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Inhibition of pulsatile luteinizing hormone release by morphine microinjected into mesencephalic dorsal raphe
Life Sciences, Vol. 30, pp. 1473-1478 Printed in the U.S.A.
Pergamon Press
INHIBITION OF PULSATILE LUTEINIZING HORMONERELEASE BY MORPHINE MICROINJECTED INTO MESENCEPHALIC DORSAL RAPHE James H. Johnson, George T. Maughan and t i l i a
Anderhub
Department of Anatomy Medical College of V i r g i n i a / V i r g i n i a Commonwealth University Richmond, V i r g i n i a 23298 (Received in final form February 16, 1982)
Summary Blood samples were collected via jugular catheters from ovariectomized rats at lO-minute i n t e r v a l s for one hour before and two hours a f t e r microinjection of 0.5 ~I of e i t h e r saline vehicle or morphine sulfate (I0 ~g) into the dorsal raphe nucleus (DRN) or adjacent periaqueductal gray by means of chronically-implanted guide cannulae. LH was measured by radioimmunoassay and mean p r e - i n j e c t i o n and mean p o s t - i n j e c t i o n values were compared for each rat (t t e s t ) as well as f o r each treatment group (paired t t e s t ) . Neither saline in DRN nor morphine at other sites s i g n i f i c a n t l y altered c i r c u l a t i n g LH. A s i g n i f i c a n t decrease in LH was observed following i n j e c t i o n of morphine into DRN. This e f f e c t of morphine was prevented by pre treatment of the animals with the narcotic antagonist naltrexone (I0 mg/kg i . v . ) , indicating the involvement of opiate receptors. These results indicate that DRN is one s i t e at which systemicallyadministered morphine might act, and suggest the p o s s i b i l i t y of p a r t i c i p a t i o n of this mechanism in modulation of LH release by endogenous opioids. Administration of morphine ( 1 , 2 ) , methadone (3) or endogenous opioids (1,2) to rats has been found to decrease c i r c u l a t i n g l u t e i n i z i n g hormone (LH). A physiological role f o r the opioids is suggested by the a b i l i t y of the narcotic antagonist naloxone to increase LH (4,5). Interaction with the serotonergic system is indicated since this e f f e c t of naloxone can be diminished by 5-hydroxytryptophan and augmented by para-chlorophenylalanine (6). Since the dorsal raphe nucleus (DRN) contains both a high concentration of serotonin and neurons which respond to opiates, selection of this s i t e for. study of e f f e c t s of neuroanatomically localized i n j e c t i o n s of morphine was obvious. Moreover, although we have reported that the phasic release of LH which induces ovulation can be blocked by microinjection of morphine at this s i t e (7), tonic LH release is subject to d i f f e r e n t control mechanisms (8). In the present report we explore the p o s s i b i l i t y that DRN may represent a s i t e of action for morphine's i n h i b i t o r y e f f e c t on tonic LH release in ovariectomized rats.
