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ACTH-immunoreactive neurons and their projections in the cat forebrain
Peptides, Vol. 7, pp. 801-807, 1986. ~: Ankho International Inc. Printed in the U.S.A.
0196-9781/86 $3.00 + .00
ACTH-Immunoreactive Neurons and Their Projections in the Cat Forebrain K. K I T A H A M A , 1 M. S A L L A N O N , C. B U D A , M. J A N I N , M. P. D U B O I S * A N D M. J O U V E T
D~partement de M~deeine Exp~rimentale, I N S E R M U52 and C N R S U A l l95 Facultt; de M~;decine, Universit~ ~ Claude Bernard, Lyon, France *Laboratoire de Neuroendocrinologie, I N R A , Nouzilly, France R e c e i v e d 5 M a y 1986 KITAHAMA, K.. M. SALLANON, C. BUDA, M. JANIN, M. P. DUBOIS AND M. JOUVET. ACTH-immunoreactive neurons and their projections in the catjorebrain. PEPT1DES 7(5)801-807, 1986.--The organization of adrenocorticotropin (ACTH)-immunoreactive (IR) cell bodies and fibers in the cat forebrain is described. ACTH-IR cell bodies are found only in and around the arcuate nucleus of the hypothalamus (ARH). They are not detected elsewhere even after pretreatment with colchicine. ACTH-IR fibers are present in discrete areas of the hypothalamus, the septo-limbic areas and in the paraventricular thalamic nucleus. Complete electrolytic lesions of the ARH destroy ACTH-IR cell bodies as well as fibers in all parts of the brain. These results suggest that, in the cat forebrain, the ARH is the only source of ACTH-IR fibers. ACTH
Cat
Arcuate nucleus
Hypothalamus
Electrolytic lesion study
C E R E B R A L adrenocorticotropin (ACTH)-immunoreactive (IR) products have been extensively investigated both by radioimmunoassay and immunohistochemistry in recent years [3, 4, 9, 10, 15, 19, 21, 23, 24, 28, 32, 34] with particular reference to the rat. In the rat, A C T H - I R cell bodies have been reported to be present in the mediobasal part of the hypothalamus (MBH), specifically in the arcuate nucleus of the hypothalamus (ARH), which extends from the retrochiasmatic area to the mamillary recess. The fiber system is widely distributed in the brain and includes both intra- and extra-hypothalamic regions. The projections from the MBH to discrete areas of the hypothalamus have also been reported using retrograde transport techniques [6, 13, 18, 27, 30, 35]. The cat is widely used in neurophysiological studies and in contrast, studies on A C T H - I R structures in the cat have not yet been effected. The present paper represents an attempt to determine the distribution of A C T H - I R cell bodies and fibers in the cat forebrain and to demonstrate that the arcuate A C T H - I R cell bodies are the sole source of the A C TH- I R fibers in the cat forebrain.
lmmunohistochemistry
A C T H - I R cells and fibers in the forebrain. This series was sacrificed with or without colchicine pretreatment. Colchicine treatment was effected intraventricularly with 200/zg of colchicine (Sigma) (N=6). Six cats did not receive colchicine for the purpose of describing the fiber distribution. In the second group (6 animals), electrolytic lesions were performed in order to destroy the A CTH - I R cell bodies in and around the ARH. The bilateral lesions of the basal hypothalamus were performed under Nembutal anesthesia by means of a monopolar electrode, stereotaxically positioned in the structures, through which a 4 mA current was passed for 10 seconds. The following coordinates were used in accordance with the atlas of Reinoso-Suarez [26]. ARH: A=9.0, L=0.5, H = - 6 . 0 ; A=10.0, L=0.7, H = - 5 . 5 and A = l l . 0 , L=0.9, H = - 6 . 0 . After continuous electrophysiological recordings, animals were sacrificed from 11 to 28 days following surgery. The lesion sites and their affected areas are shown in Fig. 5. Following extensive neurophysiological experiments, under deep Nembutal anesthesia, all the animals were perfused through the ascending aorta with 0.9~?b saline followed by an ice-cold fixative containing 2% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB, pH=7.4). The brains were cut into several blocks and immersed immediately in 0.1 M PB containing 2% paraformaldehyde and 0.2% picric acid at 4°C for 48 hours.
METHOD Eighteen adult cats were used for this study. These animals were divided into two experimental groups. The first group (12 cats) was used to describe the distribution of
~Requests for reprints should be addressed to Kunio Kitahama, Departement de Medecine Experimentale, Faculte de Medecine, Universite Claude Bernard, 8 avenue Rockefeller, 69373, Lyon, France.
