- Email: [email protected]
Release of vasopressin from the microdissected median eminence of the rat
Brain Research, 206 (1981) 469~,73 © Elsevier/North-Holland Biomedical Press
469
Release of vasopressin from the microdissected median eminence of the rat
J.-L. BI~NY and A. J. BAERTSCHI Department of Animal Biology, University of Geneva, 121l Geneva 4 (Switzerland)
(Accepted September 1lth, 1980) Key words: vasopressin - - median eminence -- hypothalamus
For over 25 years arginine vasopressin (AVP) has been suspected to play a role in adrenocorticotropin (ACTH) regulation 13. AVP has been detected in monkey 21 and rat 16 hypophysial portal blood in high concentrations (several mU/ml plasma). It is known that AVP at similar concentrations induces the release of A C T H from adenohypophysial quarters 12 or dispersed cells 18 in vitro, and also when injected into rats in vivo 1. However, it is not clearly established if portal plasma AVP is released from axon terminals of the median eminence or of the neural lobe, or of both. Neurones of the paraventricular nucleus project onto the median eminence 17,20 and the staining by immunohistochemistry of vasopressin-containing fibres of the median eminence is enhanced following adrenalectomy and reduced after corticosteroid treatmenP, 19. Furthermore, electrical stimulation of the paraventricular nuclei elicits A C T H release 7,8. On the other hand, portal plasma AVP levels are strongly diminished following posterior lobectomy 18. Therefore, the goal of this study was to determine the amount of AVP which can be released from the median eminence at rest and during stimulation. Male Sprague-Dawley rats (250-350 g body weight) were decapitated and the brain was immediately removed from the skull. A tissue piece containing the pituitary stalk and arcuate nucleus but not the optic chiasm was dissected by four cuts with iridectomy scissors. We call this tissue the medial basal hypothalamus (MBH). The tissue was fastened on its ventral surface with a needle inserted through the stalk. Under a binocular microscope, the floor of the third ventricle was dissected with a scalpel (Fig. I). These manipulations lasted for 2 min per median eminence. Five median eminences were impaled on a platinum needle, stacked between two silicon rubber pieces, and incubated in 500/zl of a Krebs-Ringer-bicarbonate buffer containing 1 mg glucose, 0.5 mg beef serum albumin and 17 /zg bacitracin. The medium was continuously gassed with 95 ~ 02 and 5 ~ CO,, and maintained at 37 °C. After a preincubation of 20 min, the median eminences were subsequently incubated for three 20 min periods. After each period, the incubation medium was collected for radioimmunoassay of AVP 6 and renewed. The median eminences were stimulated in the first 15 min
470
CQU~. needle ~r~
3rdventricle ~
scalpel
optic chiasm
Fig. 1. Microdissection of the median eminence. The whole medial basal hypothalamus is shown. After pinning down the tissue with a needle, the floor of the third ventricle (interrupted line) is dissected with a scalpel.
of the second incubation period by applying biphasic pulses (1 msec duration) of 19-22 V at a frequency of 75 Hz 4 between the platinum electrode and an indifferent electrode in the medium. The pulse trains were switched on for 5 sec and offfor 5 sec during 15 min. Separate sets of M B H or median eminence were homogenized in 50 #10.1 N HC1 and frozen. Before the AVP assays, the extracts were neutralized by 5 #1 1 N N a O H complemented with 445/~1 incubation medium and subjected to centrifugation. The AVP content of the supernatant was determined by radioimmunoassay 6 and by bioassay from the pressure effects on the isolated rat mesentery perfused at constant flow 14.