0024-3205/82/171473-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1474
Morphine in DRN Inhibits LH
Vol. 30, No. 17, 1982
Materials and Methods Sprague-Dawley rats obtained from Flow Laboratories (Dublin, Va.) or from our laboratory colony were ovariectomized at least four weeks p r i o r to use. They were housed I-5 per cage and provided Purina Laboratory Chow and water ad l i b i t u m . A 27 gauge guide cannula was implanted s t e r e o t a x i c a l l y aimed at a point 1 mm dorsal to DRN by coordinates (A 0.0, LO.I, V-I.5) modified from the atlas of Pellegrino et a l . (9) f o r use in female rats weighing 275-375 g. The guide cannula was plugged with a s t y l e t u n t i l the experiment 5-7 days l a t e r . Two to four days before the i n i t i a l experiment a polyvinyl cannula was inserted into the r i g h t external jugular vein f o r c o l l e c t i o n of blood samples from the unanesthetized animal. Beginning I0 minutes a f t e r an i n j e c t i o n of saline (0.3 ml), blood samples (200 u l ) were obtained at lO-minute i n t e r v a l s for 3 hours, and were replaced with saline. Removal of t h i s volume of blood over a 3-hour period has been found previously to r e s u l t in a decrease of 10-20% in hematocrit (unpublished observations) but neither LH (3) nor s t r e s s - s e n s i t i v e p r o l a c t i n (I0) concentrations are s i g n i f i c a n t l y affected. Following c o l l e c t i o n of the seventh sample the s t y l e t was removed and the 33 gauge i n j e c t i o n cannula was inserted. The i n j e c tion cannula was constructed so as to extend 1 mm beyond the guide cannula to the level of DRN. Injection of 0.5 ul saline vehicle or morphine s u l f a t e (I0 ug) was made via p o l y v i n y l tubing connected to a I0 ul Hamilton syringe by the method of Jacquet ( I I ) . A f t e r 2-3 minutes the i n j e c t i o n cannula was removed and the s t y l e t was replaced. In many cases i t was possible to use the same animal f o r a second experiment 10-14 days l a t e r , an i n t e r v a l at which tolerance to the morphine is not a s i g n i f i c a n t f a c t o r (12). In some animals which had previously been shown to respond to morphine, the long-acting narcotic antagonist naltrexone (I0 mg/kg i . v . ) was administered in place of saline I0 minutes p r i o r to collection of the f i r s t blood sample in order to establish that this e f f e c t of morphine is mediated by opiate receptors. LH was estimated in whole blood or in plasma by double antibody radioimmunoassay as described previously ( I 0 ) . Effectiveness of the microinjection was assessed in each animal by comparing mean p r e - i n j e c t i o n and p o s t - i n j e c t i o n hormone concentrations using the t t e s t . Proportions of each group responding were compared using the Fisher exact p r o b a b i l i t y t e s t . Significance of d i f f e r ences between p r e - i n j e c t i o n and p o s t - i n j e c t i o n means for each treatment group were assessed using the paired t t e s t since each animal served as i t s own control. At the conclusion of the f i n a l experiment each animal was k i l l e d by barbiturate overdose and perfused through the heart with 10% formalin. Frontal sections of the brain 50 um in thickness were cut frozen and stained with carbol fuchsin f o r v e r i f i c a t i o n of i n j e c t i o n s i t e s . Results As shown in Fig. ! , pulses of LH release observed i n i t i a l l y were i n h i b i t e d following the microinjection of morphine into DRN or adjacent periaqueductal gray (VPAG), and resumed a f t e r a variable i n t e r v a l of time. Mean LH concent r a t i o n s were s i g n i f i c a n t l y reduced following morphine administration in 7 ~ of 26 animals tested ( t t e s t ; significance ranged from p < .05 to p < .0001) in spite of v a r i a b i l i t y r e s u l t i n g from p u l s a t i l e release during both p r e - i n j e c t i o n and recovery periods. The robustness of the e f f e c t is reflected in the s i g n i f i cance of the decrease in LH concentrations for the group as a whole (p < .0001, paired t t e s t ) . Microinjection of saline into DRN or VPAG, or of morphine into adjacent s i t e s was i n e f f e c t i v e (Table I ) . Fig. 2 i l l u s t r a t e s placements of e f f e c t i v e and i n e f f e c t i v e i n j e c t i o n s . Pretreatment with naltrexone antagonized the e f f e c t of morphine in animals shown previously to respond to the narcotic (Table I ) .
Vol. 30, No. 17, 1982
Morphine in DRN Inhibits LH
1475
DECREASED PLASMA LH FOLLOWING MICROINJECTION OF MORPHINE INTO DORSAL RAPHE NUCLEUS IN .~ REPRESENTATIVE RATS
400
."X\\ 300
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MORPHINE
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130 140 I$0
TIME (MINUTES)
Fig. I. LH responses to morphine in 3 representative rats pretreated with saline.
