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Effect of hypophysectomy on α-melanotropin in discrete regions of the rat brain
Neuroscience Letters, 14 (1979) 271--274
271
© Elsevier/North-HollandScientific Publishers Ltd.
EFFECT OF HYPOPHYSECTOMY ON c~-MELANOTROPIN IN DISCRETE REGIONS OF THE RAT BRAIN
THOMAS L. O'DONOHUE, GUNNAR E. HOLMQUISTand DAVID M. JACOBOWITZ Histopharmacology Section, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20205 (U.S.A.)
(Received April 19th, 1979) (Accepted May 31st, 1979)
SUMMARY
The effect of hypophysectomy on a-melanotropin (aMSH) concentrations in discrete brain regions was investigated. Hypophysectomy resulted in a 38--69% decrease in aMSH concentration in aMSH terminal regions 4 weeks after surgery. In contrast, the aMSH concentration in the arcuate nucleus, site of aMSH containing perikary, was unaffected by hypophysectomy. These results indicate that the brain aMSH system is distinct from, but related to that of the pituitary. The sole source of a-melanotropin or a-melanocyte stimulating hormone (a-MSH) has been thought to be the intermediate lobe of the pituitary gland. Recently, however, an a-MSH-containing neuronal system has been described and consists of neuronal perikarya in the arcuate nucleus and processes in a number of terminal areas throughout the brain [2,4,7]. The a-MSH in neuronal processes of the brain is markedly depleted by lesion of the arcuate [7], while the number of a-MSH containing nerves seem unaffected by hypophysectomy [2,4,7]. a-MSH concentrations in the brain [8], pineal [9], pituitary gland [12], and blood [13] have all been shown to have diurnal rhythmic fluctuations. To further investigate a possible relationship between the a-MSH systems of the brain, which originates in the arcuate nucleus [7], and the pituitary gland, rats were hypophysecotmized and a-MSH concentrations were determined in major a-MSH perikarya or terminal regions. Hypophysectomized or sham hypophysectomized adult male rats, weighing 275--325 g, were obtained from a commercial source (Zivic-Miller, Allison Park, PA). Hypophysectomy was performed by a parapharyngeal approach followed by an application of formaldehyde to the region of the sella turcica to destroy additional tissue spared by the surgery [ 1]. Animals were housed 6 per cage and maintained on a 12 h light/12 h dark cycle with lights on
272
from 0600 to 180~ h. Food and tap water containing 1.0% sodium chloride were available ad lib. Rats were sacrificed by decapitation between 0900 and 1200 h at 2, 3 or 4 weeks after surgery. Brains were rapidly removed, mounted on specimen plates and frozen on dry ice. Serial 300 ~m frontal sections were cut in a cryostat at -7°C. Specific brain regions were microdissected from these sections as described previously [ 7,8,11 ]. The region of the amygdala which was microdissected contains the greatest number of a-MSH immunoreactive fibers [4,7] and consists primarily of the basal and medial amygdaloid nuclei. Tissue punches were delivered into 100 pl of 2 N acetic acid in 0.4 ml plastic microtubes. Samples were then boiled for 15 min, and homogenized by sonication. A 10--20 pl aliquot was removed for protein determination by a micromodification of the method of Lowry et al. [5]. These samples were then centrifuged at 8000 X g and duplicate 35 ~1 aliquots of the supernatant fluid were removed and lyophylized in preparation for radioimmunoassay. a-MSH in brain was determined as described previously. The antibody to