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Binding of [3H]quinuclidinyl Benzilate to regions of rat pituitary and hypothalamus
Brabl_Research Bulletin, Vol. 6, pp. 209-211, 1981. Printed in the U.S.A.
Binding of [3H]Quinuclidinyl Benzilate to Regions of Rat Pituitary and Hypothalamus I DONALD
B. H O O V E R ,
J O H N C. H A N C O C K
A N D N A N C Y S. T A L L E Y
Department of Pharmacology, Quillen-Dishner College of Medicine East Tennessee State University, Johnson City, TN 37614 R e c e i v e d 1 O c t o b e r 1980 HOOVER, D. B., J. C. HANCOCK AND N. S. TALLEY. Binding of [aH]quinuclidiny/benzi/ate to regions of rat pitttitary and hypothalamus. BRAIN RES. BULL. 6(3) 209-211, 1981.--Muscarinic ligand binding sites in fragments of rat hypo-
thalamus and pituitary were studied using [3H]quinuclidinyl benzilate (QNB). In the hypothalamus, the highest amount of specific QNB binding was to n. paraventricularis and n. dorsomedialis. Specific QNB binding in other hypothalamic regions varied within a relatively narrow range. Fragments of whole pituitary also bound QNB but to a much smaller degree than brain. Pituitary binding of QNB was blocked by atropine but not by hexamethonium or d-tubocurarine. Within the pituitary, specific QNB binding to posterior pituitary was three times greater than to anterior pituitary. These findings are consistent with the operation of cholinergic mechanisms in hypothalamic and pituitary function. Muscarinic receptors
Quinuclidinyl benzilate
Pituitary
A L T H O U G H cholinergic systems have been implicated in the hypothalamic regulation of various pituitary hormones [9,15], very little is known about the cholinergic pathways involved or their pharmacological characteristics. Previous studies have demonstrated the localization of acetylcholine (ACh), choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) in several hypothalamic nuclei and the pituitary [1, 3, 5-7]. The present study was undertaken to further describe the cholinergic systems of the hypothalamus and pituitary by establishing the presence of muscarinic binding sites. The selective muscarinic receptor antagonist, quinuclidinyl benzilate (QNB) was used to identify these sites [16]. METHOD
Male Sprague-Dawley rats (310 A.A.0g) were decapitated and their brains and pituitaries rapidly removed. Brains were frozen on specimen plates and processed for microdissection of hypothalamic regions [10,11]. The pituitaries were sectioned into small fragments which were placed in 1.5 ml polypropylene tubes and frozen. Freezing has previously been shown not to affect QNB binding [14]. Discrete regions of the hypothalamus (300-1000 /~m in diameter) were removed from 300 p.m transverse hypothalamic sections, allowed to thaw and placed in tubes containing 50/zl of 0.05 M sodium-potassium phosphate buffer (pH 7.4). The same amount of buffer was added to thawed pituitary fragments. Muscarinic binding sites were studied using a saturating con-
Hypothalamus
Median eminence
centration of [aH]QNB (Amersham Corp.; 43 Ci/mmole). Total [aH] QNB binding was determined by adding 200/~1 of buffer to hypothalamic or pituitary pellets. The buffer contained [3H]QNB in an appropriate amount to bring the final concentration of QNB to 1 nM. Non-specific binding was determined by adding 200/~1 of buffer containing [aH]QNB and atropine in appropriate amounts to bring the final concentrations to 1 nM QNB and 1 p.M atropine. Tubes were incubated at 25°C for 1 hr, the reaction was stopped by adding 750/zl of cold buffer and the tubes centrifuged at 15,000 g for 1 min. The supernatant was discarded, and the tissue washed 3 times with 1 ml aliquots of cold buffer. Fragments of tissue remained intact throughout these steps. Tissue was then homogenized in 150 /zl of distilled water using a micro-ultrasonic cell dtsrupter (Kontes). A 100 p.I aliquot of this homogenate was dissolved in Aquasoi-2, and [aH]QNB measured by liquid scintillation spectrometry. Another aliquot of the homogenate was taken for protein analysis [8]. Specific QNB binding was calculated as the difference between total and non-specific binding. RESULTS
Specific [aH]QNB binding to hypothalamic regions and pituitary is shown in Table 1. All regions of the hypothalamus contained specific [aH]QNB binding sites. The highest amounts were in the n. paraventricularis and n. dorsomedialis. Binding of QNB at the medium eminence was lower
1Portions of this work were.presented at the 62nd Annual Meeting of the Endocrine Society, 1980.
