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Effect of 6-hydroxydopamine lesions of the median eminence and neurointermediate lobe on the secretion of pituitary hormones in the male rat
B, am Re~eatch, 246 (19821 330 q3 E)~evler Biomedical Pre~
330
Effect of 6-hydroxydopamine lesions of the median eminence and neurointermodiate lobe on the secretion of pituitary hormones in the male rat G R A E M E C SMITH*, W JOHN SHEWARD** and GEORGE FINK**
Department o f Human Anatomy, South Park3 Road, Orford, OXI 3 Q X ( U K ) (Accepted May l lth, 1982)
Key words catecholamlnerg]c terminals - - medmn eminence - - neuromtermedmte lobe - - 6-hydroxydopamlne - prolactm - - cortlcosterone - - thyrotropm - - gonadotrophm - - Faglu fluorescence h~stochem]stry
The effect of 6-hydroxydopamme lesions of the catecholamme (CA) neurons of the medmn eminence and neuromtermedmte lobe (ME-NIL) on the secretion of several pltmtary hormones was studied m male rats The results show that the CA neurons of the ME-NIL facilitate prolactm release, inhibit the seeretmn of thyrotrophm and adrenocortlcotrophm and may not play an important role m the control of gonadotrophm secretion
The neurotoxm 6-hydroxydopamme (6-OHDA), when gaven intravenously, produces wathm 24 h a massave depletaon of catecholammes (CA) m the me&an eminence and neuromtermedmte lobe (MENIL) whale leaving largely unchanged the ammergac mnervatlon of the rest of the hypothalamus ~s Using this approach we stu&ed the effect of th~s specific and anatomically locahzed CA lesaon on the plasma concentration and p~tmtary content of prolactm, thyrotropan stamulatmg hormone (TSH), cortacosterone, luteamzmg hormone (LH) and folhcle stimulating hormone (FSH) The changes m the CA contents of the hypothalamus and ME-NIL were monatored by CA-fluorescence hlstochemastry All the experiments were carried out on one batch of male Wastar rats, purchased from Charles Rwer (U K ) Ltd , Margate, and housed under controlled hghtmg (hghts on 05 00-19 00 h) and temperature (22 °C) and given free access to &et 41B (E Daxon and Sons, Ware) and tap water At the beginning of the experiment the anamals weighed 250-300 g and had had 3 weeks to become accustomed to the con-
&tlons of the ammal house All experiments were carried out between 17 00 and 18 00 h The ammals were rejected 1 v (external jugular vem) under ether anesthesia with either 150 mg/kg 6-OHDA-HCI (Labkeml, Stockholm, 50 mg/mt m 0 l ~ ascorblc acid m 0 9 ~o sahne solution (vehlcle)) or with vehicle alone One group of ammals ('untreated control') was not rejected At 1, 2, 7, 14, 21 and 36 days after the rejection the anmaals were kdled by decapitation m batches of 6 at one ttme N o more than 2 mm elapsed between the tame that a cage of 6 anamals was removed qmetly from the antmal housing room and the tame that the last anamal an the group was decapitated The trunk blood was collected m heparm]zed 10 ml plastic tubes on ice, centrifuged, and the plasma stored at --20 °C untd assayed The patmtary glands were &ssected free, blotted, weaghed, and homogemzed an 10 ml 0 9 O/o saline soluUon by 15 strokes of a Potter Elvethem homogemzer Plasma and pttmtary LH was determmed by the method of Nlswender et al 13 as used previously m thas laboratory4 The plasma and p]tmtary con-
* On leave from Monash Umverslty, Department of Psychological Medtcme, Prince Henry's Hospital, Melbourne, Victoria 3004, Austraha ** Present address MRC Brain Metabohsm Umt, Umverslty Department of Pharmacology, 1 George Square, Edinburgh, Scotland EH8 9JZ, U K 0006-8993/82/0000-0000/$02
75 © 1982 Elsevier Blome&cal Press
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Fig 1 Mean ( :L S E M ) plasma concentrations of prolactln (NIAMDD-rat PrI-RP1), TSH (NIAMDD-rat TSH-RP1). cortxcosterone, LH (NIH-LH-SI8) and FSH (N1AMDD-rat FSH-RPI) in male rats killed by decapitation 1-36 days after the I v Injectionof either 6-OHDA or vehicle (0 1% ascorblc acid m 0 9 % saline) Also shown are hormone concentrations in animals treated similarly but not injected with either 6OHDA or vehicle alone The numbers of animals in each group are shown at the base of each column centratlons of FSH, prolactln and TSH were determined using the radloImmunoassay kits supplied by the Pltmtary Hormone Distribution Agency of the N I A M D D (Bethesda, MD) as used previously in this laboratory4,15 The concentration of plasma cortlcosterone was determined by competitive protern binding assay The significance of differences between means was determined by unpaired t-test Two furthei animals from each group were perfused and the brains subsequently processed for CA fluo-
