcsizing

nerve cells and that all LHRH-containing cell elements dis-

appear from this region if it is surgically disconnected from

the rest of the brain. The review further stated that among

the nearly 2000 rat brains the authors had studied over the

preceding 5 years (comprising specimens representing differ-

ent physiologic states and experimental interventions in both

sexes) with six different antibodies to synthetic LHRH, not a

single cell with immunorcactive LHRH-content could be de-

tccted in the tubcral (arcuate-ventromedial) region of the rat

brain. Except for one example, our laboratory, also using a

variety ofantisera over the years, has also been unable to detect

such LHRH-containing cells.

�i

� _

Figure 2. Adjacent sections of the rostral median eminence of the rat

using unabsorbed Kelch #12 anti-LHRH antiserum (a) and the same

antiserum absorbed with: LHRH (b), BSA (c), and both BSA and

LHRH (d). When anti-BSA is present (a, b) in the antiserum, thereis staining of cell bodies of the medial basal hypothalamus (arrows),

which disappears when the serum is absorbed with BSA (c, d). Staining

for LHRH-containing fibers (arrowheads) in the zona externa of the

median eminence, seen with unabsorbed antiserum (a), persists when

BSA is used as an absorbant (c), but disappears when LHRH is usedas an absorbant (b, d). Original magnification x 80. Bar = 125 p.m.

Nearly 10 years ago, we (Kozlowski and Zimmerman, 1974;

Zimmerman et al., 1980) became suspicious of staining of the

magnocellular system when using the Nett-Niswcndcr #42

anti-LHRH antiserum in studies ofthc sheep and mouse brain.

However, prior to reporting our work (Zimmerman et al.,

1974), we recognized the problem that anti-BSA antibodies

could pose when staining for LHRH. Subsequently, we un-

dertook to publicize this experience (Kozlowski et al., 1975;

Kozlowski et al., 1976; Zimmerman et al., 1980; Kozlowski

and Nilaver, 1983).

In answer to the question of why they have been able to

find numerous LHRH-positivc neurons, Kelly et al. (1982)

stated that they were successful because 1 ) certain unique

characteristics of the WP-l antiserum may facilitate the rec-

ognition of LHRH, 2) a limited fixation time was used (75

mm rather than 90 mm or 1 hr, which they found diminished



C D

86 KOZLOWSKI, DEES

iz,’..�: ..�. �,

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Figure 3. Adjacent sections of the middle median eminence of the

rat using unabsorbed Kelch #12 anti-LHRH antiserum (a) and the

same antiserum absorbed with: ACTH (b), BSA (c), and both BSA

and LHRH (d). There is no effect on staining when ACTH is used

as an absorbant. When anti-BSA is present (a, b) in the antiserum,

artifactual staining of albuminoid substances appears in neurons (ar-

rows) and fibers (double arrows) of the zona interna of the medianeminence as well as in ependymal surfaces (arrowheads), connective

tissue, and blood vessels. Original magnification x 70. Bar = 60 p.m.

the staining), and 3) the sagittal sectioning ofspccimcns affords

a greater opportunity to visualize the rostrocaudally oriented

neurons. In order to provide further support for their results,

they cited “physiological” evidence for the existence of MBH

neurons using other techniques including deafferentation studies

in which the content of LHRH is not totally eliminated within

the MBH island, as a result of Hal#{225}sz knife cuts (Brownstein

et al., 1977; Kordon et al., 1982), and the finding that dcc-

trochemical stimulation of the deafferented MBH resulted in

significantly increased peripheral levels oflutcinizing hormone

(Carrillo et al. , 1980). However, other studies have shown that

deafferentation can result in the complete disappearance of

LHRH-containing elements within the MBH (for a review see

Flerk#{243}et al., 1980). These different results are probably due

to the deafferentation technique. Gibbs (1973) was the first

to advocate the application of an extra downward force to the

stereotaxic knife in order to achieve complete deafferentation.

Hoffman and Gibbs (1982) demonstrated that for deaffer-

entation of the median eminence to be successful, the knife

must pass through the optic chiasm to cut the subchiasmatic

LHRH tract, and must extend far enough caudally and laterally

to cut a LHRH tract arising from caudal cells of the lateral

preoptic and lateral anterior hypothalamus.