a-MSH was generously provided by Drs M.C. Tonon and H. Vaudry, Laboratoire d'Endocrinologie, Mont-Saint-Aignan, France. The antiserum 81-0103 to a-MSH was raised in rabbits against synthetic a-MSH (Ciba-Geigy) conjugated to bovine serum albumin. Vaudry et al. [14] have demonstrated the high specificity of this antibody by showing little or no cross-reactivity with human or bovine/3-MSH, ovine/~-lipotropin, natural porcine ACTH, or the synthetic ACTH fragments ACTH 1--16, ACTH 1--24, ACTH 1--19, ACTH 1--10, ACTH 17--39, ACTH 11--19 in the.a-MSH radioimmunoassay. The specificity of this antibody for brain tissue has been further characterized by high pressure liquid chromatographic techniques [7]. Synthetic a-MSH (Beckman) was labelled with 12sI using the chloroamine-T method of Greenwood et al. [3] and was purified by QAE Sephadex column chromatography. All steps of the assay were performed at 4°C. On the first day, lyophylized samples, standards (Beckman) and blanks were resuspended in a volume of 400 ul of 1% egg albumin (Miles Laboratories) in pH 7.4 phosphate buffered saline (PBS) in 12 × 75 mm borosilicate glass test tubes. Fifty microlitres of diluted a-MSH antibody was added to achieve a dilution of 1/44,000. On day 2, approximately 3000 cpm. (4--8 pg) of ~SI-labelled a-MSH in 50 pl of PBS was added to each tube. On day 4, 200 pl of 1% normal rabbit serum in PBS and 200 ~1 of PBS containing 50 pl of sheep antiserum against rabbit 7-globulin were added to each tube simultaneously. On day 5, samples were centrifuged at 6000 × g for 30 min, supernatants aspirated and the radioactivity in the pellet counted. a-MSH concentrations were compared by analyses of variance and Duncan's New Multiple Range Tests using a computer assisted statistical packet [6]. Statistical significance was assigned to the P < 0.05 level. Rats that were sham hypophysectomized for 2, 3, or 4 weeks did n o t have a-MSH concentrations that were significantly different from each other and therefore were grouped together as sham operated animals. Hypophysectomy, however, had differential effects on a-MSH concentrations depending on
273 TABLE
I
~-MSH CONCENTRATIONS (PG/uG PROTEIN) (±) S.E.M. IN DISCRETE REGIONS OF SHAM OPERATED AND HYPOPHYSECTOMIZED RATS Area
Sham operated
H y p o p h y s e c t o m i z e d (% change f r o m S h a m o p e r a t e d ) 2 weeks
A. ~-MSH terminal regions N. I n t e r s t i t i a l i s 9.86 ± 3.97 stria terminalis Medial Preoptic 10.00 ± 2.40 N. Anterior Hypo9.00 + 1.59 t h a l a m i c N. P a m v e n t r i c u l a r N. 11.56 ± 3.34 P e r l v e n t r i c u l a r N. 13.30 ± 1.69 (thalamus) Median Eminence 11.67 + 2.74 D o r s o m e d i a l N. 8.56 + 2.56 Amygdala 5.11 ± 0.32 B. a-MSH perikarya region A r e u a t e N. 13.38 ± 4.21
BRAIN
3 weeks
4 weeks
7.80 ± 1.62 (--21)
4. 0 + 2 . 6 7 ( - - 5 9 )
3.33 ± 0.86*
(--66)
6.67 + 1.64 (--33)
7.67 + 2.78(--23)
4.90 + 0.96*
(--51)
6.33 ± 1.14 (--30)
7.25 ± 1.86(--19)
4.50 ± 0.90*
(--50)
6.67 + 1.28"(--42) 6.87 + 0.73*(--48)
7.75 ± 4.38(--32) 10.25 ± 3.55(--21)
3 . 5 6 +- 1 . 0 0 " * ( - - 6 9 ) 4 . 8 9 -+ 0 . 8 7 * * ( - - 6 3 )
1 1 . 2 5 ± 2.51 (-- 4) 6 . 8 0 + 1.53 (--20) 5 . 0 0 ± 1.03 (-- 2)
10.50 + 2.50(--10) 8.00 ± 2.02(-- 7) 4.5 + 2.40(--11)
6.63 ± 1.85 4.90 + 1.75 3.13 + 1.10
1 2 . 1 3 + 4 . 8 6 ( - - 9)
1 3 . 0 0 ± 4 . 9 5 ( - - 3)
13.36 ± 2.75
(--43) (-42) (--38)
(
0)
C. N u m b e r o f samples Sham operated: 7--10
Hypophysectomized: 2 w e e k , 5 - - 9 3 week, 3 - - 4 4 week, 8 - - 1 1 * --P < 0.05 ** --P < 0.01.