The basal AVP release from each median eminence was 0.09 4- 0.01 mU/20 min (mean 4- S.E.M., n = 9). During electrical stimulation, AVP release increased 11-fold (Fig. 2). Following electrical stimulation, AVP release returned towards control values but was significantly larger than during the first incubation period (P < 0.005), presumably because of the AVP which had filled the interstitial space during stimulation in the preceding incubation period. Prior to microdissection, the M B H contained 12.9 4- 0.2 m U AVP (mean 4S.E.M., n = 4) and wet weight was 2.1 ± 0.3 rag. After microdissection, the median eminence contained 12.4 4- 0.5 m U AVP (n = 4) and wet weight was 0.11 4- 0.01 mg (n = 4). At the end of the first incubation period, AVP content of the median eminence was 8.8 4- 0.6 m U (n = 6). Thus one median eminence releases in 20 min about 1% of its content under basal conditions and about l 1% during electrical stimulation. In order to check if radioimmunoassayable (RIA) AVP reflects bioassayable AVP, both types of assays were performed with M B H extracts. AVP content per M B H was 16.6 4- 1.0 m U (bioassay) compared to 12.9 4- 2.0 m U (n = 4) (RIA), giving a ratio ofbioassay/RIA of about 1.3. The results show that the microdissected median eminence, which represents essentially the floor of the third ventricle, weighs about 20 times less (0.11 rag) than
471 1
o E
I o 1st
2nd
3 rd
electrical stim.
Fig. 2. Release of arginine vasopressin from median eminence in vitro (mean ± S.E.M. per median eminence, n = 4-5). Incubations (lst, 2nd, 3rd) lasted 20 min each. Electrical stimulation was 75 Hz, 5 sec on and offfor 15 min. the MBH and contains the same quantity of AVP. Similar amounts of AVP, 4.3-12 mU, have been reported by othersl°, 15 for the MBH. The manipulation of the tissue including impalement on a platinum electrode and 40 min incubation results in a loss of only 25 ~ of the AVP content. This preparation may perhaps be applied to study the release of other hypothalamic hormones. The advantage over using the MBH or whole hypothalamus resides in the fact that hormones (or neuromodulators) released from the hypothalamus which are not directly involved in adenohypophysial control do not contaminate the hormones released from the median eminence. The electrical stimulation appears to be effective in promoting the release of AVP from the median eminence. A stimulation frequency of 75 Hz has previously been found to be optimal for the release of corticotropin releasing factor from the hypothalamus in vitro 4, although such high frequencies are rarely encountered when hypothalamic cells are monitored. It is unlikely that the electrical stimulation led to significant tissue damage, since the stimulus voltages were reasonably low and biphasic. Furthermore, the poststimulation AVP levels returned almost to control levels. By considering the rate of AVP release upon electrical stimulation (1 mU/20 min) and of portal blood flow (0.0041-0.0056 ml/min for a l0 mg adenohypophysisl), the concentration reached in portal plasma can be estimated to range from 18 to 24 mU/ml when AVP release is maximally stimulated and from 1.6 to 2.2 mU/ml at rest. These concentrations, if applied to a suspension of acutely dispersed adenohypophysial cells, increase the A C T H release by 70-150 ~a.
472 Previous studies suggest that the neural lobe contributes significantly to the high plasma A V P levels of the hypophysial portal circulation TM and that vasopressin mediates the increase of plasma A C T H release in response to electrical stimulation of the neural lobe in vivo 2. O u r results a n d previously published studies thus suggest that A V P can be released from both the median eminence and from the neural lobe in sufficient a m o u n t s to elicit a m a x i m u m vasopressin-mediated A C T H release from the adenohypophysis, an action which may be amplified by the c o n c o m i t a n t secretion of a p o t e n t i a t i n g factor 9. This work was supported by Swiss National Research F o u n d a t i o n G r a n t s 3.248-0.77 and 3.581-0.79. We t h a n k Ms. M. Friedli for excellent technical assistance, Ms. N. Gulati (Geneva) for i n t r o d u c i n g us to the isolated mesentery preparation, a n d Dr. R. M a t h i s o n for reviewing the manuscript. We are indebted to Drs. G. Gillies and P. Lowry, L o n d o n , for a specific vasopressin antibody.