C o p y r i g h t © 1981 A N K H O
I n t e r n a t i o n a l Inc.--0361-9230/81/030209-03500.80/0
210
HOOVER, H A N C O C K AND T A L L E Y TABLE 1 SPECIFIC BINDINGOF [aH]QNB TO REGIONSOF THE RAT HYPOTHALAMUSAND THE PITUITARY Area*
N. preopticus medialist N. preopticus lateralis't N. hypothalamicus anteriort N. supraopticus:~ N. paraventricularis:l: N. ~'entromedialis:~ N. arcuatus§ Median eminence~ N. dorsomedialis~: Pituitaryt
n
Specifically Bound [aH] QNB (fmole/p.g protein -+ S.E.M.)
11 9 10 7 5 5 5 5 9 8
0.328 _+ 0.015 0.316 - 0.018 0.356 ___0.018 0.297 --- 0.029 0.809 _ 0.116 0.375 ___0.018 0.300 _+ 0.024 0.192 --_ 0.014 0.758 _ 0.046 0.012 _+ 0.001
*For most brain regions, tissue from one side was incubated with 1 nM QNB plus atropine and the contralateral side was incubated with QNB alone. Such matching was not done with median eminence and pituitary. "tTissue from one rat for each sample. ~tTissue pooled from 2 rats for each sample. §Tissue pooled from 4 rats for each sample.
than for other regions of hypothalamus. A lower amount of specific QNB binding was found in the fragments of whole pituitary. This binding was blocked by atropine (1/zM) but not by hexamethonium (1 p.M) or d-tubocurarine (1/~M) suggesting that the sites are muscarinic (Table 2). The regional distribution of [3H]QNB binding sites in the pituitary was also examined by dissecting it into anterior and posterior lobes (Table 2). Specific [aH]QNB binding in the posterior pituitary was approximately 3 times higher than in the anterior pituitary.
TABLE 2 PHARMACOLOGICAL ANTAGONISMAND REGIONAL
DISTRIBUTIONOF [aH] QNB BINDINGTO RAT PITUITARY Antagonist
n
None 23 Atropine (1 /zM) 22 Hexamethonium (1 p.M) 8 d-Tubocurarine (1 p.M) 12
[aH] QNB Bound* (fmole/p.g protein _+ S.E.M.) 0.021 __. 0.001 0.010 __. 0.00It 0.017 __. 0.001 0.026 ___0.005
DISCUSSION Quinuclidinyl benzilate (QNB) was found to bind specifically to muscarinic receptors in regions of the rat hypothalamus and pituitary. With the exceptions of the n. paraventricularis and n. dorsomedialis, there was little variation in specific QNB binding among the hypothalamic sites examined. All of these areas have been shown to contain ACh, ChAT and AChE [3]. Accordingly, it is reasonable to assume that the specific QNB binding is a reflection of muscarinic receptors associated with cholinergic transmission. Alternatively specific binding could reflect non-innervated muscarinic receptors of vascular smooth muscle. The presence of ACh, ChAT and AChE argues against this possibility. It is noteworthy that the median eminence has a high ChAT activity [3] but does not have a correspondingly high specific QNB binding. Also, the high amount of specific QNB binding to the n. paraventricularis and n. dorsomedialis is not accompanied by high ChAT activity. All three regions have identical ACh levels [3]. These observations suggest that for these regions a poor correlation exists between ChAT activity, AChE activity, ACh concentration and specific ligand binding. There is some disagreement among investigators regarding the relative amounts of iigand binding to hypothalamic regions. Although we found substantially greater specific QNB binding to the n. paraventricularis and n. dorsomedi-
Lobe of Pituitary
n
Specifically Bound [all] QNB (fmole/p.g protein _.+ S.E.M.)