Our results indicate that LHRH-containing neurons arc

quite different in appearance from those containing albumi-

noid substances. When viewed in a longitudinal plane, two

types of LHRH-containing neurons arc seen (Krisch, 1980):

type 1 cells, which arc bipolar with a smooth surface, and type

2 cells, which are unipolar with a rough surface (Figures 7 and

8). Both types of cell have only a small rim of cytoplasm

surrounding the nucleus. In contradistinction to LHRH-con-

taming neurons, the albuminoid-containing neurons that we

observed (Figures 1 and 6) closely resembled those reported

by Kelly et al. (1982) to contain LHRH. Most of these al-



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Figure 4. Adjacent sections ofthe caudal me-

dian eminence of the rat using unabsorbed

Kelch #12 anti-LHRH antiserum (a) or thesame antiserum absorbed with BSA (b). The

intensity of LHRH staining (arrows) remains

unaffected when the absorbed antiserum is

used. The globular-staining structures of the

magnocellular system (arrowheads) seen with

the unabsorbed antiserum (a) disappear when

the antiserum is absorbed with BSA (b). Orig-inal magnification x 100. Bar = 100 p.m.

,�.

buminoid-containing cells appear spheroid or multangular, with

only a few being fusiform. It is uncommon to find processes

emanating from these cells, but when fibers are present they

are not beaded. A major problem in interpretation has been

the fact that many bonafide LHRH fibers intermingle with

neurons of the arcuate nucleus, giving beaded fibers the ap-

pearance of emanating from the cell bodies of the nucleus.

However, by carefully focusing at different planes within the

section, one can observe that in reality, the beaded LHRH

fibers only pass close to the neurons. In this regard, the cx-

amples of rat arcuate neurons giving rise to beaded fibers, as

shown by Kelly et a!. (1982), should be regarded with some

skepticism. Following immunoabsorption of anti-BSA, the WP-

1 anti-LHRH antiserum is indeed useful for immunostaining

LHRH-containing cell bodies in other areas of the brain.

Another issue brought to light by Kelly et al. (1982) con-

cerns the interpretation of the granular nature of the final

LHRH-staining-rcaction prod uct. They eq uatc the granular

appearance of the stain as seen with light-microscopic im-

munocytochcmistry with neurosecretory granules as seen with

electron-microscopic immunocytochemistry as reported by

Kozlowski et al. (1980). Likewise, Kelly et al. said that they

could distinguish LHRH-containing neurons from /3-cndor-

phin-containing neurons on the basis of whether or not the

“granules” were aggregated or evenly distributed throughout

the soma. In fact, the granules to which they refer are aggre-

gates of insoluble polymers of reduced diaminobenzidine, which

are inherent to the nature of the final LHRH-staining-reaction

product regardless of the granular or agranular nature of the

substance localized. The ultrastructural correlate of the final

reaction product seen light microscopic immunocytochemistry

for LHRH is stained peptide associated with the rough en-

doplasmic reticulum, Golgi complex, neurosecretory granules,

neurotubules, and the axonal endoplasmic reticulum (Koz-

lowski and Hostetter, 1978; Kozlowski et al., 1980). There-

fore, one must be cautious when relating the immunostaining



88 KOZLOWSKI, DEES

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Figure 5. Adjacent sections through the middle median eminence of

the rat using unabsorbed WP-1 anti-LHRH antiserum (5a), or thesame antiserum absorbed with LHRH (b), BSA (c), and both BSAand LHRH (d). There is staining of arcuate nucleus neurons (AN)when anti-BSA is present in the antiserum (a, b). Original magnifi-cation x85. Bar = 120 p.m.

seen with light microscopy to the subcellular distribution of

LHRH.

The many demonstrated examples of MBH neurons

throughout the rostral, middle, and caudal ME serves as a map

of those neurons especially laden with albuminoid substances.

In the rostral hypothalamus, elements of the magnocellular

system (i.e. , paraventricular, supraoptic, and associated neu-

rons and fibers) also react with anti-BSA. Nilaver et al. (1982)

immunostained rat-brain sections with rabbit anti-rat serum

albumin in order to describe more fully those neurons having

albuminoid substances. He found that some neurons of the

arcuate nucleus stained under these conditions (Nilaver, per-

sonal communication). These albuminoid-containing neurons

probably share the common feature of forming neurohemal

contacts with blood vessels of the hypophysial portal vessels

of the ME or the pars nervosa of the neurohypophysis. It is

at these sites that the neurons could incorporate blood-borne

albumins and transport them retrogradely to their perikaya

(Nilaver et al., 1982). The numbers of albuminoid-containing

cell bodies or the amount ofalbuminoid substance within these

cell bodies may therefore vary according to the plasma albumin

level.