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KITAHAMA ABBREVIATIONS A AC ACC ARH CA DBV DMH EN F GP HAA HDA HLA HPA IC LV MM MR MT
amygdala anterior c o m m i s s u r e nucleus a c c u m b e n s arcuate nucleus o f the h y p o t h a l a m u s caudate nucleus nucleus of the diagonal band o f Broca, vertical division dorsomedial hypothalamic nucleus e n t o p e d u n c u l a r nucleus fornix globus pallidus anterior hypothalamic area dorsal hypothalamic area lateral hypothalamic area posterior hypothalamic area internal capsule lateral ventricle medial mamillary nucleus mamillary recess mamillothalamic tract
OC OT PARA PP PVH SCN SFN Sl SLN SMN SON ST STN STR SUB TCA V3 VMH ZI
optic chiasm optic tract paraventricular nucleus of the thalamus pes pedunculi paraventricular nucleus of the h y p o t h a l a m u s suprachiasmatic nucleus septofimbrial nucleus substantia innominata lateral septal nucleus medial septal nucleus supraoptic nucleus stria terminalis triangular septal nucleus stria terminalis subthalamic nucleus area of the tuber cinereum third ventricle ventromedial hypothalamic nucleus zona incerm
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FIG. I. Photomicrograph of A C T H - i m m u n o r e a c t i v e (IR) n e u r o n s and fibers in the arcuate nucleus of the h y p o t h a l a m u s (ARH). B a r s - 100 /xm.
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These blocks were then transferred to 15% sucrose in PB for 48 hours at 4°C. Subsequently, 20 ~ m frozen frontal sections were cut on a cryostat and pretreated for at least 4 days in phosphate buffered saline (PBS) containing 0.3% Triton-X and 0.1% sodium azide, followed by incubation with antiACTH antiserum at a dilution of 1:30000 at 4°C for 96 hours. Following several rinses in PBS, sections were transferred to a 1:1000 dilution of swine antibody to rabbit immunoglobulin (DAKO) at 4°C overnight. Sections were then rinsed several times and then incubated in rabbit peroxidase antiperoxidase (rabbit PAP, DAKO) diluted 1:2000 for 2 hours at room temperature. After final rinses, sections were developed in 0.02% 3-3'-diaminobenzidine solution containing 0.003% hydrogen peroxide and 0.5% nickel ammonium sulfate for I0 minutes [7]. The reaction was terminated by washes in a Tris-saline rinse. Mounted sections were dehydrated and coverslipped using Depex c~. These sections were traced on a camera lucida to accurately locate ACTH-IR structures. The atlases of Berman [2] and Reinoso-Suarez [26] were consulted for nomenclature and identification of anatomical structures and stereotaxic plane. The preparation and immunological specificity of the antiserum used in the present study (Anti-ACTH 1-24) have been described elsewhere [1,5]. To check the specificity of the immunohistochemical reaction in the cat brain tissue, the
controls were made by (1) using a normal non-immune rabbit serum instead of anti-ACTH serum, (2) omitting the antiACTH serum, (3) incubating the antiserum with homologous or heterologous antigens. No immunohistochemical reaction was observed when the anti-ACTH serum was omitted or replaced by non-immune rabbit serum. The staining for anti-ACTH serum was inhibited only after preincubation with antigens ACTH 1-39, 1-24, 17-39. The preincubation with other antigens without any common amino acid sequence did not affect the immunohistochemical reaction (/3-LPH, a-endorphin,/~-endorphin, c~-MSH, /3-MSH). This antiserum did not stain enkephalin- nor substance P-containing neurons. RESULTS
A C T H - I R Cell Distribution
As shown in Fig. 1, the reaction product in immunoreactive neurons after staining with ACTH antiserum is uniformly distributed over the cytoplasm of the cells whose shapes are generally oval to fusiform and 20-25 microns in diameter. They generally have one or two short processes. Figure 2 illustrates a mapping of the topography of perikarya in the ACTH bed nucleus. As shown in Fig. 2a, ACTH-IR perikarya are present in the ARH surrounding the mamillary
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KITAHAMA E T A L .