Anterior Posterior
6 6
0.012 _+ 0.001 0.039 _ 0.002:~
*Tissue was incubated with [aH] QNB alone or in combination with a nicotinic or muscarinic antagonist. tSignificantly different (p~<0.05) from binding observed with [all] QNB alone; analysis of variance and Dunnett's t-test. ~tSignificantly different (p~<0.05) from binding to anterior lobe; Student's t-test.
alis, this was not observed by Kobayashi et al. [4]. The primary difference between this study and that of Kobayashi et al. [4] was in the method of ligand-receptor complex trapping. We studied binding in small pellets of thermally disrupted tissue and obtained values for bound QNB by centrifugation, while Kobayashi et al. [4] used f'dtration to isolate bound QNB. Using an autoradiographic technique, Rotter et al. [12] observed that the specific binding of the muscarinic ligand, [3H]propylbenzilylcholine mustard, was highest in the n. supraopticus and median eminence. This may also reflect differences in technique.
M U S C A R I N I C R E C E P T O R S IN P I T U I T A R Y A N D H Y P O T H A L A M U S Specific Q N B binding was also found in the pituitary, but this was lower than for hypothalamus. Within the pituitary, binding was 3 times greater in the posterior lobe c o m p a r e d with the anterior. These data are in conflict with those of other laboratories [2,14] which showed greater binding in the anterior than the posterior lobe. The reason for this discrepancy is not clear, but the present findings parallel the distribution of ACh in the rat pituitary [ 1]. S o m e A C h E - c o n t a i n i n g fibers have also been found in the posterior pituitary w h e n e x a m i n e d at the light microscopic level ([1,5], Barton and H o o v e r , unpublished observation). T h e s e findings suggest that Q N B binding to posterior pituitary may be related to a cholinergic innervation. In contrast, the anterior pituitary does not appear to receive any AChE-staining fibers, and the role of muscarinic receptors at this site is less obvious. Burt and Taylor [2] suggested that ACh may reach the anterior
211
pituitary by way of the hypophyseal portal system. In this case, A C h would function as a regulatory hormone. This view is supported by the fact that A C h and m o d e r a t e C h A T activity was found in the median e m i n a n c e [3]. The present findings d e m o n s t r a t e the p r e s e n c e o f muscarinic receptor sites in the hypothalamus and pituitary. This lends further c r e d e n c e to the postulates that cholinergic m e c h a n i s m s may have a significant influence on hypothalamic function and that they may also have direct influences on the pituitary. Functional and anatomical relationships have yet to be established. ACKNOWLEDGEMENTS This study was supported by Biomedical Research Development Grant 1-SO8-RR-09171-01. We thank Teresa Garland for secretarial work.
REFERENCES I. Bridges, T. E., A. W. Fisher, J. L. Gosbee, K. Lederis and R. C. Santolaya. Acetylcholine and cholinesterases (assays and light- and electron microscopical histochemistry) in different parts of the pituitary of rat, rabbit and domestic pig. Z. Zellforsch. 136: 1-18, 1973. 2. Burr, D. R. and R. L. Taylor. Muscarinic receptor binding in sheep anterior pituitary. Neuroendocrinology 30: 344-349, 1980. 3. Hoover, D. B., E. A. Muth and D. M. Jacobowitz. A mapping of the distribution of acetylcholine, choline acetyltransferase and acetylcholinesterase in discrete areas of rat brain. Brain Res. 153: 295-306, 1978. 4. Kobayashi, R. M., M. Palkovits, R. E. Hruska, R. Rothschild and H. I. Yamamura. Regional distribution of muscarinic cholinergic receptors in rat brain. Brain Res. 154: 13-23, 1978. 5. Koelle, G. B. and C. N. Geesey. Localization ofacetylcholinesterase in the neurohypophysis and its functional implications. Proc. Soc. exp. Biol. Med. 106: 625-628, 1961. 6. LaBella, F. S. and S. Shin. Estimation of cholinesterase and choline acetyltransferase in bovine anterior pituitary, posterior pituitary, and pineal body. J. Neurochem. 15: 335-342, 1968. 7. Lederis, K. and A. Livingston. Acetylcholine and related enzymes in the neural lobe and anterior hypothalamus of the rabbit. J. Physiol., Lond. 201: 695-709, 1969. 8. Lowry, O. H., N. Y. Rosebrough, A. L. Farr and R. J. Randall. Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275, 1951. 9. Miiller, E. E., G. Nistoc6 and U. Scapagnini. Neurotransmitters and Anterior Pituitary Fttnction. New York: Academic Press, 1977, p. 220.