whether an ~-MSH terminal or perikarya~ontaining region was analyzed. As shown in Table 1, 4 weeks after hypophysectomy, a-MSH terminal regions had ~-MSH concentrations which were 38% to 69% lower than a-MSH concentrations of sham operated rats. ~-MSH concentrations were also significantly reduced in the paraventricular (-42%) and the thalamic periventricular (-48%) nuclei two weeks after surgery. The concentrations three weeks after hypophysectomy were not significantly lower than sham operated animals, a-MSH concentrations in the arcuate nucleus, the site of a-MSH containing perikarya, were not significantly altered by hypophysectomy at any time period investigated. The fact that ~-MSH is still present in the brain after hypophysectomy is consistent with recent evidence that demonstrates intact a-MSH containing neurons in the brains of hypophysectomized rats. Furthermore, it has recently been demonstrated that the a-MSH neuronal system of the rat brain arises from the arcuate nucleus and not from the pituitary gland. However, a-MSH concentrations have been reported to decrease significantly in the hypothalamus 1 or 2 months after hypophysectomy [14] and in the thalamus, cerebellum and brain stem three weeks after surgery [10] which is in agreement with the findings of the present study. It is particularly interesting that hypophysectomy results in a decrease in ~-MSH concentrations in terminal regions, but not in the arcuate nucleus
274 f r o m which t h e fibers originate [ 7 , 2 ] . This.imbalance in n e u r o n a l localizat i o n c o u l d possibly result f r o m an increased t u r n o v e r o f a - M S H in t e r m i n a l plexuses c o u p l e d with i n s u f f i c i e n t synthesis and s u p p l y o f the p e p t i d e f r o m t h e p e r i k a r y a l region. T h e cause o f the l o n g - t e r m h y p o p h y s e c t o m y - i n d u c e d decrease in a - M S H c o n c e n t r a t i o n s in t e r m i n a l regions is a m a t t e r o f c o n j e c t u r e . Multiple e n d o crine deficits result f r o m r e m o v a l o f t h e p i t u i t a r y t r o p h i c h o r m o n e s (e.g., A C T H , MSH, T S H , LH, F S H , GH, prolactin). It is possible t h a t e l i m i n a t i o n o f a n y n u m b e r o f these h o r m o n e s c o u l d result in a deficit o f a f e e d b a c k inhib i t o r y c o m p o n e n t with a s u b s e q u e n t increase in a - M S H t u r n o v e r . Alternatively, it is possible t h a t a - M S H s y n t h e s i z e d in t h e p i t u i t a r y m a y be t h e source o f t h e a - M S H in nerves o f the brain. REFERENCES 1 Denckla, W.D. and Marcum, E., Minimal O~ consumption as an index of throid status: standardization of method, Endocrinology, 93 (1973) 61--73. 2 Dube, D., Lissitzky, J.C., Leclerc, R. and Pelletier, G., Localization of a-melanocyte stimulation hormone in rat brain and pituitary, Endocrinology, 102 (1978) 1283-1291. 3 Greenwood, F.C., Hunter, W.M. and Glover, J.S., The preparation of l~lI-labelled human growth hormone of high specific radioactivity, Biochem. J. 89 (1963) 114-123. 4 Jacobowitz, D.M. and O'Donohue, T.L., a-Melanocyte stimulating hormone: Immunocytochemical identification and mapping in neurons of the rat brain, Proc. Natl. Acad. Sci. USA, 75 (1978) 6300--6304. 5 Lowery, O., Rosebrough, M., Farr, A. and Randall, R., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265--275. 