I
,
160
170
1
180
1476
Morphine in DRN Inhibits LH
Vol. 30, No. 17, 1982
TABLE ! Effects of Mesencephalic Microinjections of Saline or Morphine on Circulating LH Substance Injected
Location
Pretreatment
% in which LH Decreased (n Tested)
Saline b Morphine Morphine Morphine
DRNa Not DRN DRN DRN
Saline Saline Saline d Naltrexone
0(6) 12.5(8) c 76.9(26) 20.0(5)
a Dorsal raphe nucleus or adjacent ventral periaqueductal gray b I0 ug morphine sulfate in 0.5 ~I physiological saline c Significantly different from responses of other groups (p < 0.026; Fisher Exact Probability) d Naltrexone, I0 mg/kg i . v .
A350u
A 620jJ
mr
FLM FOR~
AI60~
o SALINE PRETREATMENT NALTREXONE PRETREATMENT 0 I (SOLID SYMBOLS INDICATE THAT LH DECREASED )
PiOO2
2
3
3
mm
Fig. 2. Approximate sites of microinjections of morphine indicated at 4 A-P levels (u) modified from the atlas of Konig and Klippel (14). Pretreatment is coded in symbols: circles for saline, triangles for naltrexone (lOmg/kg). Solid symbols signify that mean post-injection LH was s i g n i f i c a n t l y less than the pre-injection mean.
Vol. 30, No. 17, 1982
Morphine in DRN Inhibits LH
1477
Discussion The present results indicate that the mesencephalon contains an opiatesensitive system capable of i n h i b i t i n g tonic LH release in ovariectomized rats. The s i m i l a r i t y to our e a r l i e r report that such injections of morphine i n h i b i t ovulation (7) suggests that effects on both tonic and phasic release of LH are mediated, at least in part, by the same neuroanatomical substrate. Effective i n j e c t i o n s of morphine appeared to be localized to the immediate v i c i n i t y of the dorsal raphe nucleus (Fig. 2). I n e f f e c t i v e i n j e c t i o n s located within 0.8 mm of DRN are in agreement with the findings of Lomax (14) that 90% of a 1 ~I i n j e c tion of radioactive morphine remains within a 1 mm envelope of the s i t e of application. S p e c i f i c i t y of the phenomenon is indicated by the i n a b i l i t y of saline to depress LH concentrations. Mediation of morphine's e f f e c t by opiate receptors was confirmed by blockade with the long-acting narcotic antagonist naltrexone. Although the e f f e c t of administration of naltrexone alone in the same paradigm as morphine was not tested in this experiment, the lower dosage (Img/kg) reported previously had no s i g n i f i c a n t e f f e c t (3). Increased concentrations of LH in blood obtained at a single time point 15 or 30 minutes a f t e r naloxone administration have been observed in adult male (3) as well as in prepubertal (4,5) rats. In contrast, in the present experiment, mean pre-morphine LH concentrations represented 7 samples obtained at lO-minute i n t e r v a l s , and were not s i g n i f i c a n t l y d i f f e r e n t in s a l i n e - and naltrexone-pretreated animals. Thus i t is u n l i k e l y that antagonism of morphine's e f f e c t in these experiments is clouded by an independent influence of naltrexone on the release of LH. E l e c t r i c a l stimulation of DRN has been shown to have a similar i n h i b i t o r y e f f e c t on LH release (15, 16). This i n h i b i t i o n is mediated by serotonin (15). The l o c a l i z a t i o n to DRN of morphine's e f f e c t on LH, together with i t s s i m i l a r i t y to the response to e l e c t r i c a l stimulation, strongly suggest the involvement of serotonin in this phenomenon as well. Existence of enkephalinergic inputs to ascending serotonergic neurons would be consistent with the i n a b i l i t y of n a l t r e x one to antagonize the i n h i b i t i o n of LH release r e s u l t i n g from e l e c t r i c a l stimul a t i o n of DRN (16). Modulation of the e f f e c t of naloxone on LH by manipulation of serotonin (6) f u r t h e r supports this hypothesis. Although the present experiments neither rule out other sites of action nor d e f i n i t i v e l y establish DRN as such, the implication is clear that at least one possible locus of action of morphine administered systemically has been i d e n t i f i e d . More importantly, the i n h i b i t o r y influence of endogenous opioids on LH as disclosed by naloxone (4,5) and the presence of such substances in the midbrain (17) suggest a physiological role f o r this pathway in c o n t r o l l i n g the release of LH. Acknowledgements The authors acknowledge the s k i l l e d technical assistance of Ms. Lynn Davis, Ms. Letia McDaniel and Ms. Elizabeth Jackson, and thank Ms. Ellen Hawthorne f o r preparing the manuscript. The materials used in the LH assays were generous g i f t s of Dr. A.F. Parlow and the Hormone D i s t r i b u t i o n Program of NIAMDD. We thank Mallinkrodt I n c . , St. Louis, f o r the morphine s u l f a t e . Support was received from grant HD 12165 from NICHHD. References 1.
