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Rat plasma prolactin levels in response to electrical stimulation of the ventromedial nucleus of the hypothalamus: dependence upon stimulation parameters
390
Brain Research, 147 (1978) 390-395 © Elsevier/North-Holland Biomedical Press
Rat plasma prolactin levels in response to electrical stimulation of the ventromedial nucleus of the hypothalamus: dependence upon stimulation parameters
STUART W. SMITH* and RICHARD R. GALA** Department of Physiology, Wayne State University, School of Medicine, Detroit, Mich. 48201 (U.S.A.)
(Accepted December 1st, 1977)
A neural role for prolactin homeostasis was initially suspected over 40 years ago is. Subsequent experiments demonstrated that hypothalamic lesions6,14 or electrical stimulation s resulted in increased prolactin release as determined by biological parameters. With the introduction of specific prolactin radioimmunoassays a number of studies were initiated to evaluate the effect of electrical stimulation of the brain on prolactin release1,4, s,7. A question inherent in the above studies was the influence of the anesthetic and/or the surgical procedures on the induced prolactin release. The purpose of the present experiment was to compare the effect of several electrical stimulation parameters on the plasma concentration of prolactin in conscious non-stressed rats bearing chronically implanted electrodes in the ventromedial hypothalamic nucleus. All animals were Sprague-Dawley derived female rats (Spartan Research Animals, Inc., Haslett, Mich.) and weighed 200-250 g upon arrival at the laboratory. They were housed in a constant temperature (23 °C), light controlled room (14 h ligbt: 10 h dark, lights on at 06.00 h) and given tap water and standard rat chow ad libitum. Bilateral ovariectomies were performed 3-5 days after arrival. Fourteen days later an indwelling catheter was inserted into the aorta via the common carotid by a method previously described 10. Three to seven days after catheterization, bipolar Nichrome electrodes were implanted in the hypothalamus and each rat was injected s.c. with 1 mg of Estradurin (Ayerst Laboratories; 0.5 mg polyestradiol phosphate). Most experiments were performed 6-8 days after electrode implants; however, some animals were initially stimulated after 4 or 12 days. Some animals were stimulated again 7 days after the initial experiment. All animals were conditioned to experimental procedures prior to the initiation of stimulation and blood sampling. During preliminary experiments we found that animals could give overt 'agitated' motor behavior reactions when 60 Hz stimuli were used at higher current levels. The current level was, therefore, increased from 0 / t A to * Present address: Nuclear Medicine Service, Allen Park Veterans Administration Hospital, Allen Park, Mich., 48101, U.S.A. ** To whom reprint requests should be addressed.
391 the point where increased motor activity was just noticeable. The current was then reduced slightly ( ~ 5/~A) and that level was noted as the animals' behavior limit (BL) for subsequent experiments. This testing procedure never took more than 5-10 sec. On the day of the experiment this BL was again verified, since some animals demonstrated increased sensitivity over time as indicated by overt motor behavior to the previously established electrical parameters. This verified behavioral limit was the one used for the immediate experiment. Preparations for sampling and stimulation were completed before 08.00 h after which all animals were allowed to acclimate at least 2 h before taking the first blood sample. An initial 0.4 ml blood sample was taken at 10.00 h and mixed with 0.4 ml of sterile saline containing 20--50 U/ml heparin. Subsequent samples were obtained at 10, 30, 50 and 110 min after the start of electrical stimulation. Immediately after taking the 30-min blood sample the current was turned off. The blood volume removed at each sampling was immediately replaced with warm (37 °C) sterile saline. The plasma was recovered by centrifugation and stored frozen (--20 °C) until assayed for prolactin. Electrodes were supported on a 1.0-1.3 mm length of 23 g stainless steel tubing that had parallel flat surfaces milled onto it. Two 32-g Nichrome wires, one each against the flat surfaces, extended 2-3 mm from the tubing and were adjusted to be 1 mm apart. Epoxylite (6001-M) coated wires were