1 Arimura, A., Schally, A. V. and Bowers, E. Y., Corticotropin releasing activity of lysine vasopressin analogues, Endocrinology, 84 (1969) 579 583. 2 Baertschi, A. J., Vallet, P., Baumann, J. B. and Girard, J., Neural lobe of pituitary modulates corticotropin release in the rat, Endocrinology, 106 (1980) 878-882. 3 Beny, J.-L. and Baertschi, A. J., Release of corticotropin and corticotropin-releasing factors from rat posterior pituitary in vitro, Neuroendocrinology, 30 (1980) 108-112. 4 Bradbury, M. W. B., Burden, J., Hillhouse, E. W. and Jones, M. T., Stimulation electrically and by acetylcholine of the rat hypothalamus in vitro, J. Physiol. (Lond.), 239 (1974) 269-283. 5 Burlet, A., Chauteau, M. and Czernichow, P., lnfundibular localization of vasopressin, oxytocin and neurophysins in the rat; its relationships with corticotrope function, Brain Research, 168 (1979) 275-286. 6 Dogterom, J., Van Wimersma Greidanus, Tj. B. and Swaab, D. F., Evidence for the release of vasopressin and oxytocin into cerebrospinal fluid: measurements in plasma and CSF of intact and hypophysectomized rats, Neuroendocrinology, 24 (1977) 108-118. 7 Dorenhorst, A., Carlson, D. E., Self, S. M., Robinson, A. G., Zimmerman, E. A. and Gann, D. S., Control of release of ACTH and vasopressin by supraoptic and paraventricular nuclei, Neurosci. ,4bstr., 4 0978) 344. 8 Dunn, J. and Critchlow, W., Electrically stimulated ACTH rdease in pharmacologically blocked rats, Endocrinology, 93 (1973) 835-842. 9 Gillies, G. and Lowry, P., Corticotropin releasing factor may be modulated vasopressin, Nature (Lond.), 278 0979) 463464. l0 Gillies, G., Van Wimersma Greidanus, T. B. and Lowry, P. J., Characterization of rat stalk median eminence vasopressin and its involvement in adrenocorticotropin release, Endocrinology, 103 (1978) 528-534. I 1 Goldman, H., Endocrineglandbloodflowinthe unanesthetized, unrestrained rat, J. appl. Physiol., 16 (1961) 762-764. 12 Lutz-Bucher, B., Koch, B., Mialhe, C. and Briaud, B., Involvement of vasopressin in corticotropin releasing effect of hypothalamic median eminence extract, Neuroendocrinology, 30 (1980) 178-182. 13 McCann, S. M. and Brobeck, J. R., Evidence for a role of the supraoptico-hypophyseal system in regulation of adrenocorticotropin secretion, Proc. Soc. exp. Biol. (N. F.), 87 (1954) 318-324. 14 McGregor, D. D., The effect of sympathetic nerve stimulation on vasoconstrictor responses in perfused mesenteric blood vessels of the rat, J. Physiol. (Lond.), 177 (1965) 21-30. 15 Mialhe, C., Lutz-Bucher, B., Briaud, B., Schleiffer, R. and Koch, B. B., Corticotropin-releasing factor (CRF) and vasopressin in the regulation of corticotropin (ACTH) secretion. In M. T. Jones B. Gillham, M. F. Dallman and S. Chattopadhyay (Eds.), Interaction within the Brain-PituitaryAdrenocortical System, Academic Press, London, 1979, pp. 63-74. 16 Oliver, C., Mical, R. S. and Porter, C., Hypothalamic-pituitary vasculature: evidence for retrograde blood flow in the pituitary stalk, Endocrinology, 101 (!977) 598-604.
473 17 Parry, H. B. and Livett, B. G., A new hypothalamic pathway to the median eminence containing neurophysin and its hypertrophy in sheep with natural scrapie, Nature (Lond.), 242 (1973) 63-65. 18 Portanova, R. and Sayers, G., An in vitro assay for corticotropin releasing factor(s) using suspension of isolated pituitary cells, Neuroendocrinology, 12 (1973) 236-248. 19 Stillman, M. A., Recht, L. D., Rosario, S. L., Seif, S. M., Robinson, A. G. and Zimmerman, E. A., The effects of adrenalectomy and giucocorticoid replacement on vasopressin-neurophysin in the zona externa of the median eminence of the rat, Endocrinology, 101 (1977) 42--49. 20 Vandesande, F., Dierickx, K. and De Mey, J., The origin of the vasopressinergic and oxytocinergic fibers of the external region of the median eminence of the rat hypophysis, Cell Tiss. Res., 180 (1977) 443-452. 21 Zimmerman, E. A., Carmel, P. W., Husain, M. K., Ferin, M., Tannenbaum, M., Frank, A. G. and Robinson, A. G., Vasopressin and neurophysin- high concentrations in monkey hypophyseal portal blood, Science, 182 (1973) 925-927.