10. Palkovits, M. Isolated removal of hypothalamic nuclei for neuroendocrinological and neurochemical studies. In: Anatomical Neuroendocrinology, edited by W. E. Stumpfand L. D. Grant. Basel: Karger, 1975, pp. 72-80. 11. Palkovits, M. Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 59: 449-450, 1973. 12. Rotter, A., N. J. M. Birdsall, A. S. U. Burgen, P. M. Field, E. C. Hulme and G. Raisman. Muscarinic receptors in the central nervous system of the rat. I. Technique for autoradiographic localization of the binding of [all] propylbenzilylcholine mustard and its distribution in the forebrain. Brain Res. Rev. l: 141-165, 1979. 13. Saavedra, J. M., M. Palkovits, J. S. Kizer, M. Brownstein and J. A. Zivin. Distribution of biogenic amines and related enzymes in the rat pituitary gland. J. Neurochem. 25: 257-260, 1975. 14. Schaeffer, J. M. and A. J. W. Hsueh. Acetylcholine receptors in the rat anterior pituitary gland. Endocrinology 106: 1377-1381, 1980. 15. Sladek, C. D. and R. J. Joynt. Characterization of cholinergic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system. Endocrinology 104: 659-663, 1979. 16. Yamamura, H. I. and S. H. Snyder. Muscarinic cholinergic binding in rat brain. Proc. natn. Acad. Sci. U.S.A. 71: 17251729, 1974.
Binding of [3H]Quinuclidinyl Benzilate to Regions of Rat Pituitary and Hypothalamus I DONALD
B. H O O V E R ,
J O H N C. H A N C O C K
A N D N A N C Y S. T A L L E Y
Department of Pharmacology, Quillen-Dishner College of Medicine East Tennessee State University, Johnson City, TN 37614 R e c e i v e d 1 O c t o b e r 1980 HOOVER, D. B., J. C. HANCOCK AND N. S. TALLEY. Binding of [aH]quinuclidiny/benzi/ate to regions of rat pitttitary and hypothalamus. BRAIN RES. BULL. 6(3) 209-211, 1981.--Muscarinic ligand binding sites in fragments of rat hypo-
thalamus and pituitary were studied using [3H]quinuclidinyl benzilate (QNB). In the hypothalamus, the highest amount of specific QNB binding was to n. paraventricularis and n. dorsomedialis. Specific QNB binding in other hypothalamic regions varied within a relatively narrow range. Fragments of whole pituitary also bound QNB but to a much smaller degree than brain. Pituitary binding of QNB was blocked by atropine but not by hexamethonium or d-tubocurarine. Within the pituitary, specific QNB binding to posterior pituitary was three times greater than to anterior pituitary. These findings are consistent with the operation of cholinergic mechanisms in hypothalamic and pituitary function. Muscarinic receptors
Quinuclidinyl benzilate
Pituitary
A L T H O U G H cholinergic systems have been implicated in the hypothalamic regulation of various pituitary hormones [9,15], very little is known about the cholinergic pathways involved or their pharmacological characteristics. Previous studies have demonstrated the localization of acetylcholine (ACh), choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) in several hypothalamic nuclei and the pituitary [1, 3, 5-7]. The present study was undertaken to further describe the cholinergic systems of the hypothalamus and pituitary by establishing the presence of muscarinic binding sites. The selective muscarinic receptor antagonist, quinuclidinyl benzilate (QNB) was used to identify these sites [16]. METHOD