6 Nie, N.H., HulI,C.H., Jenkins, J.G., Steinbrenner, K. and Bent, D.H., Statistical package for the social sciences, NcGraw Hill, New York, 1975. 7 0 ' D o n o h u e , T.L., Miller, R.L. and Jacobowitz, D.M., Identification, characterization and sterotaxic mapping of intraneuronal ~-melanocyte stimulating hormone-like immunoreactive peptides in discrete regions of the rat brain, Brain Res., in press. 8 0 ' D o n o h u e , T.L., Miller, R.L., Pendleton, R.C. and Jacobowitz, D.M., A diurnal rhythm of immunoreactive a-melanocyte stimulating hormone in discrete regions of the rat brain, Neuroendocrinology, in press. 9 0 ' D o n o h u e , T.L., Miller, R.L., Pendleton, R.C. and Jacobowitz, D.M., Demonstration of an endogenous circadian rhythm of ~-melanocyte stimulating hormone in the rat pineal gland, Brain Res., submitted. 10 Oliver, C. and Porter, J.C., Distribution and characterization of ~-melanocyte-stimulating hormone in the rat brain, Endocrinology, 102 (1978) 697--705. 11 Palkovits, M., Isolated removal of hypothalamic nuclei for neuroendocrinological and neurochemical studies. In W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, pp. 72--80. 12 Tilders, F.J.H. and Smelik, P.G., A diurnal rhythm in melanocyte-stimulating hormone content of the rat pituitary gland and its independence from the pineal gland, Neuroendocrinology, 17 (1975) 296--308. 13 Usategui, R., Oliver, C., Vaudry, H., Lombardi, G., Rozenberg, I. and Mourre, A.M., Immunoreactive a-MSH and ACTH levels in rat plasma and pituitary. Endocrinology, 98 (1976) 189--196. 14 Vaudry, H., Tonon, M.C., Delarue, R., Vaillant, R. and Kraicer, J., Biological and radioimmunological evidence for melanocyte stimulating hormones (MSH) of extrapituitary origin in the rat brain, Neuroendocrinology, 27 (1978) 9--24.
271
© Elsevier/North-HollandScientific Publishers Ltd.
EFFECT OF HYPOPHYSECTOMY ON c~-MELANOTROPIN IN DISCRETE REGIONS OF THE RAT BRAIN
THOMAS L. O'DONOHUE, GUNNAR E. HOLMQUISTand DAVID M. JACOBOWITZ Histopharmacology Section, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20205 (U.S.A.)
(Received April 19th, 1979) (Accepted May 31st, 1979)
SUMMARY
The effect of hypophysectomy on a-melanotropin (aMSH) concentrations in discrete brain regions was investigated. Hypophysectomy resulted in a 38--69% decrease in aMSH concentration in aMSH terminal regions 4 weeks after surgery. In contrast, the aMSH concentration in the arcuate nucleus, site of aMSH containing perikary, was unaffected by hypophysectomy. These results indicate that the brain aMSH system is distinct from, but related to that of the pituitary. The sole source of a-melanotropin or a-melanocyte stimulating hormone (a-MSH) has been thought to be the intermediate lobe of the pituitary gland. Recently, however, an a-MSH-containing neuronal system has been described and consists of neuronal perikarya in the arcuate nucleus and processes in a number of terminal areas throughout the brain [2,4,7]. The a-MSH in neuronal processes of the brain is markedly depleted by lesion of the arcuate [7], while the number of a-MSH containing nerves seem unaffected by hypophysectomy [2,4,7]. a-MSH concentrations in the brain [8], pineal [9], pituitary gland [12], and blood [13] have all been shown to have diurnal rhythmic fluctuations. To further investigate a possible relationship between the a-MSH systems of the brain, which originates in the arcuate nucleus [7], and the pituitary gland, rats were hypophysecotmized and a-MSH concentrations were determined in major a-MSH perikarya or terminal regions. Hypophysectomized or sham hypophysectomized adult male rats, weighing 275--325 g, were obtained from a commercial source (Zivic-Miller, Allison Park, PA). Hypophysectomy was performed by a parapharyngeal approach followed by an application of formaldehyde to the region of the sella turcica to destroy additional tissue spared by the surgery [ 1]. Animals were housed 6 per cage and maintained on a 12 h light/12 h dark cycle with lights on