J.F. BRUNI, D. VAN VUGT, S. MARSHALL and J. MEITES, L i f e Sciences 21 461-466 (1977).
1478
2.
Morphine in DRN Inhibits LH
Vol. 30, No. 17, 1982
3.
J. MEITES, J.F. BRUNI, D.A. VAN VUGT and A.F. SMITH, Life Sciences 24 1325-1336 (1979). J-~.H. JOHNSON and J.A. ROSECRANS, Res. Commun. Subs. Abuse, 2 305-312
4. 5.
T. IEIRI, H.T. CHEN and J. MEITES, Neuroendocrinology 29 288-292 (1979). M.S. BLANK, A.E. PANERAI and H.G. FRIESEN, J. Endocrinol. 85 307-315
6. 7.
T. IEIRI, H.T. CHEN and O. MEITES, Life Sciences 26 1269-1274 (1980). G.T. MAUGHAN, J.H. JOHNSONand J.A. ROSECRANS, Brain Res. 155 413-417 (1978). B. HALASZ, Frontiers in Neuroendocrinology, 1969, W.F. GANONGand L. MARTINI, eds., pp 307-342, Oxford University Press, New York (1969). L.S. PELLEGRINO, A.S. PELLEGRINO and A.J. CUSHMAN, A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York (1979). J.H. JOHNSON, A.R. SWAJKOSKI and L. ANDERHUB, Exp. Neurol. 74 419-429 (1981). Y.F. JACQUET, Methods in Narcotic Research, S. Ehrenpreis and A. Neidle, eds., pp 35-57, Marcel Dekker, New York (1975). T.L. YAKSH, J.C. YEUNG and T. RUDY, Brain Res. 114 83-103 (1976). P. LOMAX, Experientia 22 249-250 (1966). J.F.R. KONIG and R.A. KLIPPEL, The Rat Brain, Williams and Wilkins, Baltimore (1963). G.W. ARENDASHand R.V. GALLO, Endocrinology 102 1199-1206 (1978). J.H. JOHNSON, A.R. SWAJKOSKI and D.J. MAYER, The Anterior P i t u i t a r y , A.S. BHATNAGAR, ed., Raven Press, New York, In Press (1982). R. SIMANTOV, M.J. KUHAR, G.R. UHL and S.H. SNYDER, Proc. Natl. Acad. Sci. 74 2167-2171 (1977).
(1981).
(1980).
8. 9. I0. II. 12. 13. 14. 15. 16. 17.