Male Sprague-Dawley rats (310 A.A.0g) were decapitated and their brains and pituitaries rapidly removed. Brains were frozen on specimen plates and processed for microdissection of hypothalamic regions [10,11]. The pituitaries were sectioned into small fragments which were placed in 1.5 ml polypropylene tubes and frozen. Freezing has previously been shown not to affect QNB binding [14]. Discrete regions of the hypothalamus (300-1000 /~m in diameter) were removed from 300 p.m transverse hypothalamic sections, allowed to thaw and placed in tubes containing 50/zl of 0.05 M sodium-potassium phosphate buffer (pH 7.4). The same amount of buffer was added to thawed pituitary fragments. Muscarinic binding sites were studied using a saturating con-
Hypothalamus
Median eminence
centration of [aH]QNB (Amersham Corp.; 43 Ci/mmole). Total [aH] QNB binding was determined by adding 200/~1 of buffer to hypothalamic or pituitary pellets. The buffer contained [3H]QNB in an appropriate amount to bring the final concentration of QNB to 1 nM. Non-specific binding was determined by adding 200/~1 of buffer containing [aH]QNB and atropine in appropriate amounts to bring the final concentrations to 1 nM QNB and 1 p.M atropine. Tubes were incubated at 25°C for 1 hr, the reaction was stopped by adding 750/zl of cold buffer and the tubes centrifuged at 15,000 g for 1 min. The supernatant was discarded, and the tissue washed 3 times with 1 ml aliquots of cold buffer. Fragments of tissue remained intact throughout these steps. Tissue was then homogenized in 150 /zl of distilled water using a micro-ultrasonic cell dtsrupter (Kontes). A 100 p.I aliquot of this homogenate was dissolved in Aquasoi-2, and [aH]QNB measured by liquid scintillation spectrometry. Another aliquot of the homogenate was taken for protein analysis [8]. Specific QNB binding was calculated as the difference between total and non-specific binding. RESULTS
Specific [aH]QNB binding to hypothalamic regions and pituitary is shown in Table 1. All regions of the hypothalamus contained specific [aH]QNB binding sites. The highest amounts were in the n. paraventricularis and n. dorsomedialis. Binding of QNB at the medium eminence was lower
1Portions of this work were.presented at the 62nd Annual Meeting of the Endocrine Society, 1980.
C o p y r i g h t © 1981 A N K H O
I n t e r n a t i o n a l Inc.--0361-9230/81/030209-03500.80/0
210
HOOVER, H A N C O C K AND T A L L E Y TABLE 1 SPECIFIC BINDINGOF [aH]QNB TO REGIONSOF THE RAT HYPOTHALAMUSAND THE PITUITARY Area*
N. preopticus medialist N. preopticus lateralis't N. hypothalamicus anteriort N. supraopticus:~ N. paraventricularis:l: N. ~'entromedialis:~ N. arcuatus§ Median eminence~ N. dorsomedialis~: Pituitaryt
n
Specifically Bound [aH] QNB (fmole/p.g protein -+ S.E.M.)
11 9 10 7 5 5 5 5 9 8
0.328 _+ 0.015 0.316 - 0.018 0.356 ___0.018 0.297 --- 0.029 0.809 _ 0.116 0.375 ___0.018 0.300 _+ 0.024 0.192 --_ 0.014 0.758 _ 0.046 0.012 _+ 0.001
*For most brain regions, tissue from one side was incubated with 1 nM QNB plus atropine and the contralateral side was incubated with QNB alone. Such matching was not done with median eminence and pituitary. "tTissue from one rat for each sample. ~tTissue pooled from 2 rats for each sample. §Tissue pooled from 4 rats for each sample.
than for other regions of hypothalamus. A lower amount of specific QNB binding was found in the fragments of whole pituitary. This binding was blocked by atropine (1/zM) but not by hexamethonium (1 p.M) or d-tubocurarine (1/~M) suggesting that the sites are muscarinic (Table 2). The regional distribution of [3H]QNB binding sites in the pituitary was also examined by dissecting it into anterior and posterior lobes (Table 2). Specific [aH]QNB binding in the posterior pituitary was approximately 3 times higher than in the anterior pituitary.