272
from 0600 to 180~ h. Food and tap water containing 1.0% sodium chloride were available ad lib. Rats were sacrificed by decapitation between 0900 and 1200 h at 2, 3 or 4 weeks after surgery. Brains were rapidly removed, mounted on specimen plates and frozen on dry ice. Serial 300 ~m frontal sections were cut in a cryostat at -7°C. Specific brain regions were microdissected from these sections as described previously [ 7,8,11 ]. The region of the amygdala which was microdissected contains the greatest number of a-MSH immunoreactive fibers [4,7] and consists primarily of the basal and medial amygdaloid nuclei. Tissue punches were delivered into 100 pl of 2 N acetic acid in 0.4 ml plastic microtubes. Samples were then boiled for 15 min, and homogenized by sonication. A 10--20 pl aliquot was removed for protein determination by a micromodification of the method of Lowry et al. [5]. These samples were then centrifuged at 8000 X g and duplicate 35 ~1 aliquots of the supernatant fluid were removed and lyophylized in preparation for radioimmunoassay. a-MSH in brain was determined as described previously. The antibody to a-MSH was generously provided by Drs M.C. Tonon and H. Vaudry, Laboratoire d'Endocrinologie, Mont-Saint-Aignan, France. The antiserum 81-0103 to a-MSH was raised in rabbits against synthetic a-MSH (Ciba-Geigy) conjugated to bovine serum albumin. Vaudry et al. [14] have demonstrated the high specificity of this antibody by showing little or no cross-reactivity with human or bovine/3-MSH, ovine/~-lipotropin, natural porcine ACTH, or the synthetic ACTH fragments ACTH 1--16, ACTH 1--24, ACTH 1--19, ACTH 1--10, ACTH 17--39, ACTH 11--19 in the.a-MSH radioimmunoassay. The specificity of this antibody for brain tissue has been further characterized by high pressure liquid chromatographic techniques [7]. Synthetic a-MSH (Beckman) was labelled with 12sI using the chloroamine-T method of Greenwood et al. [3] and was purified by QAE Sephadex column chromatography. All steps of the assay were performed at 4°C. On the first day, lyophylized samples, standards (Beckman) and blanks were resuspended in a volume of 400 ul of 1% egg albumin (Miles Laboratories) in pH 7.4 phosphate buffered saline (PBS) in 12 × 75 mm borosilicate glass test tubes. Fifty microlitres of diluted a-MSH antibody was added to achieve a dilution of 1/44,000. On day 2, approximately 3000 cpm. (4--8 pg) of ~SI-labelled a-MSH in 50 pl of PBS was added to each tube. On day 4, 200 pl of 1% normal rabbit serum in PBS and 200 ~1 of PBS containing 50 pl of sheep antiserum against rabbit 7-globulin were added to each tube simultaneously. On day 5, samples were centrifuged at 6000 × g for 30 min, supernatants aspirated and the radioactivity in the pellet counted. a-MSH concentrations were compared by analyses of variance and Duncan's New Multiple Range Tests using a computer assisted statistical packet [6]. Statistical significance was assigned to the P < 0.05 level. Rats that were sham hypophysectomized for 2, 3, or 4 weeks did n o t have a-MSH concentrations that were significantly different from each other and therefore were grouped together as sham operated animals. Hypophysectomy, however, had differential effects on a-MSH concentrations depending on