held in place with a 1 mm segment of silicone tubing (Silastic Tubing, Dow-Corning, 0.50 × 0.93) at the distal end and the unit double coated with Epoxylite. The distal ends of the wires were soldered to male microconnector pins that were subsequently secured in a microconnector manifold. The solder connections and exposed wire at the distal end of the electrode was coated with a thin layer of silicone glue (Dow-Corning 891, Type A medical adhesive). Prior to implanting, 0.5 mm of insulation was scraped from the tip of each wire and the electrode tested for isolation of each pole and for conduction leaks. Electrically faulty electrodes were implanted into control animals. Coordinates for the ventromedial nucleus of the hypothalamus (VMH) were derived from the rat brain atlas of Pellegrino and Cushman 15. To reduce the possibility of rupturing the sagittal sinus, the electrodes were implantea at a 10° angle. Bidirectional constant current square-wave pulses were delivered to the bipolar electrodes with a Grass model $88 stimulator and a floating ground was provided by two photoelectric stimulus isolation units (Grass Inst. Co. PSIU6B). The current was continuously monitored with an oscilloscope. Stimulation parameters are presented in Table I. The BL current was found to range from 30 #A to 150 #A. The duration of the current pulse was 1 msec in one polarity, then instantly reversed to the opposite polarity for another 1 msec. Stimulation for each electrical pattern was continued for 30 min. Brains were fixed in a formalin-acetic acid-alcohol-water solution (0.54).5-4.54.5) for 5-14 days, imbedded in paraffin, serially sectioned at 15-20/~m and stained with crystal violet to verify the placement of the electrodes. Plasma prolactin levels were determined at two dilutions in duplicate by a double antibody radioimmunoassayL All plasma samples from a given rat were run in the same assay. The prolactin standard was NIAMDD-RP-1 which had 11 IU/mg. Dunnetts' test 2 was used to estimate the statistical significance of the data.
392 Animals were included in the study on the basis of the following criteria: (1) maintenance of body weight, (2) true square wave stimulus during the experiment, and (3) proper electrode placement as revealed by histological brain examination with at least one of the poles imbedded in the VMH. When the 60 Hz stimulus was used, no animal was observed to have agitated motor activity when the current level was below 30/,A; therefore, the low current level was routinely 20-25 # A when 60 Hz frequency was used. No behavioral reactions were evident when the 5 Hz frequency was used, thus, low and high current levels were defined in absolute terms of 20-25/.,A and 100 #A, respectively. Table I summarizes the data from this study. Control animals showed no responses to the caging or blood sampling procedures. When 5 Hz was used neither the 25 # A nor 100 #A current level induced a change in the plasma prolactin concentration. Also, no change in plasma prolactin was observed when either 60 Hz-continuous or 60 Hzintermittent stimuli were used at 25/zA; however, when the BL current was used, the plasma prolactin level was significantly elevated at 10 and 30 min after the start of the stimulus. The 50 and 110 min (20 and 80 min after terminating the stimulus) plasma prolactin levels were not significantly different from those of prestimulation. We were able to perform a second experiment with some of the animals one week after initial stimulation. This was done to determine whether our animal model could be used in sequential experiments. Some animals were stimulated a second time while others served as controls. The stimulated animals received the same electrical parameters during the second experiment as they did the first. Of the animals that served as controls for the first experiment, 3 died before the second experiment, 5 had elevated TABLE I Influence o f the stimulation o f the ventromedial hypothalamus by various electricalparameters on prolactin release Stimulation parameters Fre-
Train
Current
No. Plasma prolactin levels (ng/ml) * animals Time after initiation o f stimulation
quency
sham control - 60 Continuous Continuous 5 sec on 10 sec off 5 sec on 10 sec off Continuous Continuous
Pre-stim
+10 min
-11 < 25/~A 9 B.L.** 8 < 25/*A 7
37±4 50±7 50&8 41 ±4
33±3 32±3 40±3 524-4 404-2 514-7 1114-13§§1054-18§§ 594-7 47±5 5 2 4 - 1 0 49±9
B.L.
