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Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments
Life Sciences Vol . 19, pp . 685-690, 1976 . Printed in the II .S .A .
Pnrgamoa Press
BEHAVIORAL EFFECTS OF INTRAVENTRICULARLY ADMINISTERED VASOPRESSIN AND VASOPRESSIN FRAGMENTSe) D . de Wied Rudolf Magnus Institute for Pharmacology Medical Faculty, University of Utrecht Vondellaan 6, Utrecht, The Netherlands . (Received is final form July 8, 1976)
Summary Vasopressin is involved in memory processes . A single subcutaneous injection of arginine-8-vasopressin (AVP) increaaea resistance to extinction of a pole jumping avoidance response . This effect can also be achieved after a single intraventricular administration of much lower amounts than after systemic injection . The covalent ring of AVP, pressinamide (PA), is also highly active following intraventricular administration while the C-terminal part prolylarginyl-glycinamide (PAG) is leas active . These results indicate that the covalent ring of vasopressin contains the essential requirements for the behavioral effect of this neurohormone . A second activity site however may be present in the C-terminal portion of the molecule . The aupraoptic-neurohypophyaeal system is involved in the formation and maintenance of adaptive behavior . Removal of the posterior lobe of the pituitary interferes with the maintenance of a shuttle box avoidance response (1) while administration of vasopressin analogues increases resistance to extinction of active (2, 3, 4) and passive avoidance behavior (5, 6 . 7) . These and studies on amnesia (S, 9) indicate an effect of vasopressin on memory processes . Implantation studies point to limbic midbrain structures as the site of the behavioral action of vasopressin (10) . A number of reports suggests the presence of vasopressin in the cerebrospinal fluid (CSF)(11, 12, 13), which might indicate that the CSF is a route of transport of these principles to their central target structures . Indeed, the intracerebroventricular administration of vasopressin antiserum induces an almost complete deficit in passive avoidance behavior (14) . Indirect evidence for this assumption may also be obtained by studying the behavioral effect of vasopressin upon intracerebroventricular administration . The present report describes the influence of arginine-8-vasopressin (AVP), the covalent ring, preasinamide (PA), and the C-terminal tripeptide prolyl-arginyl-glycinamide (PAG) administered via one of the lateral ventricles, on the rate of extinction of a pole jumping avoidance response . e) Presented in part at the Fourth American Peptide Symposium in New York, 1975 (15) . 685
68 6
Vasopresein and Behavior
Vol . 19, No . 5
Materials and Methods Male Wistar strain rats weighing between 130 and 150 g were used . They were kept at near constant temperature (210 ) under controlled lighting condit~.ons (7 a.m .-6 p .m . light) . The animals had free access to food and water and were housed 5 per cage . To study the behavior, a pole jumping avoidance response was used (15) . Animals were trained to jump onto a pole placed in the middle of a box, within 5 sec after presentation of the conditioned stimulus (CS) which was a light on the transparant top of the box . Rats which failed to jump within 5 sec, received the unconditioned stimulus (US) of shock (0 .2 mA) to the feet via the grid floor of the box . Ten trials were given each day with an average interval of 60 sec for 3 consecutive days . All animals which made 7 or more positive responses (jumped within 5 sec) during the last acquisition session, were injected s .c . with AVP immediately after the session. On the 4th, 5th and 8th day, extinction sessions of 10 trials were run, in which if an animal failed to jump within 5 sec, the CS was not followed by the US of shock . Rats for intraventricular injection were equipped with a polyethylene cannula in the lateral ventricle . The operation was performed under ether anesthesia and aseptic conditions . A hole was drilled through the skull, 1 mm lateral to the midline and 0 .5 mm caudal to the bregma for insertion of the cannula . After insertion, the cannula was fixed to the skull by means of dental cement on 2 stainless steel screws . Commercially available Hamilton syringes were