Naik’s (1974, 1975a and b, 1976) localization studies for

LHRH indicate that there may be some unknown type of

interfering antibodies present in the anti-LHRH antiserum

used in those investigations. This antiserum not only stains

arcuate neurons, but also parvocellular neurons of the su-

praoptic and paraventricular nuclei (Naik, 1974). Indeed, Naik

(1976) claims to have demonstrated, with paraffin sections, a

cyclic accumulation of LHRH-positive material in the cere-

brospinal fluid ofthe infundibular recess. We have found stain-

ing of the material in the third ventricle to be due to the

presence ofanti-BSA (Figures la, 2a and b, 3a and b, and 4a)

which reacts with albuminized slides. The method of prepa-

ration of this antibody and its use for radioimmunoassay were

reported by Hirsch and Givner (1976). As Kelly et al. (1982)

remarked, the antiserum is of low titer; however, even low-

titer antisera can be used for specific localization studies (Swaab,

1982). Buffa et al. (1979a, 1979b) warn about the possible

nonspecific binding of immunoglobulins to endocrine cells

mediated by the Ciq fraction ofcomplcmcnt, which they found

can be overcome by using complement-deprived, highly di-

luted anti-hormone antisera. Naik ( 1976) reported that the

antiserum he used (Hirsch and Givncr, 1976) was diluted

1 :40- 1 : 100 for routine fluorescent and peroxidase techniques.

Additionally, Hirsch and Givncr (1976) dissolved their pep-

tide in 0.1% gelatin in physiologic saline for their booster

injections. Their rabbit may therefore have produced anti-

gelatin antibodies which in turn could have stained gelatin-

like substances in neurons of the arcuate and magnocellular

systems. Another possibility is that the rabbit they used for

actively raising anti-LHRH antisera may have had albumin

autoantibodics that cross-reacted with rat albuminoid sub-

stances. This type of autoimmunity is known to occur in pa-

tients with acute or chronic liver disease (Hauptman and To-

masi 1974, Kakumu et al., 1979). The use of preimmune

serum as a negative control is especially important whenever

a newly-produced, uncharacterized antiserum is to be used for

immunocytochemistry, and along with other controls would

obviate the possibility of such a cross-reaction. In order to



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Figure 6. Nerve-cell bodies of the me-

dial basal hypothalamus which react with

anti-BSA using the Kelch #12 anti-

LHRH antiserum range in shape fromfusiform, through spheroid, to multi-

polar. Original magnification x 500. Bar

= 20 p.m.

Figure 7. In comparison with the neu-rons shown in Figure 6, LHRH-contain-ing neurons of the preoptic area are much

smaller and are oftwo types: type 1 cells,which are smooth and bipolar (arrows);

and type 2 cells, which are rough and

unipolar (arrowheads). OVLT = or-ganum vasculosum of the lamina ter-

minalis. Original magnification x 500.

Bar = 50 p.m.

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determine the nature of any possible interfering antibodies,

one would have to perform staining experiments with appro-

priate controls using the Hirsch and Givner ( 1976) antisera,

as we have done here with WP-1. It must be emphasized that

although antisera generated against LHRH-BSA conjugates

could present a problem when used for immunocytochemistry,

their use for radioimmunoassay remains unaffected by the

presence of anti-BSA (Kozlowski et al., 1975).

Figure 8. Example of staining for LHRH using an antiserum (Koz-

lowski #705) against a hapten-conjugate with thyroglobulin as the

carrier. With 50-p.m-thick vibrating-microtome sections, the close as-

sociation of LHRH-containing fibers (arrow) to ependymal cells of

the third ventricle is easily appreciated. The lack of background stain-ing also allows easy visualization of the ventrolateral course of the

fibers (arrowheads) as they pass through the region of the arcuatenucleus, where a single type 1 LHRH-containing neuron (double-

arrow) is found. Original magnification x 120. Bar = 85 p.m.