7
49±3
63±5
81 4-15§ 61i6
594-7
_< 25/*A 100/~A
6 8
364-1 57±7
374-2 62±10
38±3 75±11
454-4 60±13
* Mean + S.E.M. ** B.L. = behavioral limits. § Significantlydifferent from pre-stimulation values, P < 0.05. §§ Significantlydifferent from pre-stimulation values, P < 0.01.
+30 min
+50 min
36±2 684-18
+110 min
37±4 454-4 464-4 454-4
393 initial prolactin values that were maintained throughout the second experiment and 5 animals maintained normal low prolactin levels throughout the experiment. Animals that had been stimulated the previous week and served as sham controls in the second experiment had a similar distribution; most had elevated initial prolactin levels but some developed and sustained high prolactin levels after the first blood sample was obtained. Three animals that were subjected to continuous 5 Hz 100 # A stimuli for a second time each maintained their initial low prolactin levels. Three out of 4 animals subjected to continuous 60 Hz behavioral limit current for the second time gave clear elevations in plasma prolactin while the fourth gave a mild rise. It became apparent that animals could not be uniformly utilized more than once in an experiment. Several initial experiments with a 60 Hz stimulus had to be terminated because animal behavioral reactions were so intense that they either tangled the electrode cable and sampling catheter extension or jumped out of the sampling cage. The stimulus to several animals exhibiting these behavioral conditions was immediately stopped; however, sampling was continued. If an increase in plasma prolactin had occurred in these animals it could be attributed to a stress condition17,19. The results in these cases did not reveal an elevated plasma prolactin, suggesting that the behavioral motor responses were not analogous to classical stress conditions 17,19. We concluded from this experience that the absolute current level may not represent the most reliable stimulus and may in fact stimulate inhibitory components to prolactin release. Thus, BL current was used routinely in experiments using 60 Hz stimuli. This definition was not needed with 5 Hz stimuli because a behavioral response with 5 Hz could not be elicited with current levels up to 250 keA, the practical range of our equipment. When a pulsed (5 sec on, 10 sec off) 60 Hz stimulus was used, animals did not develop the pronounced behavioral agitation demonstrated with continuous stimulus. To provide a means of comparison between 60 Hz stimulus groups, the high current level delivered to the pulsed stimulus group was the BL current defined by a continuous 60 Hz stimulus. Although the 60 Hz stimuli provoked an elevation of plasma prolactin when behavioral limit current was used (Table I), the mechanism for the elevation is not clear. We believe, however, that the elevation of plasma prolactin was not due to a stress reaction because animals that received a short 60 Hz continuous stimulus well over the behavioral limit and were highly agitated, were not observed to have elevated plasma prolactin levels. Further, animals that received pulsed 60 Hz behavioral limit current had elevated plasma prolactin with no evidence of behavioral motor arousal. There is evidence that both a prolactin releasing factor (PRF) and a prolactin inhibiting factor (PIF) is concentrated in the median eminence region s. Renaud and Martin 16 have demonstrated that there are direct neural connections to the median eminence (ME) originating in the VMH. It is possible, then, that some of these connections facilitate the release of a P R F from the ME and were activated by our stimulus conditions. Alternatively, the primary effect of our electrical stimulations may have activated internuncial neurons associated with the V M H that predominantly inhibited P I F associated neurons. A third mechanism may involve the release of thyrotrophin releasing hormone ( T R H ) in large enough quantities to induce prolactin release 2°. Our animal model does respond to exogenous T R H 11.