used, provided with needles specially adapted to the appropriate length for intraventricular injection . The localization of the tip of the cannula was determined at the termination of the experiment by the injection of Evans blue . The staining was then inspected macroscopically in formaldehyde fixated brain sections . The training procedure for rats for intraventricular injection was slightly different from that of rats used for subcutaneous administration of peptides . They were first trained for 3 days in the pole jumping test and operated 3 days later . Two retraining sessions of 10 trials were given at the 4th and 5th day after operation . All animals which made 7 or more positive avoidances were injected intraventricularly with the respective materials immediately after the second reacquisition session . Extinction sessions were run 1, 2 and 5 days after injection . The various peptides were given in 3 dose levels . Highly purified synthetic peptides were obtained from Organon H .V ., Oss, the Netherlands . They were stored at room temperature in dry form in bottles under dry conditions in a desiccator . Results
The s .c . injection of AVP (430-580 International pressor Units per mg) resulted in a dose dependent resistance to extinction as compared to saline treated controls (table 1) . An amount of 0 .54 ug . AVP was sufficient to maintain a high rate of responding up to 5 days after injection when saline treated rats were virtually extinguished . After intraventricular administration, the same behavioral effects were obtained (table 1) . AVP again increased resistance to extinction which was maximal after 2 .5 ng . However, 25 pg already elicited a significant effect . The amounts needed were much smaller than after systemic injection (table 1) . Pressinamide (PA) was only slightly less active since 5 ng was needed to obtain an effect comparable to that of 2 .5 ng of AVP . In contrast, doses up to
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Vsaopreeaia aad Behavior
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100 ng PAG failed to reach the same high response rate se with AVP although the effect of the 3 doses differed significantly from that of saline administration . Discussion
The present results confirm previous studies on the effect of systemically administered AVP on the maintenance of a pole jumping avoidance response in rats (15) . The data further demonstrate that similar effects can be obtained following intraventricular administration of picogram to nanogram quantities of AVP . In addition, it appears that intraventricularly administered fragments of vasopressin pressinamide (PA) and prolyl-arginyl-glycinamide (PAG) also maintain the behavior for a considerable period of time . The data suggest that pressinamide contains the essential requirements for the behavioral effect of vasopressin, being nearly as active as AVP, while PAG had less activity . Since PAG significantly increased resistance to extinction, a second activity site may be present in the C-terminal part of the vasopressin molecule . PA has only 5 percent of the behavioral activity of AVP following syst~nic injection (15) . It acts much stronger following intraventricular administration since 5 .0 nq PA equalled the effect of 2 .5 nq AVP. This may suggest that the requirements for behavioral activity are better protected in the whole molecule than in the covalent ring structure on route to the CNS . This would explain the much higher ratio PA/AVP following intraventricular administration . Studies on the interaction of these neurôpeptides with putative receptor sites however are needed to determine these requirements in the vasopressin molecule in this respect . These findings have several implications . Firstly, vasopressin may act as a prohormone for a neuropeptide with memory consolidating effects . Secondly, these principles may exert their behavioral effect via release into the CSF, which may be an efficient way of transport to midbrain limbic structures which are their site of action (4, 16) . It has been found that a membrane bound hypothalamic exopeptidase is able to generate a biologically active oligopeptide from ôxytocin which inhibits the release of MSH (17) or potentiate be havioral effects of L-Dopa (18, 19) . The same may hold for