Pergamon Press
INHIBITION OF PULSATILE LUTEINIZING HORMONERELEASE BY MORPHINE MICROINJECTED INTO MESENCEPHALIC DORSAL RAPHE James H. Johnson, George T. Maughan and t i l i a
Anderhub
Department of Anatomy Medical College of V i r g i n i a / V i r g i n i a Commonwealth University Richmond, V i r g i n i a 23298 (Received in final form February 16, 1982)
Summary Blood samples were collected via jugular catheters from ovariectomized rats at lO-minute i n t e r v a l s for one hour before and two hours a f t e r microinjection of 0.5 ~I of e i t h e r saline vehicle or morphine sulfate (I0 ~g) into the dorsal raphe nucleus (DRN) or adjacent periaqueductal gray by means of chronically-implanted guide cannulae. LH was measured by radioimmunoassay and mean p r e - i n j e c t i o n and mean p o s t - i n j e c t i o n values were compared for each rat (t t e s t ) as well as f o r each treatment group (paired t t e s t ) . Neither saline in DRN nor morphine at other sites s i g n i f i c a n t l y altered c i r c u l a t i n g LH. A s i g n i f i c a n t decrease in LH was observed following i n j e c t i o n of morphine into DRN. This e f f e c t of morphine was prevented by pre treatment of the animals with the narcotic antagonist naltrexone (I0 mg/kg i . v . ) , indicating the involvement of opiate receptors. These results indicate that DRN is one s i t e at which systemicallyadministered morphine might act, and suggest the p o s s i b i l i t y of p a r t i c i p a t i o n of this mechanism in modulation of LH release by endogenous opioids. Administration of morphine ( 1 , 2 ) , methadone (3) or endogenous opioids (1,2) to rats has been found to decrease c i r c u l a t i n g l u t e i n i z i n g hormone (LH). A physiological role f o r the opioids is suggested by the a b i l i t y of the narcotic antagonist naloxone to increase LH (4,5). Interaction with the serotonergic system is indicated since this e f f e c t of naloxone can be diminished by 5-hydroxytryptophan and augmented by para-chlorophenylalanine (6). Since the dorsal raphe nucleus (DRN) contains both a high concentration of serotonin and neurons which respond to opiates, selection of this s i t e for. study of e f f e c t s of neuroanatomically localized i n j e c t i o n s of morphine was obvious. Moreover, although we have reported that the phasic release of LH which induces ovulation can be blocked by microinjection of morphine at this s i t e (7), tonic LH release is subject to d i f f e r e n t control mechanisms (8). In the present report we explore the p o s s i b i l i t y that DRN may represent a s i t e of action for morphine's i n h i b i t o r y e f f e c t on tonic LH release in ovariectomized rats.
0024-3205/82/171473-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1474
Morphine in DRN Inhibits LH
Vol. 30, No. 17, 1982
Materials and Methods Sprague-Dawley rats obtained from Flow Laboratories (Dublin, Va.) or from our laboratory colony were ovariectomized at least four weeks p r i o r to use. They were housed I-5 per cage and provided Purina Laboratory Chow and water ad l i b i t u m . A 27 gauge guide cannula was implanted s t e r e o t a x i c a l l y aimed at a point 1 mm dorsal to DRN by coordinates (A 0.0, LO.I, V-I.5) modified from the atlas of Pellegrino et a l . (9) f o r use in female rats weighing 275-375 g. The guide cannula was plugged with a s t y l e t u n t i l the experiment 5-7 days l a t e r . Two to four days before the i n i t i a l experiment a polyvinyl cannula was inserted into the r i g h t external jugular vein f o r c o l l e c t i o n of blood samples from the unanesthetized animal. Beginning I0 minutes a f t e r an i n j e c t i o n of saline (0.3 ml), blood samples (200 u l ) were obtained at lO-minute i n t e r v a l s for 3 hours, and were replaced with saline. Removal of t h i s volume of blood over a 3-hour period has been found previously to r e s u l t in a decrease of 10-20% in hematocrit (unpublished observations) but neither LH (3) nor s t r e s s - s e n s i t i v e p r o l a c t i n (I0) concentrations are s i g n i f i c a n t l y affected. Following c o l l e c t i o n of the seventh sample the s t y l e t was removed and