rescence hlstochemlstry according to the Faglu techtuqueS, 16 The sectaons were examined by G C S and W J S without knowing the treatment to which the animal had been subjected Fig I shows that in the group InJected with 6O H D A the plasma concentrations of prolactin were not significantly different from those in the untreated control group However, in animals injected with vehicle alone the concentrations of prolactln were significantly elevated above the values in the untreated control group at 1 and 2 days and at 14 and 21 days after treatment (P < 0 05, 0 001, 0 01 and 0 025, respectively) The plasma concentration of TSH was significantly (P <~ 0 01) elevated 24 h after injecting 6-OHDA compared with the concentrations in either untreated control animals or ammals mjected with vehicle alone The plasma concentratlons of cort~costerone were slgmficantly (P < 0 02) elevated 24 h after injecting 6-OHDA compared with those in untreated control ammals On days 2 and 7 the concentrations of cortlcosterone in ammals Injected with 6-OHDA were lower (P < 0 01 and 0 02, respectively) than m vehicle treated animals In ammals treated with vehicle the concentrations of corticosterone were higher (P ~ 0 050 001 ) than m untreated control animals for all days except day l after rejection Treatment with e~ther 6O H D A or vehMe had no significant effect on the plasma concentrations of LH or FSH. except that at day 36 plasma FSH concentrations m both of these treatment groups were significantly lower than those in the untreated control group (P ~ 0 005 and P < 0 001, respectively) Fig 2 shows that for LH, FSH and TSH there were no major changes m pituitary content or concentration m the treated animals compared w~th those m the untreated control animals other than reduced values for LH and FSH on day 36 (P < 0 005-0 001) The pituitary content and concentration of prolactln m the treated animals were mostly lower than in the untreated control animals, and these changes were significant on days 1 and 7 (P < 0 05-0 005) The pituitary content and concentraUon of prolactln were sIgmficantly greater (P 0 02-0 005) at 14 and 21 days in the ammals treated with 6-OHDA compared w~th those m animals treated with vehicle alone The terminals in the ME-NIL were depleted of
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Fig 2 Mean (± S E M ) pltmtary hormone content (left) and concentration (right) m male rats (reference preparauons as m Fig 1) kdled by decapitation 1-36 days after i v rejection of either 6-OHDA or aseorbtc aod vehmlealone Values from untreated rats are also shown Number of ammals per group given at the base of each column
CA fluorescence 24 h after rejection of 6-OHDA The adjacent medlobasal hypothalamus was moderately depleted at 24 h, but was of normal appearance at 48 h, as was the rest of the hypothalamus at all times studied Regenerating terminals In the median eminence were seen by day 7 after Injecting 6O H D A , and by day 36 the fluorescence appearance of this region could not be distinguished from that in untreated animals or animals treated with vehicle alone. The plasma cortlcosterone concentrations (Fig l) in the untreated control ammals were similar to those reported by Chlappa and Fink 4 and suggest that the plasma samples were obtained under relatively 'unstressed' conditions, 1 e , m agreement with the results of studies on the prolactin response to stress 6,11,17, the time between removal of the animal cage from the housing room and decapitation was too short for a significant endocrine response to stress Fig 1 shows that injection of 6-OHDA, which produced depletion of CA terminals in the