vasopressin, although the generation of PA or PAG has not been demonstrated . Enzymes have been found in the brain which inactivate vasopressin by hydrolysis of peptide bonds in the acyclic portion of the molecule (20), but no enzymes have been identified which remove the proline residue from vasopressin to leave pressinamide . From the present results however it is tempting to speculate that vasopressin is a putative prohormone for neuropeptides involved in learning and memory processes . This might explain the efficacy with which PA affects the maintenance of avoidance behavior fol- . lowing intracerebroventricular administration . Morphological and functional evidence has accumulated which suggests that hypothalamic hormones can be released from neural tissue into the liquor of the brain ventricular system (12, 21, 22) . Vasopreasin has been detected in the CSF and evidence for a direct secretion into the CSF has been obtained (13) . In addition, Goldsmith and Zimmerman (23) with the use of electron microscopic and immunochemistry techniques, demonstrated that neuronal processes containing granules with neurophysin and vasopressin were observed protruding into the third ventricle . Indeed, neutralizing centrally circulating vasopressin antiserum prevents passive avoidance behavior and facilitates extinction of pole jumping
Vol. 19, No . 5
Vaeoprasein and Behavior
689
avoidance behavior (14, 24) . Admittedly, the CSF does not need to be the route of transport of neurohypophyseal principles . It is also possible that a direct hypothalamic-limbic pathway exists which may be an integral subsystem of the main peptidergic neurosecretory system (25) . Martin et al . (26) postulated that hypothalamic peptidergic neurons are part of a diffuse neural network that terminates in widespread areas of the brain which may be important in terms of neurobiologic regulation . Various possible functions for vasopressin in the CSF have been suggested, like a role in the feedback control of the secretion of neurosecretory principles, in osmoreceptor regulation, in the homeostasis of ionic concentration in the brain, etc .(22) . The present experiments demonstrate the importance of vasopreesin and vasopresein fragments in memory processes sugqesting a role of these principles in the maintenance of behavioral homeostasis . The physiological significance of vasopressin in this respect has been demonstrated in rats with hereditary diabetes inaipidus (DI) which have difficulties in acquisition of active avoidance behavior (27) and memory disturbances (28, 29), and in intact rats following the intracerebroventricular administration of specific vasopressin antiserum which induces similar behavioral deficits (14) . Açknowledgemente
Excellent technical assistance was obtained from Miss Andrea Siepmann and Miss Angèle Balvers, Dr . J .M . van Ree kindly evaluated the effectiveness of the implantation of the intraventricular cannulae . Organon B .V ., Oss, The Netherlands, supplied the peptides . References 1 . D . DE WIED, Int . J . Neuropharmacol . _4 157-167 (1965) . 2 . D . DE WIED, Nature (Loud .) 232 58-60 (1971), 3 . D . DE WIED, H .M . GREVEN, S .LANDE and A . WITTER, Brit . J . Pharmacol . _45 118-122 (1972) . 4 . Tj .B . VAN WIMERSMA GREIDANUS, B. BOHUS and D . DE WIED, Progx . Endocr ., Excerpts Med . (Amst .) Int . Congress Series No . 273, pp . 197-201 (1973) . 5 . R. ADER and D . DE WIED, Psychon . Sci . 29 46-48 (1972) . 6 . B . BOHUS, R. ADER and D . DE WIED, Horm, Behav . 3 191-197 (1972) . 7 . E .A . THOMPSON and D . DE WIED, Physiol . Behav . 11 377-380 (1973) . 8 . H . RIGTER, H . VAN RIEZEN and D . DE WIED, Physiol . Behav . 13 381-388 (1974) . 9 . R. WALTER, P .L . HOFFMAN, J .B . FLEXNER and L .B . FLEXNER, Proc . net . Aced, Sci . (Wash .) 72_4180-4184 (1975) . 10 . Tj .B . VAN WIMERSMA GREIDI~NUS, B . BOHUS and D . DE WIED, Anatomical Neuroendocrinology (pp . 284-289), S . Karger, Basel (1975) . 11 . H . HELLER, S .H . HASAN and A .Q . SAIFI, J . Endocr . _41 273-280 (1968) . 12 . H . VORHERR, M.W .B . BRADBi,JRY, M. HOGHOUGHI and C .R . KLEEMAN, Endocrinology 83 246-250 (1968) . 13 . S .M .A . ZAID I a~ H . HELLER, J . Endocr . 60 195-196 (1974) . 14 . Tj .B . VAN WIMERSMA GREIDANUS, J . DOGTEROM and D . DE WIED, Life Sci . 16 637-644 (1975) .