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FIG. 3. Schematic drawings of distribution of ACTH-IR fibers in the cat basal forebrain.
recess. The majority of the neurons are located in the area ventrolateral to the recess. More rostrally, at the level of A 10 (Fig. 2b), ACTH-IR cells are more widely distributed and extend more ventrally and laterally. Density of the neurons is relatively high in the floor of the third ventricle. At the level of A11, the largest population of ACTH-IR neurons is clustered in the medial portion of the ARH (Fig. 2c). The perikarya spread dorsally to the border of the ventromedial hypothalamic nucleus (VMH) and extend ventrally to the area of junction of the ARH and median eminence. At the more rostral level (AI2, Fig. 2d), corresponding to the retrochiasmatic area in the rat, ACTH-IR neurons are less densely packed in the ARH in comparison with its caudal portion. A C T H - I R F i b e r Distribution
The majority of ACTH-IR fibers shows thick or fine short processes with varicosities (Fig. 1). These fibers appear to be randomly oriented and form more or less dense plexuses. Figure 3 shows frontal aspect of schematic ACTH-IR fiber distribution in the cat forebrain. More caudally, at the level of the medial mamillary nucleus, (Fig. 3-A8), where no ACTH-IR perikarya are ob-
served, the fibers are seen in the area surrounding the medial mamillary nucleus, and extend laterally. Vertically oriented fibers are present in the supramamillary decussation, posterior hypothalamic area (HPA), the zone surrounding the third ventricle, the midline of the thalamus, and the paraventricular nucleus of the thalamus (PARA). At the level of the mamillary recess (Fig. 3-A9), ACTHIR fibers are seen in the ARH and extend laterally at the surface of the brain, or dorsally to the HPA. They are also observed in the PARA. At the level of the rostral portion of the HPA (Fig. 3-AI0), the density of the ACTH-IR fibers markedly increases, and they form dense plexuses in the ARH and the periventricular zone. They are also intensely stained in the HPA and perifornical region. Several long fibers horizontally traverse the area just ventral to the mamillo-thalamic tract and extend into the zona incerta. Fine ACTH-IR fibers are observed passing through the area just dorsal to the optic tract. More rostrally, at the level of the caudal portion of the VMH (Fig. 3-A11), ACTH-IR fiber plexuses are evident in the ARH, the dorsomedial nucleus of the hypothalamus (DMH), the periventricular zone and the perifornical region. At this level, virtually all the hypothalamic regions are moderately stained by ACTH-IR fibers, including the VMH
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where a moderately dense plexus is seen in the cat, whereas this nucleus has been reported to be free of ACTH-IR fibers in the rat [10,15]. The fibers extend laterally to the lateral hypothalamic area (HLA) and further to the area dorsal to the optic tract, and seem to enter the amygdala. At the level of the rostral part of the VMH (Fig. 3-A12), ACTH-IR fibers are densely packed in the ARH and periventricular zone. They are observed in the VMH and supraoptic nucleus but fewer in number. At the level of A13 (Fig. 3-A13), ACTH-IR fibers are densely distributed in the periventricular zone, and in the medial portion of the PVH. The long processes are detected in the perifornical region and the basal part of the HLA. Laterally oriented fibers noted in the area dorsal to the supraoptic nucleus appear to invade the amygdala. Ascending fibers are evident in the area between the third ventricle and PARA. More rostrally, at the level of A14 and A15 (Fig. 3), ACTH-IR fibers are concentrated in the periventricular
zone, the preoptic portion of the anterior hypothalamic area (HAA), the PVH and the basal part of the HLA. They are fewer in number in the supraoptic nucleus and few, if any, are present in the suprachiasmatic nucleus. ACTH-IR fibers form a dense plexus in the septofimbrial nucleus, in the area medial, ventral and dorsal to the anterior commissure and in the bed nucleus of the stria terminalis (BNST). The fibers are always detected in the stria terminalis from A8 plane to this level. We observed ventrolaterally oriented fibers in the HLA. At the level of A16, ACTH-IR fibers ascend to the lateral edge of the lateral septal nucleus, passing through the BNST. They are also abundant in the nucleus accumbens. Lesion Studies
Figure 4 shows the diagrams of lesion studies in the hypothalamus of the cats utilized for various physiological experiments. In two cases (V94 and B95), a complete arcuate lesion
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KITAHAMA E T A L .