469
Release of vasopressin from the microdissected median eminence of the rat
J.-L. BI~NY and A. J. BAERTSCHI Department of Animal Biology, University of Geneva, 121l Geneva 4 (Switzerland)
(Accepted September 1lth, 1980) Key words: vasopressin - - median eminence -- hypothalamus
For over 25 years arginine vasopressin (AVP) has been suspected to play a role in adrenocorticotropin (ACTH) regulation 13. AVP has been detected in monkey 21 and rat 16 hypophysial portal blood in high concentrations (several mU/ml plasma). It is known that AVP at similar concentrations induces the release of A C T H from adenohypophysial quarters 12 or dispersed cells 18 in vitro, and also when injected into rats in vivo 1. However, it is not clearly established if portal plasma AVP is released from axon terminals of the median eminence or of the neural lobe, or of both. Neurones of the paraventricular nucleus project onto the median eminence 17,20 and the staining by immunohistochemistry of vasopressin-containing fibres of the median eminence is enhanced following adrenalectomy and reduced after corticosteroid treatmenP, 19. Furthermore, electrical stimulation of the paraventricular nuclei elicits A C T H release 7,8. On the other hand, portal plasma AVP levels are strongly diminished following posterior lobectomy 18. Therefore, the goal of this study was to determine the amount of AVP which can be released from the median eminence at rest and during stimulation. Male Sprague-Dawley rats (250-350 g body weight) were decapitated and the brain was immediately removed from the skull. A tissue piece containing the pituitary stalk and arcuate nucleus but not the optic chiasm was dissected by four cuts with iridectomy scissors. We call this tissue the medial basal hypothalamus (MBH). The tissue was fastened on its ventral surface with a needle inserted through the stalk. Under a binocular microscope, the floor of the third ventricle was dissected with a scalpel (Fig. I). These manipulations lasted for 2 min per median eminence. Five median eminences were impaled on a platinum needle, stacked between two silicon rubber pieces, and incubated in 500/zl of a Krebs-Ringer-bicarbonate buffer containing 1 mg glucose, 0.5 mg beef serum albumin and 17 /zg bacitracin. The medium was continuously gassed with 95 ~ 02 and 5 ~ CO,, and maintained at 37 °C. After a preincubation of 20 min, the median eminences were subsequently incubated for three 20 min periods. After each period, the incubation medium was collected for radioimmunoassay of AVP 6 and renewed. The median eminences were stimulated in the first 15 min
470
CQU~. needle ~r~
3rdventricle ~
scalpel
optic chiasm
Fig. 1. Microdissection of the median eminence. The whole medial basal hypothalamus is shown. After pinning down the tissue with a needle, the floor of the third ventricle (interrupted line) is dissected with a scalpel.
of the second incubation period by applying biphasic pulses (1 msec duration) of 19-22 V at a frequency of 75 Hz 4 between the platinum electrode and an indifferent electrode in the medium. The pulse trains were switched on for 5 sec and offfor 5 sec during 15 min. Separate sets of M B H or median eminence were homogenized in 50 #10.1 N HC1 and frozen. Before the AVP assays, the extracts were neutralized by 5 #1 1 N N a O H complemented with 445/~1 incubation medium and subjected to centrifugation. The AVP content of the supernatant was determined by radioimmunoassay 6 and by bioassay from the pressure effects on the isolated rat mesentery perfused at constant flow 14.
The basal AVP release from each median eminence was 0.09 4- 0.01 mU/20 min (mean 4- S.E.M., n = 9). During electrical stimulation, AVP release increased 11-fold (Fig. 2). Following electrical stimulation, AVP release returned towards control values but was significantly larger than during the first incubation period (P < 0.005), presumably because of the AVP which had filled the interstitial space during stimulation in the preceding incubation period. Prior to microdissection, the M B H contained 12.9 4- 0.2 m U AVP (mean 4S.E.M., n = 4) and wet weight was 2.1 ± 0.3 rag. After microdissection, the median eminence contained 12.4 4- 0.5 m U AVP (n = 4) and wet weight was 0.11 4- 0.01 mg (n = 4). At the end of the first incubation period, AVP content of the median eminence was 8.8 4- 0.6 m U (n = 6). Thus one median eminence releases in 20 min about 1% of its content under basal conditions and about l 1% during electrical stimulation. In order to check if radioimmunoassayable (RIA) AVP reflects bioassayable AVP, both types of assays were performed with M B H extracts. AVP content per M B H was 16.6 4- 1.0 m U (bioassay) compared to 12.9 4- 2.0 m U (n = 4) (RIA), giving a ratio ofbioassay/RIA of about 1.3. The results show that the microdissected median eminence, which represents essentially the floor of the third ventricle, weighs about 20 times less (0.11 rag) than
471 1
o E
I o 1st
2nd
3 rd
electrical stim.