273 TABLE
I
~-MSH CONCENTRATIONS (PG/uG PROTEIN) (±) S.E.M. IN DISCRETE REGIONS OF SHAM OPERATED AND HYPOPHYSECTOMIZED RATS Area
Sham operated
H y p o p h y s e c t o m i z e d (% change f r o m S h a m o p e r a t e d ) 2 weeks
A. ~-MSH terminal regions N. I n t e r s t i t i a l i s 9.86 ± 3.97 stria terminalis Medial Preoptic 10.00 ± 2.40 N. Anterior Hypo9.00 + 1.59 t h a l a m i c N. P a m v e n t r i c u l a r N. 11.56 ± 3.34 P e r l v e n t r i c u l a r N. 13.30 ± 1.69 (thalamus) Median Eminence 11.67 + 2.74 D o r s o m e d i a l N. 8.56 + 2.56 Amygdala 5.11 ± 0.32 B. a-MSH perikarya region A r e u a t e N. 13.38 ± 4.21
BRAIN
3 weeks
4 weeks
7.80 ± 1.62 (--21)
4. 0 + 2 . 6 7 ( - - 5 9 )
3.33 ± 0.86*
(--66)
6.67 + 1.64 (--33)
7.67 + 2.78(--23)
4.90 + 0.96*
(--51)
6.33 ± 1.14 (--30)
7.25 ± 1.86(--19)
4.50 ± 0.90*
(--50)
6.67 + 1.28"(--42) 6.87 + 0.73*(--48)
7.75 ± 4.38(--32) 10.25 ± 3.55(--21)
3 . 5 6 +- 1 . 0 0 " * ( - - 6 9 ) 4 . 8 9 -+ 0 . 8 7 * * ( - - 6 3 )
1 1 . 2 5 ± 2.51 (-- 4) 6 . 8 0 + 1.53 (--20) 5 . 0 0 ± 1.03 (-- 2)
10.50 + 2.50(--10) 8.00 ± 2.02(-- 7) 4.5 + 2.40(--11)
6.63 ± 1.85 4.90 + 1.75 3.13 + 1.10
1 2 . 1 3 + 4 . 8 6 ( - - 9)
1 3 . 0 0 ± 4 . 9 5 ( - - 3)
13.36 ± 2.75
(--43) (-42) (--38)
(
0)
C. N u m b e r o f samples Sham operated: 7--10
Hypophysectomized: 2 w e e k , 5 - - 9 3 week, 3 - - 4 4 week, 8 - - 1 1 * --P < 0.05 ** --P < 0.01.
whether an ~-MSH terminal or perikarya~ontaining region was analyzed. As shown in Table 1, 4 weeks after hypophysectomy, a-MSH terminal regions had ~-MSH concentrations which were 38% to 69% lower than a-MSH concentrations of sham operated rats. ~-MSH concentrations were also significantly reduced in the paraventricular (-42%) and the thalamic periventricular (-48%) nuclei two weeks after surgery. The concentrations three weeks after hypophysectomy were not significantly lower than sham operated animals, a-MSH concentrations in the arcuate nucleus, the site of a-MSH containing perikarya, were not significantly altered by hypophysectomy at any time period investigated. The fact that ~-MSH is still present in the brain after hypophysectomy is consistent with recent evidence that demonstrates intact a-MSH containing neurons in the brains of hypophysectomized rats. Furthermore, it has recently been demonstrated that the a-MSH neuronal system of the rat brain arises from the arcuate nucleus and not from the pituitary gland. However, a-MSH concentrations have been reported to decrease significantly in the hypothalamus 1 or 2 months after hypophysectomy [14] and in the thalamus, cerebellum and brain stem three weeks after surgery [10] which is in agreement with the findings of the present study. It is particularly interesting that hypophysectomy results in a decrease in ~-MSH concentrations in terminal regions, but not in the arcuate nucleus