abolished aH the ACTH-IR cell bodies and consequently no ACTH-IR fibers were detected in any part of the brain. These animals survived for 28 and 16 days after surgery, In cases I94 and 094, which survived for 14 and 18 days after surgery, ARH was incompletely destroyed and we observed a marked decrease in the number of the fibers mostly in the caudal segments, and no fibers are detected in the area lateral to the lesion site at the level of A9. The remaining fibers, however, were diffusely distributed in the forebrain. In case 093, in which the ARH was almost entirely destroyed except for its anterior portion, there was a severe decrease in the fibers after a survival of 24 days. The loss of the large cell component of the ARH was obtained in case T95 which survived for 14 days. A group of ACTH-IR cells were preserved only in the anterior portion of the nucleus. In all the brain, almost all the fibers disappeared. However, we detected occasional fibers in the periventricular region of the hypothalamus. DISCUSSION Our present immunohistochemical method enabled us to demonstrate a widespread distribution of ACTH-IR neuronal structures in the cat forebrain. The results obtained here in the cat are almost identical in terms of distribution of staining for ACTH immunoreactivity within cells and fibers of the rat brain with several exceptions mentioned in the Results section. It is likely that the source of the cerebral ACTH-IR fibers, from the present results, is the ARH. Incomplete lesions of the ARH induce a decrease in ACTH-IR fibers according to the dimensions and site of lesion. This result confirms the almost total disappearance of ACTH-IR fibers in the rat after systemic neonatal injections of monosodium glutamate [17, 21, 24]. This treatment failed to abolish completely ACTH-IR cells in the MBH, and preserved their fibers even after 10 daily consecutive injections of high doses in the rat [21] and in the cat (in preparation). Microinjection of a neurotoxin such as kainic acid in the ARH induces the death of the animal. Thus, only complete electrolytic coagulation of the cat mediobasal hypothalamus leads to their total disappearance. Recent results obtained with ACTH immunohistochemistry have reported the presence of ACTH-IR cell bodies within the pars commissuralis of the nucleus of the tractus solitarius of the rat [11,31], and we also confirmed them in the rat by our present technique using the antiserum ACTH 1-39 (in preparation). In the cat, however, we could not detect them in the medulla oblongata with the same antiserum even after colchicine pretreatment in situ and/or intraventricular administration. The absence of a medullary group has also been reported in the lizard [12]. Since we observed also the total disappearance of ACTH-IR fibers in the brain stem of the pontile cat three weeks after total removal of the tele-diencephalon (in preparation), it is most likely that no lower brain stem perikarya exist in the cat. As far as the organization of ACTH-IR fibers is concerned, the present results may lead us to postulate the possible trajectory of ACTH-IR fiber pathways.
Arcuate cells project diffusely to the various hypothalamic nuclei, such as DMH, VMH, perifornical region, PVH, HAA and periventricular stratum making up dense fiber plexuses. In the rat, DMH and VMH are reported to receive the fibers from the ARH [13,18], and fibers in the PVH come from the ARH according to retrograde transport [30] and electrophysiological [8} studies. The fiber plexuses in the preoptic portion of the HAA, which originate mainly in the middle and caudal portion of the ARH revealed by our HRP study (in preparation), might diffusely course through the ventral part of the hypothalamus. They may continue to ascend in the area rostral to the anterior commissure and enter into the lateral septum and/or BNST and stria terminalis. In the rat, they project from several arcuate cells to the BNST [35]. They continue to extend in a dorsocaudal direction up to the level of A6. The fibers in the stria terminalis located laterally to the optic tract require further investigation in order to determine their pathway. Projection into the amygdala is more complicated. From HRP study, the cells of the middle portion of the ARH are labeled by this tracer when injected into the central amygdala in the cat [29]. Our preliminary data revealed that the cells doubly labeled with HRP and ACTH can be observed in the bilateral ARH. The fibers may course diffusely into the HLA and extend to the area dorsal to the optic tract (AI0-A12) to invade the amygdala. Now it is in progress to determine the origin of these projections by employing the retrograde cell marker HRP coupled with the present immunohistochemical method. Finally, as arcuate fibers appear to bifurcate with one branch leaving the nucleus and the other terminating within it as described by Van den Pol [33], ACTH-IR neurons may reciprocally exchange information between the ARH and the terminal fields. Recent immunohistochemical studies have demonstrated the remarkable concordance of localization of corticotropin releasing factor-IR perikarya and ACTH-IR fibers within discrete hypothalamic, limbic and brain stem autonomic regions in the rat [25], suggesting the reciprocal influences between the two systems. In addition, several ACTH-IR neurons in the ARH project directly to the locus coeruleus [14] rich in noradrenergic cells, lontophoretically applied ACTH in the rat locus coeruleus were excitatory on the majority of the neurons [22}, from which evidence we can speculate that ACTH-IR neurons may also have an influence upon the other terminal fields. These types of neurons might constitute the neuronal component of the ultra-short feedback in the hypothalamus [20] controlling the outputs of these regions. This problem requires further studies in order to be elucidated.
ACKNOWLEDGEMENTS The investigation in this report was supported by INSERM (U52), CNRS (UAII95), DRET (Grant 84-160) and Biologie Humaine. The authors thank N. Gay for her skillful technical assistance and Dr. J. Carew for correction of the EngLish.
