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Proc. Natl. Acad. Scs. USA Vol. 76, No. 6, pp.3006-3009, June 1979 Neurobiology and endorphins modulate turnover in rat median eminence (///opiates) SCOTT N. DEYO*, ROBERT M. SWIFT*t, AND RICHARD J. MILLER*t *Department of Pharmacological and Physiological Sciences and the tPritzker School of Medicine, The University of Chicago, Chicago, Illinois 60637 Communicated by Elwood V. Jensen, March 5, 1979

ABSTRACT There is evidence that some of the actions of compounds on pituitary prolactin are mediated by effects on both endogenous and exogenous (e.g., stimulation of hypothalamic dopaminergic systems. In this paper we report prolactin release) are mediated by interaction with catechol- aminergic systems. Morphine (1.67, 5, and 15 mg/kg of body that the endorphins ond morphine are able to slow the activity weight, intraperitoneally) altered dopamine turnover as mea- of hypothalamic dopaminergic neurons as reflected in mea- sured by the a-methyl-p-tyrosine method in the median emi- surements of hypothalamic dopamine turnover, and we hy- nence, neostriatum, and frontal cortex of male Sprague-Dawley pothesize that this action of is responsible for their rats. The turnover rate of dopamine was reduced in the median releasing effect on pituitary prolactin. eminence and frontal cortexbut accelerated in the neostriatum. In the frontal cortex all doses were effective in decreasing do- pamine turnover; however, in the median eminence the lowest METHODS AND MATERIALS dose of morphine did not significantly alter dopamine turnover. All three doses accelerated dopamine turnover in the neostri- Experiments were performed on male Sprague-Dawley rats atum. Naloxone effectively reversed the effects of morphine at between 240 and 290 grams. Animals were housed in a 12-hr all doses in all areas, whereas it had no effect on turnover light-dark cycle with food and water supplied ad lib. All drugs when given alone. In the median eminence, neostriatum, and were prepared in physiological saline. Morphine sulfate was frontal cortex, intraventricular injection of D-Ala2,D-Leu5J prepared in three concentrations: 1.67, 5.0, and 15.0 mg/ml. enkephalin (25 Mug) or fl-endorphin (15 ,g) produced the same a-Methyl-p-tyrosine methyl ester (AMT) was prepared in a effects on dopamine turnover as morphine. The actions of these were blocked by naloxone. It is hypothesized that op- concentration of 200 mg/ml. [D-Ala2,D-Leu5]enkephalin was iates and peptides increase prolactin release by reducing prepared in a concentration of 5 mg/ml and f3-endorphin was the activity of the tuberoinfundibular dopaminergic system. prepared in a concentration of 3 mg/ml. Fresh solutions were prepared each day of the experiment. Since the discovery of the endorphins and the , Morphine sulfate (1.67,5.0, and 15.0 mg/kg of body weight), peptides with opiate activity, intensive investigations naloxone hydrochloride-(5.0, 10.0, and 20.0 mg/kg), and AMT have been carried out in order to define the functions of these (400mg/kg) were injected intraperitoneally. [D-Ala2,D-Leu5]- peptides in the nervous system. It is becoming evident that one enkephalin (25 Mg) and f3-endorphin (15 Mg) were injected such important role may be in the control of the release of through polyethylene cannulas that had been previously im- from the . It is well known from planted in the lateral ventricle. Because of the short half-life previous investigations on the of narcotics that of the naturally occurring enkephalins, we employed the analog drugs affect the secretion of several pituitary hormones, [D-Ala2,D-Leu5]enkephalin, which is less susceptible to enzy- including prolactin, growth , , and adre- matic inactivation (11-13) but is pharmacologically similar to nocorticotropic hormone (ACTH). Morphine is a potent releaser the native forms of enkephalin (13). All animals, except for the of prolactin, , and vasopressin (1-4). Recently, saline controls, were injected with AMT. Injections of the op- it has been shown that enkephalin analogs can also increase ioids followed the AMT injection by 10 min and naloxone was blood prolactin levels (5). injected 10 min prior to AMT. All animals that did not receive Immunohistochemical studies on enkephalin distribution a given drug were injected with the saline vehicle at the cor- within the brain have defined several potential enkephalinergic responding time points. The animals were sacrificed by de- pathways that may serve to regulate the release of these hor- capitation at 15 min after injection of the endorphins and at 30, mones (6). Thus, an enkephalin-containing neuronal system 