the 33 gauge i n j e c t i o n cannula was inserted. The i n j e c tion cannula was constructed so as to extend 1 mm beyond the guide cannula to the level of DRN. Injection of 0.5 ul saline vehicle or morphine s u l f a t e (I0 ug) was made via p o l y v i n y l tubing connected to a I0 ul Hamilton syringe by the method of Jacquet ( I I ) . A f t e r 2-3 minutes the i n j e c t i o n cannula was removed and the s t y l e t was replaced. In many cases i t was possible to use the same animal f o r a second experiment 10-14 days l a t e r , an i n t e r v a l at which tolerance to the morphine is not a s i g n i f i c a n t f a c t o r (12). In some animals which had previously been shown to respond to morphine, the long-acting narcotic antagonist naltrexone (I0 mg/kg i . v . ) was administered in place of saline I0 minutes p r i o r to collection of the f i r s t blood sample in order to establish that this e f f e c t of morphine is mediated by opiate receptors. LH was estimated in whole blood or in plasma by double antibody radioimmunoassay as described previously ( I 0 ) . Effectiveness of the microinjection was assessed in each animal by comparing mean p r e - i n j e c t i o n and p o s t - i n j e c t i o n hormone concentrations using the t t e s t . Proportions of each group responding were compared using the Fisher exact p r o b a b i l i t y t e s t . Significance of d i f f e r ences between p r e - i n j e c t i o n and p o s t - i n j e c t i o n means for each treatment group were assessed using the paired t t e s t since each animal served as i t s own control. At the conclusion of the f i n a l experiment each animal was k i l l e d by barbiturate overdose and perfused through the heart with 10% formalin. Frontal sections of the brain 50 um in thickness were cut frozen and stained with carbol fuchsin f o r v e r i f i c a t i o n of i n j e c t i o n s i t e s . Results As shown in Fig. ! , pulses of LH release observed i n i t i a l l y were i n h i b i t e d following the microinjection of morphine into DRN or adjacent periaqueductal gray (VPAG), and resumed a f t e r a variable i n t e r v a l of time. Mean LH concent r a t i o n s were s i g n i f i c a n t l y reduced following morphine administration in 7 ~ of 26 animals tested ( t t e s t ; significance ranged from p < .05 to p < .0001) in spite of v a r i a b i l i t y r e s u l t i n g from p u l s a t i l e release during both p r e - i n j e c t i o n and recovery periods. The robustness of the e f f e c t is reflected in the s i g n i f i cance of the decrease in LH concentrations for the group as a whole (p < .0001, paired t t e s t ) . Microinjection of saline into DRN or VPAG, or of morphine into adjacent s i t e s was i n e f f e c t i v e (Table I ) . Fig. 2 i l l u s t r a t e s placements of e f f e c t i v e and i n e f f e c t i v e i n j e c t i o n s . Pretreatment with naltrexone antagonized the e f f e c t of morphine in animals shown previously to respond to the narcotic (Table I ) .
Vol. 30, No. 17, 1982
Morphine in DRN Inhibits LH
1475
DECREASED PLASMA LH FOLLOWING MICROINJECTION OF MORPHINE INTO DORSAL RAPHE NUCLEUS IN .~ REPRESENTATIVE RATS
400
."X\\ 300
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MORPHINE
2o
!
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(10/ag, 0.5 pl)
IO
!
i
i
i
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!
30
40
50
60
1
I
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|
70
80
90
100
I!0
120
I
I
t
130 140 I$0
TIME (MINUTES)
Fig. I. LH responses to morphine in 3 representative rats pretreated with saline.
I
,
160
170
1
180
1476
Morphine in DRN Inhibits LH
Vol. 30, No. 17, 1982
TABLE ! Effects of Mesencephalic Microinjections of Saline or Morphine on Circulating LH Substance Injected
Location
Pretreatment
% in which LH Decreased (n Tested)
Saline b Morphine Morphine Morphine
DRNa Not DRN DRN DRN
Saline Saline Saline d Naltrexone
0(6) 12.5(8) c 76.9(26) 20.0(5)
a Dorsal raphe nucleus or adjacent ventral periaqueductal gray b I0 ug morphine sulfate in 0.5 ~I physiological saline c Significantly different from responses of other groups (p < 0.026; Fisher Exact Probability) d Naltrexone, I0 mg/kg i . v .