690
Vaeopreeain aad Behavior
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15 . D . DE WIED, B.BOHUS, I .URBAN, Tj .B . VAN WIMERSMA GREIDANUS and W .H . GISPEN, Peptides : Chemistry, Structure and Biology (pp. 635-643), Ann Arbor Science Publ . Inc ., Ann Arbor (1975), 16 . Tj,H . VAN WIMERSMA GREIDANTJS and D . DE WIED, Neuroendocrinology 7 291-301 (1971) . 17 . M.E . CELIS, S . TALEISNIK and R . WALTER, Proc . nat . Acad . Sci . (Wash .) _68 1428-1433 (1971) . 18 . N .P . PLOTNIKOFF, A .J . KASTIN, M,S . ANDERSON and A .V . SCHALLY, Life Sci. 10 1279-1283 (1971) . 19, A.J . RASTIN, A. BARBEAU, R,H . EHRENSING, N.P . PLOTNIKOFF and A.V . SCHALLY, Adv . Neurol . 5 225-229 (1974) . 20 . N . MARKS, L. AHRASH and R . WALTER, Proc . Soc . exp . Biol .(N .Y .) 142 455-460 (1973) . 21 . G .STERBA, Ependyma and Neurohormonal Regulation (pp.' 143-173) Veda Publ . House Slovak Acad . Sci ., Bratislava (1974), 22, E .M . RODRIGUEZ, Aspects in Neuroendocrinology (pp . 352-365) Springer Verlag, Berlin (1970) . 23 . P .C . GOLDSMITH and A .E . ZIMMERMAN, Endocrinology Suppl . to vol . 96, A-377 (1975) . 24 . Tj .B . VAN WIMERSMA GREIDANUS and D . DE WIED, Biochemical Correlates of Brain Structure and Function, Academic Press, London (in press) 25 . G . STERBA, Neurosecretion - The Final Neuroendocrine Pathway, (pp . 38-47) Springer Verlag, Berlin (1974) . 26 . J .B . MARTIN, L .P, RENAUD and P . BRAZEAU, The Lancet II, No . 7931 393-395 (1975) . 27 . J,F .CELESTIAN, R .J . CAREY and M .MILLER, Physiol . Behav ._15 707711 (1975) . 28 . D . DE WIED, B .BOHUS and Tj,B .van WIMERSMA GREIDANUS, Brain Res . 85, 152-156 (1975) . 29 . H . BOHUS, Tj .B .van WIMERSMA GREIDANUS and D . DE WIED, Physiol . Behav . 14 609-615 (1975) .
Pnrgamoa Press
BEHAVIORAL EFFECTS OF INTRAVENTRICULARLY ADMINISTERED VASOPRESSIN AND VASOPRESSIN FRAGMENTSe) D . de Wied Rudolf Magnus Institute for Pharmacology Medical Faculty, University of Utrecht Vondellaan 6, Utrecht, The Netherlands . (Received is final form July 8, 1976)
Summary Vasopressin is involved in memory processes . A single subcutaneous injection of arginine-8-vasopressin (AVP) increaaea resistance to extinction of a pole jumping avoidance response . This effect can also be achieved after a single intraventricular administration of much lower amounts than after systemic injection . The covalent ring of AVP, pressinamide (PA), is also highly active following intraventricular administration while the C-terminal part prolylarginyl-glycinamide (PAG) is leas active . These results indicate that the covalent ring of vasopressin contains the essential requirements for the behavioral effect of this neurohormone . A second activity site however may be present in the C-terminal portion of the molecule . The aupraoptic-neurohypophyaeal system is involved in the formation and maintenance of adaptive behavior . Removal of the posterior lobe of the pituitary interferes with the maintenance of a shuttle box avoidance response (1) while administration of vasopressin analogues increases resistance to extinction of active (2, 3, 4) and passive avoidance behavior (5, 6 . 7) . These and studies on amnesia (S, 9) indicate an effect of vasopressin on memory processes . Implantation studies point to limbic midbrain structures as the site of the behavioral action of vasopressin (10) . A number of reports suggests the presence of vasopressin in the cerebrospinal fluid (CSF)(11, 12, 13), which might indicate that the CSF is a route of transport of these principles to their central target structures . Indeed, the intracerebroventricular administration of vasopressin antiserum induces an almost complete deficit in passive avoidance behavior (14) . Indirect evidence for this assumption may also be obtained by studying the behavioral effect of vasopressin upon intracerebroventricular administration . The present report describes the influence of arginine-8-vasopressin (AVP), the covalent ring, preasinamide (PA), and the C-terminal tripeptide prolyl-arginyl-glycinamide (PAG) administered via one of the lateral ventricles, on the rate of extinction of a pole jumping avoidance response . e) Presented in part at the Fourth American Peptide Symposium in New York, 1975 (15) . 685