45, and 60 min after morphine injection. projecting from the paraventricular nucleus of the hypothala- After sacrifice, were immediately removed and dis- mus to the pars nervosa of the pituitary may serve to regulate section of the various regions was performed on an ice-cooled the release of vasopressin (7). In addition, it has been shown that glass plate. The median eminence was dissected as described the median eminence contains a large number of enkephalin- by Moore and Gudelsky (14), and the neostriatum was dissected ergic nerve endings (6). Because it is known that endorphins as described by Hoffman et al. (15). The frontal cortex was and morphine do not produce their releasing actions on pitu- dissected by making a cut about 2 mm anterior to the junction itary prolactin by direct action on pituitary lactotrophs (8, 9), of the olfactory tubercle. The underlying olfactory tubercle was it therefore appears that this action of these opioid compounds removed. is mediated at the level of the hypothalamus. Dopamine was assayed by the method of Ben-Jonathan and Because the main hypothalamic control of prolactin secretion Porter (16), which has a sensitivity limit of 5-10 pg. The con- is mediated by hypothalamic dopaminergic neurons (10), we centration of protein in the homogenates was determined by investigated the possibility that the releasing effects of opioid the method of Lowry et al. (17). Statistical analysis of the data was by a two-tailed Student's t test (18). The publication costs of this article were defrayed in part by page were the kind charge payment. This article must therefore be hereby marked "ad- Morphine sulfate and naloxone hydrochloride vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: AMT, a-methyl-p-tyrosine methyl ester. 3006 Neurobiology: Deyo et al. Proc. Natl. Acad. Sci. USA 76 (1979) 3007 gifts of Bruce Wainer (Department of Pediatrics, Uniyv rrity dopamine concentration was significantly less at all time points of Chicago). AMT and catechol O-methyltra-isferas&'-4e exaiiiVed.This action of morphine showed dose dependency purchased from Sigma. ,B-Endorphin was obtained from Pen- in that both of the higher doses had significantly greater effects insula Labs (San Carlos, CA), while [D-Ala2,D-Leu5]enkephalin than the low dose. However, the effects of the two higher doses was the kind gift of Sam Wilkinson (Burroughs-Wellcome were not significantly different. The action of morphine was Laboratories, Research Triangle Park, NC). S-Adenosyl- blocked by pretreatment with the narcotic antagonist naloxone, [methyl-3H]methionine (11 Ci/mmol) and S-adenosyl- whereas naloxone alone had no detectable effect.on dopamine were obtained from turnover in the median eminence (Fig. 1B). Imethyl-14C]methionine (76 mCi/mmol) with AMT New Nuclear (1 Ci = 3.7 X 1010 becquerels). In Fig. 2A it can be seen that animals treated England showed a decline to 42% in dopamine concentration in the RESULTS median eminence at 25 min after AMT. It can also be seen that Animals treated with AMT showed a time-dependent decrease treatment with either [D-Ala2,D-Leu5]enkephalin or fl-en- in dopamine concentration in the median eminence (Fig. 1A). dorphin significantly reduced the decline in dopamine con- In morphine-treated animals the magnitude of the decline in centration at the same time point (15 min after injection of the opioid). Similar doses of opioids have been shown to increase blood prolactin levels in rats (2, 3, 5). As is the case for morphine, 100' A 100 B the actions of the endorphins were blocked by naloxone pre- treatment, but in this case the blockade was not total at the 80 80- naloxone dose used. As a comparison to the actions of the opioid in the median eminence, we also studied dopamine turnover in the frontal ~60 * 60- cortex and the neostriatum. In the frontal cortex, morphine (Fig. 0 1C) and [D-Ala2,D-Leu5]enkephalin and f3-endorphin (Fig. 2B) g40- ~~~~40- act as they do in the median eminence, to decrease the rate of dopamine turnover. However, in the neostriatum, morphine (Fig. 1D) and the opioid peptides (Fig. 2C) increase dopamine turnover. The actions of morphine and of the opioid peptides in the frontal cortex and neostriatum were also greatly reduced by naloxone pretreatment. DISCUSSION The regulation of prolactin release by the pituitary has now been well studied; the release is apparently under complex control. Several putative agents-including 760. 