A350u
A 620jJ
mr
FLM FOR~
AI60~
o SALINE PRETREATMENT NALTREXONE PRETREATMENT 0 I (SOLID SYMBOLS INDICATE THAT LH DECREASED )
PiOO2
2
3
3
mm
Fig. 2. Approximate sites of microinjections of morphine indicated at 4 A-P levels (u) modified from the atlas of Konig and Klippel (14). Pretreatment is coded in symbols: circles for saline, triangles for naltrexone (lOmg/kg). Solid symbols signify that mean post-injection LH was s i g n i f i c a n t l y less than the pre-injection mean.
Vol. 30, No. 17, 1982
Morphine in DRN Inhibits LH
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Discussion The present results indicate that the mesencephalon contains an opiatesensitive system capable of i n h i b i t i n g tonic LH release in ovariectomized rats. The s i m i l a r i t y to our e a r l i e r report that such injections of morphine i n h i b i t ovulation (7) suggests that effects on both tonic and phasic release of LH are mediated, at least in part, by the same neuroanatomical substrate. Effective i n j e c t i o n s of morphine appeared to be localized to the immediate v i c i n i t y of the dorsal raphe nucleus (Fig. 2). I n e f f e c t i v e i n j e c t i o n s located within 0.8 mm of DRN are in agreement with the findings of Lomax (14) that 90% of a 1 ~I i n j e c tion of radioactive morphine remains within a 1 mm envelope of the s i t e of application. S p e c i f i c i t y of the phenomenon is indicated by the i n a b i l i t y of saline to depress LH concentrations. Mediation of morphine's e f f e c t by opiate receptors was confirmed by blockade with the long-acting narcotic antagonist naltrexone. Although the e f f e c t of administration of naltrexone alone in the same paradigm as morphine was not tested in this experiment, the lower dosage (Img/kg) reported previously had no s i g n i f i c a n t e f f e c t (3). Increased concentrations of LH in blood obtained at a single time point 15 or 30 minutes a f t e r naloxone administration have been observed in adult male (3) as well as in prepubertal (4,5) rats. In contrast, in the present experiment, mean pre-morphine LH concentrations represented 7 samples obtained at lO-minute i n t e r v a l s , and were not s i g n i f i c a n t l y d i f f e r e n t in s a l i n e - and naltrexone-pretreated animals. Thus i t is u n l i k e l y that antagonism of morphine's e f f e c t in these experiments is clouded by an independent influence of naltrexone on the release of LH. E l e c t r i c a l stimulation of DRN has been shown to have a similar i n h i b i t o r y e f f e c t on LH release (15, 16). This i n h i b i t i o n is mediated by serotonin (15). The l o c a l i z a t i o n to DRN of morphine's e f f e c t on LH, together with i t s s i m i l a r i t y to the response to e l e c t r i c a l stimulation, strongly suggest the involvement of serotonin in this phenomenon as well. Existence of enkephalinergic inputs to ascending serotonergic neurons would be consistent with the i n a b i l i t y of n a l t r e x one to antagonize the i n h i b i t i o n of LH release r e s u l t i n g from e l e c t r i c a l stimul a t i o n of DRN (16). Modulation of the e f f e c t of naloxone on LH by manipulation of serotonin (6) f u r t h e r supports this hypothesis. Although the present experiments neither rule out other sites of action nor d e f i n i t i v e l y establish DRN as such, the implication is clear that at least one possible locus of action of morphine administered systemically has been i d e n t i f i e d . More importantly, the i n h i b i t o r y influence of endogenous opioids on LH as disclosed by naloxone (4,5) and the presence of such substances in the midbrain (17) suggest a physiological role f o r this pathway in c o n t r o l l i n g the release of LH. Acknowledgements The authors acknowledge the s k i l l e d technical assistance of Ms. Lynn Davis, Ms. Letia McDaniel and Ms. Elizabeth Jackson, and thank Ms. Ellen Hawthorne f o r preparing the manuscript. The materials used in the LH assays were generous g i f t s of Dr. A.F. Parlow and the Hormone D i s t r i b u t i o n Program of NIAMDD. We thank Mallinkrodt I n c . , St. Louis, f o r the morphine s u l f a t e . Support was received from grant HD 12165 from NICHHD. References 1.
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