King et al. (1980) and Kawano and Daikoku (1981) regard

the single LHRH-containing cell bodies that they report in

the vicinity of the arcuate nucleus or median eminence of the

rat, respectively, as a rarity rather than a significant finding.

Our example of a single LHRH-containing cell body in the

rat arcuate nucleus should also be regarded as a capricious

finding. Since immunocytochemical techniques cannot prove

that LHRH does not exist in neurons ofthe rat arcuate nucleus,

the question of their possible existence may be considered as

unanswered. Nevertheless, future methodologic develop-

ments and immunoprobes such as monoclonal antibodies or

antisera to an LHRH precursor molecule may help to answer

this important question.

This report has given evidence of false-positive results in

the immunocytochemical localization of LHRH with antisera

generated against LHRH-BSA haptcn-conj ugates. Likewise,

investigators intending to localize other substances or peptides

should take into account the potential danger of using such

antisera.

Acknowledgments

We thank Drs. Robert P. Kelch (Ann Arbor, Mfl. Torrance Net,’ (Fort

Collins. C0. Gaj Nilazer (Nei� York. NY. andJohn C. Porter (Dallas.

TX; and Mr. Richard Hyfler (Arnel Products Co. . inc. . Brooklyn. NY)

for their gifts of antisera.

References

Barry J ( 1976): Characterization and topography of LHRH neurons

in the human brain. Neurosci Lett 3:287

BarryJ, Carette B ( 1975): Immunofluorescence study ofLRF neurons

in primates. Cell Tiss Res 164:163



90 KOZLOWSKI, DEES

Nilaver G, Brem H, Zimmerman EA (1982): Immunocytochemical

localization of albumin in rat brain. Neurology 32:A 107

Barry J, Dubois MP (1975): Immunofluorescence study of LRF-pro-ducing neurons in the cat and dog. Neuroendocrinology 18:290

Barry J, Dubois MP, Carette B ( 1974): Immunofluorescence study of

the preoptico-infundibular LRF neurosecretory pathway in the nor-mal, castrated or testosterone-treated male guinea pig. Endocrinology95:1416

Brownstein MJ, Arimura A, Schally AV, Palkovits M, KizerJS (1977):The effect of surgical isolation of the hypothalamus on its luteinizinghormone-releasing hormone content. Endocrinology 98:662

Buffa R, Solcia E, Fiocca R, Crivelli 0, Pera A (1979a): Complement-mediated binding of immunoglobulins to some endocrine cells of the

pancreas and gut. J Histochem Cytochem 27:1279

Buffa R, Crivelli 0, Fioica R, Fontana P, Solcia E (1979b): Comple-ment-mediated unspecific binding of immunoglobulins to some en-docrine cells. Histochemistry 63: IS

Bugnon C, Block B, Lenys D, Fellman D (1977): Ultrastructural studyof the LH-RH neurons in the human fetus. Brain Res 137:175

Carillo AJ, Hagino N, Setalo G (1980): Plasma LH levels following

electrochemical stimulation of the deafferented medial basal hypo-

thalamus. Acta Endocrinol 94:1 1

Clayton CJ, Hoffman GE (1979): Immunocytochemical evidence foranti-LHRH and anti-ACTH activity in the “F’ antiserum. Am J Anat1��:139

Dees WL, McArthur NH (1981): Immunohistochemical localizationof gonadotropin releasing hormone (GnRH) in the bovine brain andinfundibulum. Anat Rec 200:281

Dees WL, SorensenJr AM, Kemp WM, McArthur NH (1981a): GnRHlocalization in the equine brain and infundibulum: an immunohisto-chemical study. Brain Res 208:123

Dees WL, Sorensen Jr AM, Kemp WM, McArthur NH (1981b):Immunohistochemical localization ofgonadotropin releasing hormone(GnRH) in the brain and infundibulum of the sheep. Cell Tiss Res2 15: 181

Flerk#{243}B, S#{233}talOG, Vigh 5, Merchenthaler I (1980): Recent immu-nohistological findings on the LH-RH neuron system of the rat. MaterMed Pol 12:119

Fraser MO, Kelly MJ, Ronnekleiv OK (1982): Identification of tub-eroinfundibular luteinizing hormone-releasing hormone neurons inthe rat through fluorescence dye tracing and immunocytochemistry.Soc Neurosci (Abstr) 8:807