ME-NIL, prevented the increases in the concentraUons of plasma prolactln that occurred in animals rejected with vehicle alone The increase in the plasma prolactin concentrations in the antmals treated with vehicle above those m untreated control animals may have been due to the long-term effects of the stress of the injection, a phenomenon which has been reported for other types of stressl,e,10,12 Iftlus is the case, the 6 - O H D A data (Fag. 1) suggest that the mechanism requires the functional integrity of the M E - N I L CA terminals, and that although DA inhibits prolactln releaseT, xg, the overall role of the M E - N I L CA neurons is facihtatory to prolactln release The stress of injection may also explain the increased concentrations of plasma corticosterone in animals treated with vehicle compared with those in untreated animals, although in this case 6-OHDA prevented the increase only on days 2 and 7 The finding of Increased plasma concentration of corticosterone 24 h after 6 - O H D A rejection agrees with that of Cuello et al 5, who interpreted this to mean
333
that the secretion of a d r e n o c o r t i c o t r o p h l n ( A C T H )
T S H (Fig 1) prowdes another example of the fact
was inhibited by hypothalam~c N A n e u r o n s that were destroyed by 6 - O H D A The marked increase in the plasma c o n c e n t r a t i o n of T S H 24 h after 6 - O H D A
that although the secretton of b o t h h o r m o n e s m a y be influenced by D A a n d t h y r o t r o p h l n releasing h o r m o n e the secretion of the two h o r m o n e s is fre-
suggests that the secretion of T S H may also be
quently dlssocmted The plasma LH a n d F S H con-
moderated by M E - N I L C A systems Several studtes have shown that, u n d e r stress, the
centrat~ons show that the C A n e u r o n s that terminate m the M E - N I L may have httle to do w~th
secretion of T S H ~s inversely related to the secretion of A C T H ~,9 However, the release of T S H and
controlling the basal secretion of the g o n a d o t r o p h m in the male
A C T H is not always Inversely related 14, a n d this ~s dlustrated by our results obtained 24 h after the mject~on of 6 - O H D A Stmdarly, although stress stimulates the release of both prolactm a n d A C T H z,~°, our data show that u n d e r relatwely unstressed cond~txons the secretion of these two h o r m o n e s ~s d~ssocrated The plasma concentrations of prolactm and
1 Ajlka, K S, Kalra, S P, Fawcett, C P and McCann, S M , The effect of stress and Nembutal on plasma levels of gonadotropm and prolactm m ovanectom~zed rats, Endocrmology, 90 (1972) 707-715 2 Brown, G M and Martin, J B, Cort~costerone, prolactin, and growth hormone responses to handhng and new environment m the rat, Prvehosom M e d , 36 (1974) 241-247 3 Brown-Grant, K , The hypothalamus and the thyroid gland, Brlt reed Bull, 16 (1960) 165-169 4 Chlappa, S A and Fmk, G , Hypothalamle lutemlzmg hormone releasing factor and cortlcotropm releasing act~vlty in relation to p~tultary and plasma hormone levels in male and female rats, J Endo~t, 72 (1977) 195-210 5 Cuello, A C, Shoemaker, W J and Ganong, W F , Effect o| 6-hydroxydopamlne on hypothalamlc norepmephrme and dopamme content, ultrastructure of the median eminence, and plasma cortlcosterone, Brain Re~ealch, 78 (1974) 57-69 6 Fenske, M and Wuttke, W , Effects of mtraventrlcular 6-hydroxydopamme mjectxonson serum prolactln and LH levels, absence of stress-Induced pRultary prolactm release, Btam Reseateh, 104 (1976) 63-70 7 Flnk, G and Geffen, L B, The hypothalamo-hypophysial system model for central peptlderglc and monoammergic transmission In R Porter (Ed), International Revtew o f Phvstology, Neurophystology 111, Vol
17,
University Park Press, Baltimore, MD, 1978, pp 148 8 Furness, J B, Heath, J W and Costa, M, Aqueous aldehyde (Faglu) methods lor the fluorescence hlstochemlcal locahzatlon of catecholammes and for ultrastructural studies of central nervous tissue, Htstochemtstr), 57 (1978) 285 295 9 Gufllemln, R , Hypothalamlc control of concomitant secretion of ACTH and TSH, Mem Soc Endoct , 17 (1968) 19-27
We are grateful to Drs G D Nlswender, L E Relchert, Jr a n d A F Parlow a n d the Pituitary H o r m o n e D i s t r i b u t i o n Agency of the N I A M D D (MD) for materials used m the radtoammunoassays, a n d the M R C (UK)andtheNH and M R C (Austraha) for financial support