274 f r o m which t h e fibers originate [ 7 , 2 ] . This.imbalance in n e u r o n a l localizat i o n c o u l d possibly result f r o m an increased t u r n o v e r o f a - M S H in t e r m i n a l plexuses c o u p l e d with i n s u f f i c i e n t synthesis and s u p p l y o f the p e p t i d e f r o m t h e p e r i k a r y a l region. T h e cause o f the l o n g - t e r m h y p o p h y s e c t o m y - i n d u c e d decrease in a - M S H c o n c e n t r a t i o n s in t e r m i n a l regions is a m a t t e r o f c o n j e c t u r e . Multiple e n d o crine deficits result f r o m r e m o v a l o f t h e p i t u i t a r y t r o p h i c h o r m o n e s (e.g., A C T H , MSH, T S H , LH, F S H , GH, prolactin). It is possible t h a t e l i m i n a t i o n o f a n y n u m b e r o f these h o r m o n e s c o u l d result in a deficit o f a f e e d b a c k inhib i t o r y c o m p o n e n t with a s u b s e q u e n t increase in a - M S H t u r n o v e r . Alternatively, it is possible t h a t a - M S H s y n t h e s i z e d in t h e p i t u i t a r y m a y be t h e source o f t h e a - M S H in nerves o f the brain. REFERENCES 1 Denckla, W.D. and Marcum, E., Minimal O~ consumption as an index of throid status: standardization of method, Endocrinology, 93 (1973) 61--73. 2 Dube, D., Lissitzky, J.C., Leclerc, R. and Pelletier, G., Localization of a-melanocyte stimulation hormone in rat brain and pituitary, Endocrinology, 102 (1978) 1283-1291. 3 Greenwood, F.C., Hunter, W.M. and Glover, J.S., The preparation of l~lI-labelled human growth hormone of high specific radioactivity, Biochem. J. 89 (1963) 114-123. 4 Jacobowitz, D.M. and O'Donohue, T.L., a-Melanocyte stimulating hormone: Immunocytochemical identification and mapping in neurons of the rat brain, Proc. Natl. Acad. Sci. USA, 75 (1978) 6300--6304. 5 Lowery, O., Rosebrough, M., Farr, A. and Randall, R., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265--275. 6 Nie, N.H., HulI,C.H., Jenkins, J.G., Steinbrenner, K. and Bent, D.H., Statistical package for the social sciences, NcGraw Hill, New York, 1975. 7 0 ' D o n o h u e , T.L., Miller, R.L. and Jacobowitz, D.M., Identification, characterization and sterotaxic mapping of intraneuronal ~-melanocyte stimulating hormone-like immunoreactive peptides in discrete regions of the rat brain, Brain Res., in press. 8 0 ' D o n o h u e , T.L., Miller, R.L., Pendleton, R.C. and Jacobowitz, D.M., A diurnal rhythm of immunoreactive a-melanocyte stimulating hormone in discrete regions of the rat brain, Neuroendocrinology, in press. 9 0 ' D o n o h u e , T.L., Miller, R.L., Pendleton, R.C. and Jacobowitz, D.M., Demonstration of an endogenous circadian rhythm of ~-melanocyte stimulating hormone in the rat pineal gland, Brain Res., submitted. 10 Oliver, C. and Porter, J.C., Distribution and characterization of ~-melanocyte-stimulating hormone in the rat brain, Endocrinology, 102 (1978) 697--705. 11 Palkovits, M., Isolated removal of hypothalamic nuclei for neuroendocrinological and neurochemical studies. In W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, pp. 72--80. 12 Tilders, F.J.H. and Smelik, P.G., A diurnal rhythm in melanocyte-stimulating hormone content of the rat pituitary gland and its independence from the pineal gland, Neuroendocrinology, 17 (1975) 296--308. 13 Usategui, R., Oliver, C., Vaudry, H., Lombardi, G., Rozenberg, I. and Mourre, A.M., Immunoreactive a-MSH and ACTH levels in rat plasma and pituitary. Endocrinology, 98 (1976) 189--196. 14 Vaudry, H., Tonon, M.C., Delarue, R., Vaillant, R. and Kraicer, J., Biological and radioimmunological evidence for melanocyte stimulating hormones (MSH) of extrapituitary origin in the rat brain, Neuroendocrinology, 27 (1978) 9--24.