Fig. 2. Release of arginine vasopressin from median eminence in vitro (mean ± S.E.M. per median eminence, n = 4-5). Incubations (lst, 2nd, 3rd) lasted 20 min each. Electrical stimulation was 75 Hz, 5 sec on and offfor 15 min. the MBH and contains the same quantity of AVP. Similar amounts of AVP, 4.3-12 mU, have been reported by othersl°, 15 for the MBH. The manipulation of the tissue including impalement on a platinum electrode and 40 min incubation results in a loss of only 25 ~ of the AVP content. This preparation may perhaps be applied to study the release of other hypothalamic hormones. The advantage over using the MBH or whole hypothalamus resides in the fact that hormones (or neuromodulators) released from the hypothalamus which are not directly involved in adenohypophysial control do not contaminate the hormones released from the median eminence. The electrical stimulation appears to be effective in promoting the release of AVP from the median eminence. A stimulation frequency of 75 Hz has previously been found to be optimal for the release of corticotropin releasing factor from the hypothalamus in vitro 4, although such high frequencies are rarely encountered when hypothalamic cells are monitored. It is unlikely that the electrical stimulation led to significant tissue damage, since the stimulus voltages were reasonably low and biphasic. Furthermore, the poststimulation AVP levels returned almost to control levels. By considering the rate of AVP release upon electrical stimulation (1 mU/20 min) and of portal blood flow (0.0041-0.0056 ml/min for a l0 mg adenohypophysisl), the concentration reached in portal plasma can be estimated to range from 18 to 24 mU/ml when AVP release is maximally stimulated and from 1.6 to 2.2 mU/ml at rest. These concentrations, if applied to a suspension of acutely dispersed adenohypophysial cells, increase the A C T H release by 70-150 ~a.
472 Previous studies suggest that the neural lobe contributes significantly to the high plasma A V P levels of the hypophysial portal circulation TM and that vasopressin mediates the increase of plasma A C T H release in response to electrical stimulation of the neural lobe in vivo 2. O u r results a n d previously published studies thus suggest that A V P can be released from both the median eminence and from the neural lobe in sufficient a m o u n t s to elicit a m a x i m u m vasopressin-mediated A C T H release from the adenohypophysis, an action which may be amplified by the c o n c o m i t a n t secretion of a p o t e n t i a t i n g factor 9. This work was supported by Swiss National Research F o u n d a t i o n G r a n t s 3.248-0.77 and 3.581-0.79. We t h a n k Ms. M. Friedli for excellent technical assistance, Ms. N. Gulati (Geneva) for i n t r o d u c i n g us to the isolated mesentery preparation, a n d Dr. R. M a t h i s o n for reviewing the manuscript. We are indebted to Drs. G. Gillies and P. Lowry, L o n d o n , for a specific vasopressin antibody.