60 dopamine (10, 19), 5-hydroxytryptamine (19, 20), substance C P (21), (21), y-aminobutyric acid (22), thyrotro- 0 been shown ~4Q0 40- pin-releasing factor (23), and the opioids-have to modify prolactin release. The presence of dopamine in the median eminence (24) and in the hypothalamic-hypophyseal 20- 20 portal blood (25) and the increases in blood prolactin after le- sions of the tuberoinfundibular dopaminergic neurons (26, 27) suggest that this is one physiological system controlling prolactin 0.50 0.75 1.00 0 0. O.75 1.00 release. In addition, it has recently been reported that the Time, hr Time, hr concentration of dopamine found in the portal blood is suffi- FIG. 1. (A) Effects of morphine on the AMT-induced decline cient to inhibit prolactin release from pituitary lactotrophs (27), in dopamine concentration in the median eminence. Values are ex- is pressed as a percentage of the median eminence dopamine concen- suggesting that the primary regulator of prolactin release tration of untreated animals, 124.1 ± 8.8 (SEM) ng/mg of protein (n indeed this inhibitory, tuberoinfundibular, dopaminergic = 10). Animals were injected with saline, AMT, and then saline or system. morphine as described in the text. 0, Saline (n = 7); morphine at Although it has long been observed that morphine and other 1.67 mg/kg (n = 3); 0, morphine at 5 mg/kg (n = 7); O, morphine at narcotic elevate blood prolactin levels (1-4), the 15 mg/kg (n = 6). (B) Effects of naloxone + morphine on the AMT- mechanism for this action remains unknown at this time. induced decline in median eminence dopamine concentration. 0, = = Whereas opioids are able to increase prolactin release in vivo, Saline (n 7); &, morphine at 15 mg/kg (n 7); 0, naloxone at 20 mg/kg (n = 4); 0, morphine at 15 mg/kg + naloxone at 20 mg/kg (n they are ineffective in the isolated pituitary (8, 9), which indi- = 5). (C) Effects of morphine on the AMT-induced decline in frontal cates that the action of opioids is mediated at a higher level. cortex dopamine concentration. Values are expressed as a percentage Because recent evidence has localized enkephalinergic nerve of the dopamine concentration in the frontal cortex of untreated an- terminals in the rat median eminence (5), an area that also imals, 0.412 + 0.012 ng/mg of protein. 0, Saline (n = 6); A, morphine possesses a large number of dopaminergic terminals (24), we at 1.67 mg/kg (n = 4); 0, morphine at 5 mg/kg (n = 7); o, morphine at 15 mg/kg (n = 6). (D) Effects of morphine on the AMT-induced hypothesized that the effects of opioids in this structure may decline in neostriatal dopamine concentration. Values are expressed be mediated via an interaction with this dopaminergic system. as a percentage of the dopamine concentration in the neostriatum of An interaction between opioids and dopamine systems in the = untreated animals, 91.7 ± 7.6 ng/mg of protein. 0, Saline (n 6); A, striatum and elsewhere has recently been demonstrated both = = morphine at 1.67 mg/kg (n 3); 0, morphine at 5 mg/kg (n 6); 0, neurochemically and immunohistochemically (28-30). morphine at 15 mg/kg (n = 6). LaBrie et al. (31) have shown that the endorphins are able In all cases, the SEM was 4% or less of control value. *, Data are significantly different (P < 0.05) from the corresponding AMT-sa- to increase serum prolactin levels and that these levels are not line-treated control. **, Data are significantly different (P < 0.01) augmented by administration of haloperidol. The implication from the corresponding AMT-saline-treated control. of this report is that opioids act via the dopaminergic system 3008 Neurobiology: Deyo et al. Proc. Natl. Acad. Sci. USA 76 (1979)

A B C Fu;. 2. Effect of [i)-Ala2,D-Leu5jenkephalin (ENK) and 1001 -endorphin (END) on the AMT-induced decline in do- p)amine concentration in rat brain. SAL, saline; NAL, nal- 80 oxone. (A) Median eminence. Values ± SEM are expressed as a percentage of the median eminence dopamine con- -1-- (entration of animals, 124.1 ± 8.8 ng/mg of protein (n = g 60- 44 4-8). (B) Frontal cortex. Values ± SEM are expressed as + T -4- -1-i a l)ercentage of the frontal cortex dopamine concentration T I 40 I of untreated animals, 0.412 i 0.012 ng/mg of protein (n = 6). (C) Neostriatum. Values ± SEM are expressed as a percentage of the neostriatal dopamine concentration of 20- untreated animals, 91.7 + 7.6 ng/mg of protein (n = 6). *, Data are significantly different (P <0.05) from the cor- n I I responding saline-AMT-saline-treated control. **, Data AMT, AMT, NAL# AMT! NAL+ NAL+ AMT* AMT+ AMT. AMT* are SAL ENK AMT! END AMT. AMT! SAL ENK END SAL ENMT ENDM significantly different (P <0.01) from the corresponding t25, g!) ENK (15pgl END SAL (2?5g) I59g) (254g) (O5*g) saline-AMT-saline-treated control. of the medial basal hypothalamus. Our report shows that pamine turnover in the median