ACTH-IMMUNOREACTIVE
N E U R O N S IN C A T F O R E B R A I N
807
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0196-9781/86 $3.00 + .00
ACTH-Immunoreactive Neurons and Their Projections in the Cat Forebrain K. K I T A H A M A , 1 M. S A L L A N O N , C. B U D A , M. J A N I N , M. P. D U B O I S * A N D M. J O U V E T
D~partement de M~deeine Exp~rimentale, I N S E R M U52 and C N R S U A l l95 Facultt; de M~;decine, Universit~ ~ Claude Bernard, Lyon, France *Laboratoire de Neuroendocrinologie, I N R A , Nouzilly, France R e c e i v e d 5 M a y 1986 KITAHAMA, K.. M. SALLANON, C. BUDA, M. JANIN, M. P. DUBOIS AND M. JOUVET. ACTH-immunoreactive neurons and their projections in the catjorebrain. PEPT1DES 7(5)801-807, 1986.--The organization of adrenocorticotropin (ACTH)-immunoreactive (IR) cell bodies and fibers in the cat forebrain is described. ACTH-IR cell bodies are found only in and around the arcuate nucleus of the hypothalamus (ARH). They are not detected elsewhere even after pretreatment with colchicine. ACTH-IR fibers are present in discrete areas of the hypothalamus, the septo-limbic areas and in the paraventricular thalamic nucleus. Complete electrolytic lesions of the ARH destroy ACTH-IR cell bodies as well as fibers in all parts of the brain. These results suggest that, in the cat forebrain, the ARH is the only source of ACTH-IR fibers. ACTH
Cat
Arcuate nucleus
Hypothalamus
Electrolytic lesion study
C E R E B R A L adrenocorticotropin (ACTH)-immunoreactive (IR) products have been extensively investigated both by radioimmunoassay and immunohistochemistry in recent years [3, 4, 9, 10, 15, 19, 21, 23, 24, 28, 32, 34] with particular reference to the rat. In the rat, A C T H - I R cell bodies have been reported to be present in the mediobasal part of the hypothalamus (MBH), specifically in the arcuate nucleus of the hypothalamus (ARH), which extends from the retrochiasmatic area to the mamillary recess. The fiber system is widely distributed in the brain and includes both intra- and extra-hypothalamic regions. The projections from the MBH to discrete areas of the hypothalamus have also been reported using retrograde transport techniques [6, 13, 18, 27, 30, 35]. The cat is widely used in neurophysiological studies and in contrast, studies on A C T H - I R structures in the cat have not yet been effected. The present paper represents an attempt to determine the distribution of A C T H - I R cell bodies and fibers in the cat forebrain and to demonstrate that the arcuate A C T H - I R cell bodies are the sole source of the A C TH- I R fibers in the cat forebrain.
lmmunohistochemistry
A C T H - I R cells and fibers in the forebrain. This series was sacrificed with or without colchicine pretreatment. Colchicine treatment was effected intraventricularly with 200/zg of colchicine (Sigma) (N=6). Six cats did not receive colchicine for the purpose of describing the fiber distribution. In the second group (6 animals), electrolytic lesions were performed in order to destroy the A CTH - I R cell bodies in and around the ARH. The bilateral lesions of the basal hypothalamus were performed under Nembutal anesthesia by means of a monopolar electrode, stereotaxically positioned in the structures, through which a 4 mA current was passed for 10 seconds. The following coordinates were used in accordance with the atlas of Reinoso-Suarez [26]. ARH: A=9.0, L=0.5, H = - 6 . 0 ; A=10.0, L=0.7, H = - 5 . 5 and A = l l . 0 , L=0.9, H = - 6 . 0 . After continuous electrophysiological recordings, animals were sacrificed from 11 to 28 days following surgery. The lesion sites and their affected areas are shown in Fig. 5. Following extensive neurophysiological experiments, under deep Nembutal anesthesia, all the animals were perfused through the ascending aorta with 0.9~?b saline followed by an ice-cold fixative containing 2% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB, pH=7.4). The brains were cut into several blocks and immersed immediately in 0.1 M PB containing 2% paraformaldehyde and 0.2% picric acid at 4°C for 48 hours.
METHOD Eighteen adult cats were used for this study. These animals were divided into two experimental groups. The first group (12 cats) was used to describe the distribution of
~Requests for reprints should be addressed to Kunio Kitahama, Departement de Medecine Experimentale, Faculte de Medecine, Universite Claude Bernard, 8 avenue Rockefeller, 69373, Lyon, France.