68 6
Vasopresein and Behavior
Vol . 19, No . 5
Materials and Methods Male Wistar strain rats weighing between 130 and 150 g were used . They were kept at near constant temperature (210 ) under controlled lighting condit~.ons (7 a.m .-6 p .m . light) . The animals had free access to food and water and were housed 5 per cage . To study the behavior, a pole jumping avoidance response was used (15) . Animals were trained to jump onto a pole placed in the middle of a box, within 5 sec after presentation of the conditioned stimulus (CS) which was a light on the transparant top of the box . Rats which failed to jump within 5 sec, received the unconditioned stimulus (US) of shock (0 .2 mA) to the feet via the grid floor of the box . Ten trials were given each day with an average interval of 60 sec for 3 consecutive days . All animals which made 7 or more positive responses (jumped within 5 sec) during the last acquisition session, were injected s .c . with AVP immediately after the session. On the 4th, 5th and 8th day, extinction sessions of 10 trials were run, in which if an animal failed to jump within 5 sec, the CS was not followed by the US of shock . Rats for intraventricular injection were equipped with a polyethylene cannula in the lateral ventricle . The operation was performed under ether anesthesia and aseptic conditions . A hole was drilled through the skull, 1 mm lateral to the midline and 0 .5 mm caudal to the bregma for insertion of the cannula . After insertion, the cannula was fixed to the skull by means of dental cement on 2 stainless steel screws . Commercially available Hamilton syringes were used, provided with needles specially adapted to the appropriate length for intraventricular injection . The localization of the tip of the cannula was determined at the termination of the experiment by the injection of Evans blue . The staining was then inspected macroscopically in formaldehyde fixated brain sections . The training procedure for rats for intraventricular injection was slightly different from that of rats used for subcutaneous administration of peptides . They were first trained for 3 days in the pole jumping test and operated 3 days later . Two retraining sessions of 10 trials were given at the 4th and 5th day after operation . All animals which made 7 or more positive avoidances were injected intraventricularly with the respective materials immediately after the second reacquisition session . Extinction sessions were run 1, 2 and 5 days after injection . The various peptides were given in 3 dose levels . Highly purified synthetic peptides were obtained from Organon H .V ., Oss, the Netherlands . They were stored at room temperature in dry form in bottles under dry conditions in a desiccator . Results
The s .c . injection of AVP (430-580 International pressor Units per mg) resulted in a dose dependent resistance to extinction as compared to saline treated controls (table 1) . An amount of 0 .54 ug . AVP was sufficient to maintain a high rate of responding up to 5 days after injection when saline treated rats were virtually extinguished . After intraventricular administration, the same behavioral effects were obtained (table 1) . AVP again increased resistance to extinction which was maximal after 2 .5 ng . However, 25 pg already elicited a significant effect . The amounts needed were much smaller than after systemic injection (table 1) . Pressinamide (PA) was only slightly less active since 5 ng was needed to obtain an effect comparable to that of 2 .5 ng of AVP . In contrast, doses up to
687
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100 ng PAG failed to reach the same high response rate se with AVP although the effect of the 3 doses differed significantly from that of saline administration . Discussion