eminence and in other brain morphine and endogenous opioids are able to reduce dopamine regions suggests that the actions of the endogenous opioid sys- turnover in the median eminence, suggesting that the mecha- tems may frequently be mediated by interaction with the cat- nism for the opioid-induced increase in pituitary prolactin re- echolamine systems of the brain. In particular, we suggest that lease is inhibition of tonic dopaminergic control. opiates and opioid peptides increase prolactin release by in- In order to determine dopamine turnover in the brain, we hibiting the release of dopamine from nerve terminals in the utilized the synthesis inhibitor AMT to block production of new median eminence. Such inhibition might be mediated by neurotransmitter. This is one of the best characterized methods axo-axonic contacts such as those thought to mediate enk- for the determination of neurotransmitter turnover and is ephalin-induced inhibition of release in the spinal particularly applicable to the median eminence. Current evi- cord (36). dence suggests that in these neurons there is no short-term feedback mechanism We thank J. Krystal for technical assistance. This work is supported regulating activity in the dopaminergic by National Institutes of Health Grant DA 02121. S.N.D. is supported neurons that might be affected by decreasing dopamine levels by U.S. Public Health Service Predoctoral Training Grant S-732- during the course of the experiment (14). Thus, the rate of de- GM-07151-04. cline in transmitter concentration after synthesis inhibition is probably an accurate reflection of neuronal activity and do- 1. Ferland, L., Labrie, F., Coy, D., Arimura, A. & Schally, A. (1976) pamine release. In contrast to the effect of opioids in decreasing Mol. Cell. Endocrinol. 41, 797-800. dopamine turnover in the median eminence and frontal cortex, 2. Kokka, N., Garcia, J., George, R. & Elliot, H. (1972) Endocri- the opioids increase turnover in the neostriatum. This effect is nology 90,735-743. 3. Tolis,,G., Hickey, J. & Gujda, H. (1975) J. Clin. Endocrinol. similar to findings reported by Algeri et al. (28), who argued, Metab. 41, 797-800. on the basis of the pattern of dopamine metabolites produced 4. Witzman, R. E., Fisher, D. A., Minich, S., Ling, W. & Guillemin, by the opioids, that the stimulating effect of opioids on dopa- R. (1977) Endocrinology 101, 1643-1646. mine turnover in the neostriatum is due to feedback activation 5. Meltzer, H. Y., Miller, R. J., Fessler, R. G., Simonovic, M. & Fang, of striatal tyrosine hydroxylase. In agreement with this inter- V. (1978) Life Sci. 22, 1931-1938. pretation, Loh et al. (32) reported that f3-endorphin inhibits 6. Sar, M., Stumpf, W. E., Miller, R. J., Chang, K.-J. & Cuatrecasas, release of dopamine from striatal slices. However, reports that P. (1978) J. Comp. Neurol. 182, 17-37. a similar feedback mechanism exists in the frontal cortex make 7. Rossier, J., Battenberg, E., Pittman, Q., Bayon, A., Koda, L., Miller, R., Guillemin, R. & Bloom, F. (1979) Nature (London) such an interpretation less tenable (33). 277,653-655. The fact that naloxone, when given alone, had no effect on 8. Grandison, L. & Guidotti, A. (1977) Nature (London) 270, dopamine turnover, would seem paradoxical if a tonic en- 357-359. kephalin-induced control of prolactin release existed. However, 9. Rivier, C., Vale, W., Ling, R., Brown, M. & Guillemin, R. (1977) there are a number of possible explanations for this. For ex- Endocrinology 106, 238-241. ample, it seems that in some preparations (34) naloxone is a 10. Langer, G., Terin, M. & Sacher, E. (1978) Endocrinology 102, much poorer antagonist of the actions of the endogenous opioids 367-370. than of the actions of morphine-like narcotic analgesics. In our 11. Coy, D., Kastin, A., Schally, A., Martin, O., Garon, M., Labrie, it seems that naloxone is more effective in F., Walker, T., Fertel, R., Blindein, G. & Sandman, C. (1976) study blocking the Biochem. Biophys. Res. Commun. 73,632-638. action of morphine in the median eminence (see Fig. 1B) than 12. Walker, J., Bernston, G., Sandman, C., Coy, D., Schally, A. & it is in blocking the action of the endorphins (see Fig. 2A). In Kastin, A. (1977) Science 196,85-87. addition, the action of an antagonist depends on the activity of 13. Bedell, C., Clark, B., Hardy, G., Lowe, R., Ubatuba, F., Vane, the system whose action it antagonizes, so that the lack of effect J., Wilkinson, S., Chang, K., Cuatrecasas, P. & Miller, R. (1977) of naloxone may reflect a lack of activity of the enkephalinergic Proc. R. Soc. Lond. Ser. 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