801
802
ET AL
KITAHAMA ABBREVIATIONS A AC ACC ARH CA DBV DMH EN F GP HAA HDA HLA HPA IC LV MM MR MT
amygdala anterior c o m m i s s u r e nucleus a c c u m b e n s arcuate nucleus o f the h y p o t h a l a m u s caudate nucleus nucleus of the diagonal band o f Broca, vertical division dorsomedial hypothalamic nucleus e n t o p e d u n c u l a r nucleus fornix globus pallidus anterior hypothalamic area dorsal hypothalamic area lateral hypothalamic area posterior hypothalamic area internal capsule lateral ventricle medial mamillary nucleus mamillary recess mamillothalamic tract
OC OT PARA PP PVH SCN SFN Sl SLN SMN SON ST STN STR SUB TCA V3 VMH ZI
optic chiasm optic tract paraventricular nucleus of the thalamus pes pedunculi paraventricular nucleus of the h y p o t h a l a m u s suprachiasmatic nucleus septofimbrial nucleus substantia innominata lateral septal nucleus medial septal nucleus supraoptic nucleus stria terminalis triangular septal nucleus stria terminalis subthalamic nucleus area of the tuber cinereum third ventricle ventromedial hypothalamic nucleus zona incerm
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FIG. I. Photomicrograph of A C T H - i m m u n o r e a c t i v e (IR) n e u r o n s and fibers in the arcuate nucleus of the h y p o t h a l a m u s (ARH). B a r s - 100 /xm.
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These blocks were then transferred to 15% sucrose in PB for 48 hours at 4°C. Subsequently, 20 ~ m frozen frontal sections were cut on a cryostat and pretreated for at least 4 days in phosphate buffered saline (PBS) containing 0.3% Triton-X and 0.1% sodium azide, followed by incubation with antiACTH antiserum at a dilution of 1:30000 at 4°C for 96 hours. Following several rinses in PBS, sections were transferred to a 1:1000 dilution of swine antibody to rabbit immunoglobulin (DAKO) at 4°C overnight. Sections were then rinsed several times and then incubated in rabbit peroxidase antiperoxidase (rabbit PAP, DAKO) diluted 1:2000 for 2 hours at room temperature. After final rinses, sections were developed in 0.02% 3-3'-diaminobenzidine solution containing 0.003% hydrogen peroxide and 0.5% nickel ammonium sulfate for I0 minutes [7]. The reaction was terminated by washes in a Tris-saline rinse. Mounted sections were dehydrated and coverslipped using Depex c~. These sections were traced on a camera lucida to accurately locate ACTH-IR structures. The atlases of Berman [2] and Reinoso-Suarez [26] were consulted for nomenclature and identification of anatomical structures and stereotaxic plane. The preparation and immunological specificity of the antiserum used in the present study (Anti-ACTH 1-24) have been described elsewhere [1,5]. To check the specificity of the immunohistochemical reaction in the cat brain tissue, the
controls were made by (1) using a normal non-immune rabbit serum instead of anti-ACTH serum, (2) omitting the antiACTH serum, (3) incubating the antiserum with homologous or heterologous antigens. No immunohistochemical reaction was observed when the anti-ACTH serum was omitted or replaced by non-immune rabbit serum. The staining for anti-ACTH serum was inhibited only after preincubation with antigens ACTH 1-39, 1-24, 17-39. The preincubation with other antigens without any common amino acid sequence did not affect the immunohistochemical reaction (/3-LPH, a-endorphin,/~-endorphin, c~-MSH, /3-MSH). This antiserum did not stain enkephalin- nor substance P-containing neurons. RESULTS
A C T H - I R Cell Distribution
As shown in Fig. 1, the reaction product in immunoreactive neurons after staining with ACTH antiserum is uniformly distributed over the cytoplasm of the cells whose shapes are generally oval to fusiform and 20-25 microns in diameter. They generally have one or two short processes. Figure 2 illustrates a mapping of the topography of perikarya in the ACTH bed nucleus. As shown in Fig. 2a, ACTH-IR perikarya are present in the ARH surrounding the mamillary
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KITAHAMA E T A L .