10 Harms, P G , Langller, P and McCann, S M, Modification of stressqnduced prolactm release by dexamethasone or adrenalectomy, Endocrinology, 96 (1975) 475478 11 Mattheu, J A M and Van Pukeren, T A, Plasma prolactln m undisturbed cannulated male rats effects of perphenazme, frequent sampling, stress and castratton plus oestrone treatment, Acta endocJ 84 (1977) 51-6l 12 Nelll, J D , Comparison of plasma prolactm levels m cannulated and decapitated rats, Endoct mology, 90 (1972) 568-572 13 Nlswender, G D , Mldgley, A R , Jr, Monroe, S E and Relchert, L E, Jr, Radlolmmunoassay for rat lutemlzlng hormone with ant~ovlne LH serum and ovlne LH13q, Proc Soc erp Btol M e d , 128 (1968)807 811 14 Pamenter, R W and Hedge, G A, Inhibition of thyrotropm secretion by physiological levels of cortlcosterone, Lndottmolog}, 106 (1980) 162-166 15 Picketing, A J M C and Flnk, G , Do hypothalam~c regulating factors other than lutem~zmg hormone releasing tactors exert a priming effect9 J Endocr, 83 (1979) 53 59 16 Sarkar, D K , Smith, G C and Fmk, G , Effect ofmampulatmg central catecholammes on puberty and the surge of lutemlzmg hormone and lutem~zinghormone releasing hormone reduced by pregnant mare serum gonadotropm m female rats, Brain Research, 213 (1981) 335-349 17 Seggle, J A and Brown, G M, Twenty-four-hour lestmg prolactm levels m male rats the effect of septal les~onsand order of sacrifice, LndocHnology, 98 (1976) 1516-1522 18 Smith, G C, Courtney, P G , Wreford, N G M and Walker, M McD, Further studies on the effects o1 intravenously administered 6-hydroxydopamlne on the median eminence of the rat, Btam Research, 234 (1982) 101-110 19 Wemer, R I and Ganong, W 1-, Role of brain monoamines and h~stamme in regulation of anterior pRultary secretion, Ph)~lol Re~ , 58 (1978) 905-976
330
Effect of 6-hydroxydopamine lesions of the median eminence and neurointermodiate lobe on the secretion of pituitary hormones in the male rat G R A E M E C SMITH*, W JOHN SHEWARD** and GEORGE FINK**
Department o f Human Anatomy, South Park3 Road, Orford, OXI 3 Q X ( U K ) (Accepted May l lth, 1982)
Key words catecholamlnerg]c terminals - - medmn eminence - - neuromtermedmte lobe - - 6-hydroxydopamlne - prolactm - - cortlcosterone - - thyrotropm - - gonadotrophm - - Faglu fluorescence h~stochem]stry
The effect of 6-hydroxydopamme lesions of the catecholamme (CA) neurons of the medmn eminence and neuromtermedmte lobe (ME-NIL) on the secretion of several pltmtary hormones was studied m male rats The results show that the CA neurons of the ME-NIL facilitate prolactm release, inhibit the seeretmn of thyrotrophm and adrenocortlcotrophm and may not play an important role m the control of gonadotrophm secretion
The neurotoxm 6-hydroxydopamme (6-OHDA), when gaven intravenously, produces wathm 24 h a massave depletaon of catecholammes (CA) m the me&an eminence and neuromtermedmte lobe (MENIL) whale leaving largely unchanged the ammergac mnervatlon of the rest of the hypothalamus ~s Using this approach we stu&ed the effect of th~s specific and anatomically locahzed CA lesaon on the plasma concentration and p~tmtary content of prolactm, thyrotropan stamulatmg hormone (TSH), cortacosterone, luteamzmg hormone (LH) and folhcle stimulating hormone (FSH) The changes m the CA contents of the hypothalamus and ME-NIL were monatored by CA-fluorescence hlstochemastry All the experiments were carried out on one batch of male Wastar rats, purchased from Charles Rwer (U K ) Ltd , Margate, and housed under controlled hghtmg (hghts on 05 00-19 00 h) and temperature (22 °C) and given free access to &et 41B (E Daxon and Sons, Ware) and tap water At the beginning of the experiment the anamals weighed 250-300 g and had had 3 weeks to become accustomed to the con-
&tlons of the ammal house All experiments were carried out between 17 00 and 18 00 h The ammals were rejected 1 v (external jugular vem) under ether anesthesia with either 150 mg/kg 6-OHDA-HCI (Labkeml, Stockholm, 50 mg/mt m 0 l ~ ascorblc acid m 0 9 ~o sahne solution (vehlcle)) or with vehicle alone One group of ammals ('untreated control') was not rejected At 1, 2, 7, 14, 21 and 36 days after the rejection the anmaals were kdled by decapitation m batches of 6 at one ttme N o more than 2 mm elapsed between the tame that a cage of 6 anamals was removed qmetly from the antmal housing room and the tame that the last anamal an the group was decapitated The trunk blood was collected m heparm]zed 10 ml plastic tubes on ice, centrifuged, and the plasma stored at --20 °C untd assayed The patmtary glands were &ssected free, blotted, weaghed, and homogemzed an 10 ml 0 9 O/o saline soluUon by 15 strokes of a Potter Elvethem homogemzer Plasma and pttmtary LH was determmed by the method of Nlswender et al 13 as used previously m thas laboratory4 The plasma and p]tmtary con-