Gibbs FP (1973): Complete hypothalamic deafferentation and ACTHsecretion. Proc Fed Soc Exp Biol 32:295

Hal#{225}szB (1969): The endocrine effects of isolation of the hypothal-

amus from the rest of the brain. In Frontiers in Neuroendocrinology,Vol 2. Edited by L Martini, WF Ganong. Oxford University Press,New York, pp 307-342

Hauptman 5, Tomasi Jr TB (1974): Antibodies to human albumin in

cirrhotic sera. J Clin Invest 54:122

Hirsch MA, Givner ML(1976): Blockade oILHRH-induced ovulationin immature rats by LH-RH antiserum. CanJ Physiol Pharmacol 54:405

Hoffman GE, Gibbs FP (1982): LHRH pathways in rat brain: “deaf-ferentation” spares a sub-chiasmatic LHRH projection to the medianeminence. Neuroscience 7:1979

Hoffman GE, Melnyk V, Hayes T, Bennett-Clarke C, Fowler E (1978):Immunocytology of LHRH neurons. In Brain-Endocrine Interaction

III. Neural Hormones and Reproduction. Edited by DE Scott, GPKozlowski, A Weindl. S Karger, Basel, pp 67-82

Kakumu 5, ArakawaY, Groji H, KashioT, YataK(1979): Occurrenceand significance of antibody to liver-specific membrane lipoprotein bydouble-antibody immunoprecipitation method in sera of patients withacute and chronic liver diseases. Gastroenterology 76:665

Kawano H, Daikoku S (1981): Immunohistochemical demonstrationof LHRH neurons and their pathways in the rat hypothalamus. Neu-roendocrinology 32:179

Kelly MJ, Ronnekleiv OK (1981): Identification of luteinizing hor-mone-releasing hormone (LHRH) in the arcuate nucleus of hypotha-lamic slices. Soc Neurosci (Abstr) 7:20

Kelly MJ, Ronnekleiv OK, Eskay RI (1982): Immunocytochemicallocalization of luteinizing hormone-releasing hormone in neurons inthe medial basal hypothalamus of the female rat. Exp Brain Res 48:97

King JC, Tobet SA, Svarely FL, Arimura AA (1980): The LHRHsystem in normal and neonatally androgenized female rats. Peptides1 (Suppl 1):85

Kordon C, Patton E, Herman JP, Palkovits M (1982): Mapping ofLHRH-containing projections to the mediobasal hypothalamus by dif-ferential transection experiments. Brain Res (in press)

Kozlowski GP, Chu L, Hostetter G, Kerdelhu#{233} B (1980): Cellularcharacteristics of immunolabeled luteinizing hormone releasing hor-mone (LHRH) neurons. Peptides 1:37

Kozlowski GP, Frenk 5, McNeillTH, Scott DE, Hsu KC, ZimmermanEA (1976): Light and electron microscopic localization of gonadotro-pin releasing hormone (Gn-RH) in rat median eminence by immu-

noperoxidase technique. In First International Symposium on Im-munoenzymatic Techniques INSERM Symposium No. 2. Edited byP Feldmann, J Druet, S Bignon, S Avrameas. North-Holland, Am-sterdam, pp 329-332

Kozlowski GP, Hostetter G (1978): Cellular and subcellular localiza-tion and behavioral effects of gonadotropin-releasing hormone (Gn-RH) in the rat. In Neural Hormones and Reproduction. Brain-En-docrine Interaction, vol. III. Edited by DE Scott, GP Kozlowski, AWeindl. S Karger, New York, p 138-153

Kozlowski GP, NettTM, Zimmerman EA (1975): Immunocytochem-ical localization ofgonadotropin-releasing hormone (Gn-RH) and ncu-

rophysin in the brain. In Anatomical Neuroendocrinology. Edited by

WE Stumpf, LD Grant. S Karger, Basel, p. 185-191

Kozlowski GP, Nilaver G ( 1983): Immunoelectron microscopy ofneuropeptides: theoretical and technical considerations. In CurrentMethods in Cellular Neurobiology. Vol. 1: Anatomical Techniques.Edited byJL Barker,JF McKelvy.John Wiley and Sons, Poughkeepsie,NewYorkp 133-174

Kozlowski GP, Zimmerman EA (1974): Localization of gonadotropin-releasing hormone (Gn-RH) in sheep and mouse brain. Anat Rec