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FIG. 3. Schematic drawings of distribution of ACTH-IR fibers in the cat basal forebrain.
recess. The majority of the neurons are located in the area ventrolateral to the recess. More rostrally, at the level of A 10 (Fig. 2b), ACTH-IR cells are more widely distributed and extend more ventrally and laterally. Density of the neurons is relatively high in the floor of the third ventricle. At the level of A11, the largest population of ACTH-IR neurons is clustered in the medial portion of the ARH (Fig. 2c). The perikarya spread dorsally to the border of the ventromedial hypothalamic nucleus (VMH) and extend ventrally to the area of junction of the ARH and median eminence. At the more rostral level (AI2, Fig. 2d), corresponding to the retrochiasmatic area in the rat, ACTH-IR neurons are less densely packed in the ARH in comparison with its caudal portion. A C T H - I R F i b e r Distribution
The majority of ACTH-IR fibers shows thick or fine short processes with varicosities (Fig. 1). These fibers appear to be randomly oriented and form more or less dense plexuses. Figure 3 shows frontal aspect of schematic ACTH-IR fiber distribution in the cat forebrain. More caudally, at the level of the medial mamillary nucleus, (Fig. 3-A8), where no ACTH-IR perikarya are ob-
served, the fibers are seen in the area surrounding the medial mamillary nucleus, and extend laterally. Vertically oriented fibers are present in the supramamillary decussation, posterior hypothalamic area (HPA), the zone surrounding the third ventricle, the midline of the thalamus, and the paraventricular nucleus of the thalamus (PARA). At the level of the mamillary recess (Fig. 3-A9), ACTHIR fibers are seen in the ARH and extend laterally at the surface of the brain, or dorsally to the HPA. They are also observed in the PARA. At the level of the rostral portion of the HPA (Fig. 3-AI0), the density of the ACTH-IR fibers markedly increases, and they form dense plexuses in the ARH and the periventricular zone. They are also intensely stained in the HPA and perifornical region. Several long fibers horizontally traverse the area just ventral to the mamillo-thalamic tract and extend into the zona incerta. Fine ACTH-IR fibers are observed passing through the area just dorsal to the optic tract. More rostrally, at the level of the caudal portion of the VMH (Fig. 3-A11), ACTH-IR fiber plexuses are evident in the ARH, the dorsomedial nucleus of the hypothalamus (DMH), the periventricular zone and the perifornical region. At this level, virtually all the hypothalamic regions are moderately stained by ACTH-IR fibers, including the VMH
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where a moderately dense plexus is seen in the cat, whereas this nucleus has been reported to be free of ACTH-IR fibers in the rat [10,15]. The fibers extend laterally to the lateral hypothalamic area (HLA) and further to the area dorsal to the optic tract, and seem to enter the amygdala. At the level of the rostral part of the VMH (Fig. 3-A12), ACTH-IR fibers are densely packed in the ARH and periventricular zone. They are observed in the VMH and supraoptic nucleus but fewer in number. At the level of A13 (Fig. 3-A13), ACTH-IR fibers are densely distributed in the periventricular zone, and in the medial portion of the PVH. The long processes are detected in the perifornical region and the basal part of the HLA. Laterally oriented fibers noted in the area dorsal to the supraoptic nucleus appear to invade the amygdala. Ascending fibers are evident in the area between the third ventricle and PARA. More rostrally, at the level of A14 and A15 (Fig. 3), ACTH-IR fibers are concentrated in the periventricular
zone, the preoptic portion of the anterior hypothalamic area (HAA), the PVH and the basal part of the HLA. They are fewer in number in the supraoptic nucleus and few, if any, are present in the suprachiasmatic nucleus. ACTH-IR fibers form a dense plexus in the septofimbrial nucleus, in the area medial, ventral and dorsal to the anterior commissure and in the bed nucleus of the stria terminalis (BNST). The fibers are always detected in the stria terminalis from A8 plane to this level. We observed ventrolaterally oriented fibers in the HLA. At the level of A16, ACTH-IR fibers ascend to the lateral edge of the lateral septal nucleus, passing through the BNST. They are also abundant in the nucleus accumbens. Lesion Studies
Figure 4 shows the diagrams of lesion studies in the hypothalamus of the cats utilized for various physiological experiments. In two cases (V94 and B95), a complete arcuate lesion
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KITAHAMA E T A L .