The present results confirm previous studies on the effect of systemically administered AVP on the maintenance of a pole jumping avoidance response in rats (15) . The data further demonstrate that similar effects can be obtained following intraventricular administration of picogram to nanogram quantities of AVP . In addition, it appears that intraventricularly administered fragments of vasopressin pressinamide (PA) and prolyl-arginyl-glycinamide (PAG) also maintain the behavior for a considerable period of time . The data suggest that pressinamide contains the essential requirements for the behavioral effect of vasopressin, being nearly as active as AVP, while PAG had less activity . Since PAG significantly increased resistance to extinction, a second activity site may be present in the C-terminal part of the vasopressin molecule . PA has only 5 percent of the behavioral activity of AVP following syst~nic injection (15) . It acts much stronger following intraventricular administration since 5 .0 nq PA equalled the effect of 2 .5 nq AVP. This may suggest that the requirements for behavioral activity are better protected in the whole molecule than in the covalent ring structure on route to the CNS . This would explain the much higher ratio PA/AVP following intraventricular administration . Studies on the interaction of these neurôpeptides with putative receptor sites however are needed to determine these requirements in the vasopressin molecule in this respect . These findings have several implications . Firstly, vasopressin may act as a prohormone for a neuropeptide with memory consolidating effects . Secondly, these principles may exert their behavioral effect via release into the CSF, which may be an efficient way of transport to midbrain limbic structures which are their site of action (4, 16) . It has been found that a membrane bound hypothalamic exopeptidase is able to generate a biologically active oligopeptide from ôxytocin which inhibits the release of MSH (17) or potentiate be havioral effects of L-Dopa (18, 19) . The same may hold for vasopressin, although the generation of PA or PAG has not been demonstrated . Enzymes have been found in the brain which inactivate vasopressin by hydrolysis of peptide bonds in the acyclic portion of the molecule (20), but no enzymes have been identified which remove the proline residue from vasopressin to leave pressinamide . From the present results however it is tempting to speculate that vasopressin is a putative prohormone for neuropeptides involved in learning and memory processes . This might explain the efficacy with which PA affects the maintenance of avoidance behavior fol- . lowing intracerebroventricular administration . Morphological and functional evidence has accumulated which suggests that hypothalamic hormones can be released from neural tissue into the liquor of the brain ventricular system (12, 21, 22) . Vasopreasin has been detected in the CSF and evidence for a direct secretion into the CSF has been obtained (13) . In addition, Goldsmith and Zimmerman (23) with the use of electron microscopic and immunochemistry techniques, demonstrated that neuronal processes containing granules with neurophysin and vasopressin were observed protruding into the third ventricle . Indeed, neutralizing centrally circulating vasopressin antiserum prevents passive avoidance behavior and facilitates extinction of pole jumping
Vol. 19, No . 5
Vaeoprasein and Behavior
689