TABLE 2 PHARMACOLOGICAL ANTAGONISMAND REGIONAL
DISTRIBUTIONOF [aH] QNB BINDINGTO RAT PITUITARY Antagonist
n
None 23 Atropine (1 /zM) 22 Hexamethonium (1 p.M) 8 d-Tubocurarine (1 p.M) 12
[aH] QNB Bound* (fmole/p.g protein _+ S.E.M.) 0.021 __. 0.001 0.010 __. 0.00It 0.017 __. 0.001 0.026 ___0.005
DISCUSSION Quinuclidinyl benzilate (QNB) was found to bind specifically to muscarinic receptors in regions of the rat hypothalamus and pituitary. With the exceptions of the n. paraventricularis and n. dorsomedialis, there was little variation in specific QNB binding among the hypothalamic sites examined. All of these areas have been shown to contain ACh, ChAT and AChE [3]. Accordingly, it is reasonable to assume that the specific QNB binding is a reflection of muscarinic receptors associated with cholinergic transmission. Alternatively specific binding could reflect non-innervated muscarinic receptors of vascular smooth muscle. The presence of ACh, ChAT and AChE argues against this possibility. It is noteworthy that the median eminence has a high ChAT activity [3] but does not have a correspondingly high specific QNB binding. Also, the high amount of specific QNB binding to the n. paraventricularis and n. dorsomedialis is not accompanied by high ChAT activity. All three regions have identical ACh levels [3]. These observations suggest that for these regions a poor correlation exists between ChAT activity, AChE activity, ACh concentration and specific ligand binding. There is some disagreement among investigators regarding the relative amounts of iigand binding to hypothalamic regions. Although we found substantially greater specific QNB binding to the n. paraventricularis and n. dorsomedi-
Lobe of Pituitary
n
Specifically Bound [all] QNB (fmole/p.g protein _.+ S.E.M.)
Anterior Posterior
6 6
0.012 _+ 0.001 0.039 _ 0.002:~
*Tissue was incubated with [aH] QNB alone or in combination with a nicotinic or muscarinic antagonist. tSignificantly different (p~<0.05) from binding observed with [all] QNB alone; analysis of variance and Dunnett's t-test. ~tSignificantly different (p~<0.05) from binding to anterior lobe; Student's t-test.
alis, this was not observed by Kobayashi et al. [4]. The primary difference between this study and that of Kobayashi et al. [4] was in the method of ligand-receptor complex trapping. We studied binding in small pellets of thermally disrupted tissue and obtained values for bound QNB by centrifugation, while Kobayashi et al. [4] used f'dtration to isolate bound QNB. Using an autoradiographic technique, Rotter et al. [12] observed that the specific binding of the muscarinic ligand, [3H]propylbenzilylcholine mustard, was highest in the n. supraopticus and median eminence. This may also reflect differences in technique.
M U S C A R I N I C R E C E P T O R S IN P I T U I T A R Y A N D H Y P O T H A L A M U S Specific Q N B binding was also found in the pituitary, but this was lower than for hypothalamus. Within the pituitary, binding was 3 times greater in the posterior lobe c o m p a r e d with the anterior. These data are in conflict with those of other laboratories [2,14] which showed greater binding in the anterior than the posterior lobe. The reason for this discrepancy is not clear, but the present findings parallel the distribution of ACh in the rat pituitary [ 1]. S o m e A C h E - c o n t a i n i n g fibers have also been found in the posterior pituitary w h e n e x a m i n e d at the light microscopic level ([1,5], Barton and H o o v e r , unpublished observation). T h e s e findings suggest that Q N B binding to posterior pituitary may be related to a cholinergic innervation. In contrast, the anterior pituitary does not appear to receive any AChE-staining fibers, and the role of muscarinic receptors at this site is less obvious. Burt and Taylor [2] suggested that ACh may reach the anterior
211
pituitary by way of the hypophyseal portal system. In this case, A C h would function as a regulatory hormone. This view is supported by the fact that A C h and m o d e r a t e C h A T activity was found in the median e m i n a n c e [3]. The present findings d e m o n s t r a t e the p r e s e n c e o f muscarinic receptor sites in the hypothalamus and pituitary. This lends further c r e d e n c e to the postulates that cholinergic m e c h a n i s m s may have a significant influence on hypothalamic function and that they may also have direct influences on the pituitary. Functional and anatomical relationships have yet to be established. ACKNOWLEDGEMENTS This study was supported by Biomedical Research Development Grant 1-SO8-RR-09171-01. We thank Teresa Garland for secretarial work.