1 Arimura, A., Schally, A. V. and Bowers, E. Y., Corticotropin releasing activity of lysine vasopressin analogues, Endocrinology, 84 (1969) 579 583. 2 Baertschi, A. J., Vallet, P., Baumann, J. B. and Girard, J., Neural lobe of pituitary modulates corticotropin release in the rat, Endocrinology, 106 (1980) 878-882. 3 Beny, J.-L. and Baertschi, A. J., Release of corticotropin and corticotropin-releasing factors from rat posterior pituitary in vitro, Neuroendocrinology, 30 (1980) 108-112. 4 Bradbury, M. W. B., Burden, J., Hillhouse, E. W. and Jones, M. T., Stimulation electrically and by acetylcholine of the rat hypothalamus in vitro, J. Physiol. (Lond.), 239 (1974) 269-283. 5 Burlet, A., Chauteau, M. and Czernichow, P., lnfundibular localization of vasopressin, oxytocin and neurophysins in the rat; its relationships with corticotrope function, Brain Research, 168 (1979) 275-286. 6 Dogterom, J., Van Wimersma Greidanus, Tj. B. and Swaab, D. F., Evidence for the release of vasopressin and oxytocin into cerebrospinal fluid: measurements in plasma and CSF of intact and hypophysectomized rats, Neuroendocrinology, 24 (1977) 108-118. 7 Dorenhorst, A., Carlson, D. E., Self, S. M., Robinson, A. G., Zimmerman, E. A. and Gann, D. S., Control of release of ACTH and vasopressin by supraoptic and paraventricular nuclei, Neurosci. ,4bstr., 4 0978) 344. 8 Dunn, J. and Critchlow, W., Electrically stimulated ACTH rdease in pharmacologically blocked rats, Endocrinology, 93 (1973) 835-842. 9 Gillies, G. and Lowry, P., Corticotropin releasing factor may be modulated vasopressin, Nature (Lond.), 278 0979) 463464. l0 Gillies, G., Van Wimersma Greidanus, T. B. and Lowry, P. J., Characterization of rat stalk median eminence vasopressin and its involvement in adrenocorticotropin release, Endocrinology, 103 (1978) 528-534. I 1 Goldman, H., Endocrineglandbloodflowinthe unanesthetized, unrestrained rat, J. appl. Physiol., 16 (1961) 762-764. 12 Lutz-Bucher, B., Koch, B., Mialhe, C. and Briaud, B., Involvement of vasopressin in corticotropin releasing effect of hypothalamic median eminence extract, Neuroendocrinology, 30 (1980) 178-182. 13 McCann, S. M. and Brobeck, J. R., Evidence for a role of the supraoptico-hypophyseal system in regulation of adrenocorticotropin secretion, Proc. Soc. exp. Biol. (N. F.), 87 (1954) 318-324. 14 McGregor, D. D., The effect of sympathetic nerve stimulation on vasoconstrictor responses in perfused mesenteric blood vessels of the rat, J. Physiol. (Lond.), 177 (1965) 21-30. 15 Mialhe, C., Lutz-Bucher, B., Briaud, B., Schleiffer, R. and Koch, B. B., Corticotropin-releasing factor (CRF) and vasopressin in the regulation of corticotropin (ACTH) secretion. In M. T. Jones B. Gillham, M. F. Dallman and S. Chattopadhyay (Eds.), Interaction within the Brain-PituitaryAdrenocortical System, Academic Press, London, 1979, pp. 63-74. 16 Oliver, C., Mical, R. S. and Porter, C., Hypothalamic-pituitary vasculature: evidence for retrograde blood flow in the pituitary stalk, Endocrinology, 101 (!977) 598-604.
473 17 Parry, H. B. and Livett, B. G., A new hypothalamic pathway to the median eminence containing neurophysin and its hypertrophy in sheep with natural scrapie, Nature (Lond.), 242 (1973) 63-65. 18 Portanova, R. and Sayers, G., An in vitro assay for corticotropin releasing factor(s) using suspension of isolated pituitary cells, Neuroendocrinology, 12 (1973) 236-248. 19 Stillman, M. A., Recht, L. D., Rosario, S. L., Seif, S. M., Robinson, A. G. and Zimmerman, E. A., The effects of adrenalectomy and giucocorticoid replacement on vasopressin-neurophysin in the zona externa of the median eminence of the rat, Endocrinology, 101 (1977) 42--49. 20 Vandesande, F., Dierickx, K. and De Mey, J., The origin of the vasopressinergic and oxytocinergic fibers of the external region of the median eminence of the rat hypophysis, Cell Tiss. Res., 180 (1977) 443-452. 21 Zimmerman, E. A., Carmel, P. W., Husain, M. K., Ferin, M., Tannenbaum, M., Frank, A. G. and Robinson, A. G., Vasopressin and neurophysin- high concentrations in monkey hypophyseal portal blood, Science, 182 (1973) 925-927.