avoidance behavior (14, 24) . Admittedly, the CSF does not need to be the route of transport of neurohypophyseal principles . It is also possible that a direct hypothalamic-limbic pathway exists which may be an integral subsystem of the main peptidergic neurosecretory system (25) . Martin et al . (26) postulated that hypothalamic peptidergic neurons are part of a diffuse neural network that terminates in widespread areas of the brain which may be important in terms of neurobiologic regulation . Various possible functions for vasopressin in the CSF have been suggested, like a role in the feedback control of the secretion of neurosecretory principles, in osmoreceptor regulation, in the homeostasis of ionic concentration in the brain, etc .(22) . The present experiments demonstrate the importance of vasopreesin and vasopresein fragments in memory processes sugqesting a role of these principles in the maintenance of behavioral homeostasis . The physiological significance of vasopressin in this respect has been demonstrated in rats with hereditary diabetes inaipidus (DI) which have difficulties in acquisition of active avoidance behavior (27) and memory disturbances (28, 29), and in intact rats following the intracerebroventricular administration of specific vasopressin antiserum which induces similar behavioral deficits (14) . Açknowledgemente
Excellent technical assistance was obtained from Miss Andrea Siepmann and Miss Angèle Balvers, Dr . J .M . van Ree kindly evaluated the effectiveness of the implantation of the intraventricular cannulae . Organon B .V ., Oss, The Netherlands, supplied the peptides . References 1 . D . DE WIED, Int . J . Neuropharmacol . _4 157-167 (1965) . 2 . D . DE WIED, Nature (Loud .) 232 58-60 (1971), 3 . D . DE WIED, H .M . GREVEN, S .LANDE and A . WITTER, Brit . J . Pharmacol . _45 118-122 (1972) . 4 . Tj .B . VAN WIMERSMA GREIDANUS, B. BOHUS and D . DE WIED, Progx . Endocr ., Excerpts Med . (Amst .) Int . Congress Series No . 273, pp . 197-201 (1973) . 5 . R. ADER and D . DE WIED, Psychon . Sci . 29 46-48 (1972) . 6 . B . BOHUS, R. ADER and D . DE WIED, Horm, Behav . 3 191-197 (1972) . 7 . E .A . THOMPSON and D . DE WIED, Physiol . Behav . 11 377-380 (1973) . 8 . H . RIGTER, H . VAN RIEZEN and D . DE WIED, Physiol . Behav . 13 381-388 (1974) . 9 . R. WALTER, P .L . HOFFMAN, J .B . FLEXNER and L .B . FLEXNER, Proc . net . Aced, Sci . (Wash .) 72_4180-4184 (1975) . 10 . Tj .B . VAN WIMERSMA GREIDI~NUS, B . BOHUS and D . DE WIED, Anatomical Neuroendocrinology (pp . 284-289), S . Karger, Basel (1975) . 11 . H . HELLER, S .H . HASAN and A .Q . SAIFI, J . Endocr . _41 273-280 (1968) . 12 . H . VORHERR, M.W .B . BRADBi,JRY, M. HOGHOUGHI and C .R . KLEEMAN, Endocrinology 83 246-250 (1968) . 13 . S .M .A . ZAID I a~ H . HELLER, J . Endocr . 60 195-196 (1974) . 14 . Tj .B . VAN WIMERSMA GREIDANUS, J . DOGTEROM and D . DE WIED, Life Sci . 16 637-644 (1975) .
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15 . D . DE WIED, B.BOHUS, I .URBAN, Tj .B . VAN WIMERSMA GREIDANUS and W .H . GISPEN, Peptides : Chemistry, Structure and Biology (pp. 635-643), Ann Arbor Science Publ . Inc ., Ann Arbor (1975), 16 . Tj,H . VAN WIMERSMA GREIDANTJS and D . DE WIED, Neuroendocrinology 7 291-301 (1971) . 17 . M.E . CELIS, S . TALEISNIK and R . WALTER, Proc . nat . Acad . Sci . (Wash .) _68 1428-1433 (1971) . 18 . N .P . PLOTNIKOFF, A .J . KASTIN, M,S . ANDERSON and A .V . SCHALLY, Life Sci. 10 1279-1283 (1971) . 19, A.J . RASTIN, A. BARBEAU, R,H . EHRENSING, N.P . PLOTNIKOFF and A.V . SCHALLY, Adv . Neurol . 5 225-229 (1974) . 20 . N . MARKS, L. AHRASH and R . WALTER, Proc . Soc . exp . Biol .(N .Y .) 142 455-460 (1973) . 21 . G .STERBA, Ependyma and Neurohormonal Regulation (pp.' 143-173) Veda Publ . House Slovak Acad . Sci ., Bratislava (1974), 22, E .M . RODRIGUEZ, Aspects in Neuroendocrinology (pp . 352-365) Springer Verlag, Berlin (1970) . 23 . P .C . GOLDSMITH and A .E . ZIMMERMAN, Endocrinology Suppl . to vol . 96, A-377 (1975) . 24 . Tj .B . VAN WIMERSMA GREIDANUS and D . DE WIED, Biochemical Correlates of Brain Structure and Function, Academic Press, London (in press) 25 . G . STERBA, Neurosecretion - The Final Neuroendocrine Pathway, (pp . 38-47) Springer Verlag, Berlin (1974) . 26 . J .B . MARTIN, L .P, RENAUD and P . BRAZEAU, The Lancet II, No . 7931 393-395 (1975) . 27 . J,F .CELESTIAN, R .J . CAREY and M .MILLER, Physiol . Behav ._15 707711 (1975) . 28 . D . DE WIED, B .BOHUS and Tj,B .van WIMERSMA GREIDANUS, Brain Res . 85, 152-156 (1975) . 29 . H . BOHUS, Tj .B .van WIMERSMA GREIDANUS and D . DE WIED, Physiol . Behav . 14 609-615 (1975) .