* On leave from Monash Umverslty, Department of Psychological Medtcme, Prince Henry's Hospital, Melbourne, Victoria 3004, Austraha ** Present address MRC Brain Metabohsm Umt, Umverslty Department of Pharmacology, 1 George Square, Edinburgh, Scotland EH8 9JZ, U K 0006-8993/82/0000-0000/$02
75 © 1982 Elsevier Blome&cal Press
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Fig 1 Mean ( :L S E M ) plasma concentrations of prolactln (NIAMDD-rat PrI-RP1), TSH (NIAMDD-rat TSH-RP1). cortxcosterone, LH (NIH-LH-SI8) and FSH (N1AMDD-rat FSH-RPI) in male rats killed by decapitation 1-36 days after the I v Injectionof either 6-OHDA or vehicle (0 1% ascorblc acid m 0 9 % saline) Also shown are hormone concentrations in animals treated similarly but not injected with either 6OHDA or vehicle alone The numbers of animals in each group are shown at the base of each column centratlons of FSH, prolactln and TSH were determined using the radloImmunoassay kits supplied by the Pltmtary Hormone Distribution Agency of the N I A M D D (Bethesda, MD) as used previously in this laboratory4,15 The concentration of plasma cortlcosterone was determined by competitive protern binding assay The significance of differences between means was determined by unpaired t-test Two furthei animals from each group were perfused and the brains subsequently processed for CA fluo-
rescence hlstochemlstry according to the Faglu techtuqueS, 16 The sectaons were examined by G C S and W J S without knowing the treatment to which the animal had been subjected Fig I shows that in the group InJected with 6O H D A the plasma concentrations of prolactin were not significantly different from those in the untreated control group However, in animals injected with vehicle alone the concentrations of prolactln were significantly elevated above the values in the untreated control group at 1 and 2 days and at 14 and 21 days after treatment (P < 0 05, 0 001, 0 01 and 0 025, respectively) The plasma concentration of TSH was significantly (P <~ 0 01) elevated 24 h after injecting 6-OHDA compared with the concentrations in either untreated control animals or ammals mjected with vehicle alone The plasma concentratlons of cort~costerone were slgmficantly (P < 0 02) elevated 24 h after injecting 6-OHDA compared with those in untreated control ammals On days 2 and 7 the concentrations of cortlcosterone in ammals Injected with 6-OHDA were lower (P < 0 01 and 0 02, respectively) than m vehicle treated animals In ammals treated with vehicle the concentrations of corticosterone were higher (P ~ 0 050 001 ) than m untreated control animals for all days except day l after rejection Treatment with e~ther 6O H D A or vehMe had no significant effect on the plasma concentrations of LH or FSH. except that at day 36 plasma FSH concentrations m both of these treatment groups were significantly lower than those in the untreated control group (P ~ 0 005 and P < 0 001, respectively) Fig 2 shows that for LH, FSH and TSH there were no major changes m pituitary content or concentration m the treated animals compared w~th those m the untreated control animals other than reduced values for LH and FSH on day 36 (P < 0 005-0 001) The pituitary content and concentration of prolactln m the treated animals were mostly lower than in the untreated control animals, and these changes were significant on days 1 and 7 (P < 0 05-0 005) The pituitary content and concentraUon of prolactln were sIgmficantly greater (P 0 02-0 005) at 14 and 21 days in the ammals treated with 6-OHDA compared w~th those m animals treated with vehicle alone The terminals in the ME-NIL were depleted of
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Fig 2 Mean (± S E M ) pltmtary hormone content (left) and concentration (right) m male rats (reference preparauons as m Fig 1) kdled by decapitation 1-36 days after i v rejection of either 6-OHDA or aseorbtc aod vehmlealone Values from untreated rats are also shown Number of ammals per group given at the base of each column