I 78:396

Krisch B (1980): Two types of luliberin-immunorcactive perikarya in

the preoptic area of the rat. Cell Tiss Res 2 12:443

Marshall PE, Goldsmith PC (1980): Neuroregulatory and neuroen-docrine GnRH pathways in the hypothalamus and forebrain of the

baboon. Brain Res 193:353

Naik DV (1974): Immunohistochemical and immunofluorescent lo-calization of LH-RF neurons in the hypothalamus of rat. Anat Rec

178:424

Naik DV (1975a): Immunoreactive LHRH neurons in the hypothal-amus identified by light and fluorescent microscopy. Cell Tiss Res157:423

Naik DV (1975b): Immuno-electron microscopic localization of lu-teinizing hormone-releasing hormone in the arcuate nuclei and medianeminence of the rat. Cell Tiss Res 157:437

Naik DV (1976): Immunohistochemical localization ofLH-RH duringdifferent phases of estrus cycle of rat, with reference to the preopticand arcuate neurons, and the ependymal cells. Cell Tiss Res 173:143




Palkovits M, Arimura A, Brownstein M, Schally AV, Saavedra JM

( 1974): Luteinizing hormone-releasing hormone (LH-RH) content of

the hypothalamic nuclei in rat. Endocrinology 96:554

Paulin C, Dubois MP, Barry J, Dubois PM (1977): Immunofluores-cence study of LH-RH producing cells in the human fetal hypothal-amus. Cell Tiss Res 182:341

Pickard GE, Silverman AJ (1976): Distribution of luteinizing hor-

mone-releasing hormone (LH-RH) in the brain of the adult goldenhamster. Soc Neurosci 2:975

Ronnekleiv OK, Kaul 5, Eskay RI (1981): f.3-endorphin (END) andluteinizing hormone (LHRH) immunoreactive neurons in the medial

basal hypothalamus (MBH) of the rat. Soc Neurosci (Abstr) 7:9 15

Ronnekleiv OK, Kelly MJ (1982): Topographical organization of lu-teinizing hormone releasing hormone (LHRH) and B-endorphin (B-

END) in the medial basal hypothalamus of the rat. First World Con-gress ofthe International Brain Research Organization. (Abstr) Lausanne

S#{233}t#{225}l#{243}G, Flerk#{243}B, Vigh 5, Arimura A, Schally AV (1978): Local-ization of LHRH-containing neurons in the rabbit brain. In Neurose-

cretion and Neuroendocrine Activity. Evolution, Structure and Func-tion. Edited by W Bargmann, A Oksche, A Polenov, B Scharses.

Springer-Verlag, Berlin, pp 287-289

Silverman AJ (1976): Distribution of luteinizing hormone-releasinghormone (LHRH) in the guinea pig brain. Endocrinology 99:30

Silverman AJ, AntunesJL, Abrams GM, Nilaver G, Thaw R, RobinsonJA, Ferin M, Krey LC (1982): The luteinizing hormone-releasing hor-

mone pathways in Rhesus (macaca mulatta) and Pigtailed (macacanemestrina) monkeys: New observations on thick unembedded sec-tions. J Comp Neurol 211:309

Sternbcrger LA, Hardy Jr PH, Cuculis JJ, Meyer HG (1970): Theunlabeled antibody enzyme method of immunohistochemistry: prep-aration and properties of soluble antigen-antibody complex (horse-radish peroxidase-antihorseradish peroxidase) and its use in identifi-cation of spirochetes. J Histochem Cytochem 18:31S

Swaab DF ( 1982): Comments on the validity of immunocytochemical

methods. In Cytochemical Methods in Neuroanatomy. Edited by SLPalay, V Chan-Palay. Alan R Liss, New York, pp 423-440

Zimmerman EA, Hsu KG, Ferin M, Kozlowski GP (1974): Localiza-

tion ofgonadotropin releasing hormone (Gn-RH) in the hypothalamusof the mouse by immunoperoxidase technique. Endocrinology 9�:1

Zimmerman EA, Krupp L, Hoffman DL, Matthew E, Nilaver G (1980):

Exploration of peptidergic pathways in brain by immunocytochem-istry: a ten-year perspective. Peptides 1 (Suppl 1 ): 3



� Immunocytochemistry for LHRH Neurons in the � Arcuate Nucleus Area of the Rat

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