abolished aH the ACTH-IR cell bodies and consequently no ACTH-IR fibers were detected in any part of the brain. These animals survived for 28 and 16 days after surgery, In cases I94 and 094, which survived for 14 and 18 days after surgery, ARH was incompletely destroyed and we observed a marked decrease in the number of the fibers mostly in the caudal segments, and no fibers are detected in the area lateral to the lesion site at the level of A9. The remaining fibers, however, were diffusely distributed in the forebrain. In case 093, in which the ARH was almost entirely destroyed except for its anterior portion, there was a severe decrease in the fibers after a survival of 24 days. The loss of the large cell component of the ARH was obtained in case T95 which survived for 14 days. A group of ACTH-IR cells were preserved only in the anterior portion of the nucleus. In all the brain, almost all the fibers disappeared. However, we detected occasional fibers in the periventricular region of the hypothalamus. DISCUSSION Our present immunohistochemical method enabled us to demonstrate a widespread distribution of ACTH-IR neuronal structures in the cat forebrain. The results obtained here in the cat are almost identical in terms of distribution of staining for ACTH immunoreactivity within cells and fibers of the rat brain with several exceptions mentioned in the Results section. It is likely that the source of the cerebral ACTH-IR fibers, from the present results, is the ARH. Incomplete lesions of the ARH induce a decrease in ACTH-IR fibers according to the dimensions and site of lesion. This result confirms the almost total disappearance of ACTH-IR fibers in the rat after systemic neonatal injections of monosodium glutamate [17, 21, 24]. This treatment failed to abolish completely ACTH-IR cells in the MBH, and preserved their fibers even after 10 daily consecutive injections of high doses in the rat [21] and in the cat (in preparation). Microinjection of a neurotoxin such as kainic acid in the ARH induces the death of the animal. Thus, only complete electrolytic coagulation of the cat mediobasal hypothalamus leads to their total disappearance. Recent results obtained with ACTH immunohistochemistry have reported the presence of ACTH-IR cell bodies within the pars commissuralis of the nucleus of the tractus solitarius of the rat [11,31], and we also confirmed them in the rat by our present technique using the antiserum ACTH 1-39 (in preparation). In the cat, however, we could not detect them in the medulla oblongata with the same antiserum even after colchicine pretreatment in situ and/or intraventricular administration. The absence of a medullary group has also been reported in the lizard [12]. Since we observed also the total disappearance of ACTH-IR fibers in the brain stem of the pontile cat three weeks after total removal of the tele-diencephalon (in preparation), it is most likely that no lower brain stem perikarya exist in the cat. As far as the organization of ACTH-IR fibers is concerned, the present results may lead us to postulate the possible trajectory of ACTH-IR fiber pathways.
Arcuate cells project diffusely to the various hypothalamic nuclei, such as DMH, VMH, perifornical region, PVH, HAA and periventricular stratum making up dense fiber plexuses. In the rat, DMH and VMH are reported to receive the fibers from the ARH [13,18], and fibers in the PVH come from the ARH according to retrograde transport [30] and electrophysiological [8} studies. The fiber plexuses in the preoptic portion of the HAA, which originate mainly in the middle and caudal portion of the ARH revealed by our HRP study (in preparation), might diffusely course through the ventral part of the hypothalamus. They may continue to ascend in the area rostral to the anterior commissure and enter into the lateral septum and/or BNST and stria terminalis. In the rat, they project from several arcuate cells to the BNST [35]. They continue to extend in a dorsocaudal direction up to the level of A6. The fibers in the stria terminalis located laterally to the optic tract require further investigation in order to determine their pathway. Projection into the amygdala is more complicated. From HRP study, the cells of the middle portion of the ARH are labeled by this tracer when injected into the central amygdala in the cat [29]. Our preliminary data revealed that the cells doubly labeled with HRP and ACTH can be observed in the bilateral ARH. The fibers may course diffusely into the HLA and extend to the area dorsal to the optic tract (AI0-A12) to invade the amygdala. Now it is in progress to determine the origin of these projections by employing the retrograde cell marker HRP coupled with the present immunohistochemical method. Finally, as arcuate fibers appear to bifurcate with one branch leaving the nucleus and the other terminating within it as described by Van den Pol [33], ACTH-IR neurons may reciprocally exchange information between the ARH and the terminal fields. Recent immunohistochemical studies have demonstrated the remarkable concordance of localization of corticotropin releasing factor-IR perikarya and ACTH-IR fibers within discrete hypothalamic, limbic and brain stem autonomic regions in the rat [25], suggesting the reciprocal influences between the two systems. In addition, several ACTH-IR neurons in the ARH project directly to the locus coeruleus [14] rich in noradrenergic cells, lontophoretically applied ACTH in the rat locus coeruleus were excitatory on the majority of the neurons [22}, from which evidence we can speculate that ACTH-IR neurons may also have an influence upon the other terminal fields. These types of neurons might constitute the neuronal component of the ultra-short feedback in the hypothalamus [20] controlling the outputs of these regions. This problem requires further studies in order to be elucidated.
ACKNOWLEDGEMENTS The investigation in this report was supported by INSERM (U52), CNRS (UAII95), DRET (Grant 84-160) and Biologie Humaine. The authors thank N. Gay for her skillful technical assistance and Dr. J. Carew for correction of the EngLish.
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