CA fluorescence 24 h after rejection of 6-OHDA The adjacent medlobasal hypothalamus was moderately depleted at 24 h, but was of normal appearance at 48 h, as was the rest of the hypothalamus at all times studied Regenerating terminals In the median eminence were seen by day 7 after Injecting 6O H D A , and by day 36 the fluorescence appearance of this region could not be distinguished from that in untreated animals or animals treated with vehicle alone. The plasma cortlcosterone concentrations (Fig l) in the untreated control ammals were similar to those reported by Chlappa and Fink 4 and suggest that the plasma samples were obtained under relatively 'unstressed' conditions, 1 e , m agreement with the results of studies on the prolactin response to stress 6,11,17, the time between removal of the animal cage from the housing room and decapitation was too short for a significant endocrine response to stress Fig 1 shows that injection of 6-OHDA, which produced depletion of CA terminals in the
ME-NIL, prevented the increases in the concentraUons of plasma prolactln that occurred in animals rejected with vehicle alone The increase in the plasma prolactin concentrations in the antmals treated with vehicle above those m untreated control animals may have been due to the long-term effects of the stress of the injection, a phenomenon which has been reported for other types of stressl,e,10,12 Iftlus is the case, the 6 - O H D A data (Fag. 1) suggest that the mechanism requires the functional integrity of the M E - N I L CA terminals, and that although DA inhibits prolactln releaseT, xg, the overall role of the M E - N I L CA neurons is facihtatory to prolactln release The stress of injection may also explain the increased concentrations of plasma corticosterone in animals treated with vehicle compared with those in untreated animals, although in this case 6-OHDA prevented the increase only on days 2 and 7 The finding of Increased plasma concentration of corticosterone 24 h after 6 - O H D A rejection agrees with that of Cuello et al 5, who interpreted this to mean
333
that the secretion of a d r e n o c o r t i c o t r o p h l n ( A C T H )
T S H (Fig 1) prowdes another example of the fact
was inhibited by hypothalam~c N A n e u r o n s that were destroyed by 6 - O H D A The marked increase in the plasma c o n c e n t r a t i o n of T S H 24 h after 6 - O H D A
that although the secretton of b o t h h o r m o n e s m a y be influenced by D A a n d t h y r o t r o p h l n releasing h o r m o n e the secretion of the two h o r m o n e s is fre-
suggests that the secretion of T S H may also be
quently dlssocmted The plasma LH a n d F S H con-
moderated by M E - N I L C A systems Several studtes have shown that, u n d e r stress, the
centrat~ons show that the C A n e u r o n s that terminate m the M E - N I L may have httle to do w~th
secretion of T S H ~s inversely related to the secretion of A C T H ~,9 However, the release of T S H and
controlling the basal secretion of the g o n a d o t r o p h m in the male
A C T H is not always Inversely related 14, a n d this ~s dlustrated by our results obtained 24 h after the mject~on of 6 - O H D A Stmdarly, although stress stimulates the release of both prolactm a n d A C T H z,~°, our data show that u n d e r relatwely unstressed cond~txons the secretion of these two h o r m o n e s ~s d~ssocrated The plasma concentrations of prolactm and
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University Park Press, Baltimore, MD, 1978, pp 148 8 Furness, J B, Heath, J W and Costa, M, Aqueous aldehyde (Faglu) methods lor the fluorescence hlstochemlcal locahzatlon of catecholammes and for ultrastructural studies of central nervous tissue, Htstochemtstr), 57 (1978) 285 295 9 Gufllemln, R , Hypothalamlc control of concomitant secretion of ACTH and TSH, Mem Soc Endoct , 17 (1968) 19-27
We are grateful to Drs G D Nlswender, L E Relchert, Jr a n d A F Parlow a n d the Pituitary H o r m o n e D i s t r i b u t i o n Agency of the N I A M D D (MD) for materials used m the radtoammunoassays, a n d the M R C (UK)andtheNH and M R C (Austraha) for financial support
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