REFERENCES I. Bridges, T. E., A. W. Fisher, J. L. Gosbee, K. Lederis and R. C. Santolaya. Acetylcholine and cholinesterases (assays and light- and electron microscopical histochemistry) in different parts of the pituitary of rat, rabbit and domestic pig. Z. Zellforsch. 136: 1-18, 1973. 2. Burr, D. R. and R. L. Taylor. Muscarinic receptor binding in sheep anterior pituitary. Neuroendocrinology 30: 344-349, 1980. 3. Hoover, D. B., E. A. Muth and D. M. Jacobowitz. A mapping of the distribution of acetylcholine, choline acetyltransferase and acetylcholinesterase in discrete areas of rat brain. Brain Res. 153: 295-306, 1978. 4. Kobayashi, R. M., M. Palkovits, R. E. Hruska, R. Rothschild and H. I. Yamamura. Regional distribution of muscarinic cholinergic receptors in rat brain. Brain Res. 154: 13-23, 1978. 5. Koelle, G. B. and C. N. Geesey. Localization ofacetylcholinesterase in the neurohypophysis and its functional implications. Proc. Soc. exp. Biol. Med. 106: 625-628, 1961. 6. LaBella, F. S. and S. Shin. Estimation of cholinesterase and choline acetyltransferase in bovine anterior pituitary, posterior pituitary, and pineal body. J. Neurochem. 15: 335-342, 1968. 7. Lederis, K. and A. Livingston. Acetylcholine and related enzymes in the neural lobe and anterior hypothalamus of the rabbit. J. Physiol., Lond. 201: 695-709, 1969. 8. Lowry, O. H., N. Y. Rosebrough, A. L. Farr and R. J. Randall. Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275, 1951. 9. Miiller, E. E., G. Nistoc6 and U. Scapagnini. Neurotransmitters and Anterior Pituitary Fttnction. New York: Academic Press, 1977, p. 220.
10. Palkovits, M. Isolated removal of hypothalamic nuclei for neuroendocrinological and neurochemical studies. In: Anatomical Neuroendocrinology, edited by W. E. Stumpfand L. D. Grant. Basel: Karger, 1975, pp. 72-80. 11. Palkovits, M. Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 59: 449-450, 1973. 12. Rotter, A., N. J. M. Birdsall, A. S. U. Burgen, P. M. Field, E. C. Hulme and G. Raisman. Muscarinic receptors in the central nervous system of the rat. I. Technique for autoradiographic localization of the binding of [all] propylbenzilylcholine mustard and its distribution in the forebrain. Brain Res. Rev. l: 141-165, 1979. 13. Saavedra, J. M., M. Palkovits, J. S. Kizer, M. Brownstein and J. A. Zivin. Distribution of biogenic amines and related enzymes in the rat pituitary gland. J. Neurochem. 25: 257-260, 1975. 14. Schaeffer, J. M. and A. J. W. Hsueh. Acetylcholine receptors in the rat anterior pituitary gland. Endocrinology 106: 1377-1381, 1980. 15. Sladek, C. D. and R. J. Joynt. Characterization of cholinergic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system. Endocrinology 104: 659-663, 1979. 16. Yamamura, H. I. and S. H. Snyder. Muscarinic cholinergic binding in rat brain. Proc. natn. Acad. Sci. U.S.A. 71: 17251729, 1974.