394 In previous experiments on unanesthetized sheep, electrical s t i m u l a t i o n o f the p r e o p t i c a r e a resulted in an increase in p l a s m a p r o l a c t i n 12 a n d i n d u c e d b e h a v i o r a l activity were e n c o u n t e r e d with certain stimulus currents similar to t h a t r e p o r t e d here in the rat for V M H stimulation. O t h e r a u t h o r s have also r e p o r t e d t h a t some stimulus p a r a m e t e r s are m o r e effective t h a n others lz in inducing the release o f p i t u i t a r y h o r m o nes; the physiological i n t e r p r e t a t i o n o f their results, however, m a y be c o n f o u n d e d by the presence o f anesthesia a n d high stimulus currents. The different efficiency o f various stimulation p a r a m e t e r s to elicit a change o f p l a s m a p r o l a c t i n in conscious animals underscores the i m p o r t a n c e o t careful e x a m i n a tion o f stimulating p a r a m e t e r s when studying the d i s t r i b u t i o n o f neural influences over p i t u i t a r y h o r m o n e levels in plasma. W e w o u l d like to express our a p p r e c i a t i o n to Mrs. C y n t h i a Van D e W a l l e for her excellent technical assistance in the p e r f o r m a n c e o f the p r o l a c t i n r a d i o i m m u n o a s s a y . The a u t h o r s also a p p r e c i a t e receiving as a gift from the R a t Pituitary D i s t r i b u t i o n A g e n c y o f N I A M D D the r a t p r o l a c t i n used for i o d i n a t i o n and standards. S u p p o r t e d in p a r t by N S F Research G r a n t No. 74-17332.
1 Clemens, J. A., Sharr, C. J., Tandy, W. A. and Roush, M. E., Effects of hypothalamic stimulation on prolactin secretion in steroid treated rats, Endocrinology, 89 (1971) 1317-1322. 2 Dunnett, C. W., A multiple comparison procedure for comparing several treatments with a control, J. Amer. stat. Ass., 50 (1955) 1096-1121. 3 Everett, J. W. and Quinn, D. L., Differential hypothalarnic mechanisms initiating ovulation and pseudopregnancy in the rat, Endocrinology, 78 (1966) 141-150. 4 Gala, R. R., Janson, P. A. and Kuo, E. Y. H., The influence of neural blocking agents injected into the third ventricle of the rat brain and hypothalamic electrical stimulation on serum prolactin, Proc. Soc. exp. Biol. ( N. Y. ) . 140 (1972) 569-572. 5 Gala, R. R. and Lawson, D. M., The influence of electrical stimulation of the rat brain on serum prolactin levels, Experientia (Basel), 29 (1973) 355-356. 6 Haun, C. K. and Sawyer, C. H., The role of the hypothalamus in initiation of milk secretion, Acta endocr. (Kbh.), 38 (1961) 99-106. 7 Kalra, S. P., Ajika, K., Krulich, L., Fawcett, C. P., Quijada, M. and McCann, S. M., Effects of hypothalamic and preoptic electrochemical stimulation on gonadotropin and prolactin release in proestrus rats, Endocrinology, 88 (1971) 1150-1158. 8 Krulich, L., Quijada, M. and Illner, P., Localization of prolactin inhibiting factor (P1F), P-releasing factor (PRF), growth hormone RF (GRF) and G1F activities in the hypothalamus of the rat. In Proc. 53rd Nat. Endocr. Soc., 1971, p. 83. 9 Kuo, E. Y. H. and Gala, R. R., Radioimmunoassay of rat prolactin comparing prolactin obtained from anterior pituitary organ culture with that distributed by the National Institute of Arthritis and Metabolic Diseases, Biochim. biophys. Acta (Amst.), 264 (1972) 462-471. 10 Lawson, D. M. and Gala, R. R., The influence of surgery, time of day, blood volume reduction and anesthetics on plasma prolactin in ovariectomized rats, J. Endocr., 62 (1974) 75-83. 11 Lawson, D. M. and Gala, R. R., Influence of anesthetics on basal, perphenazine-induced and thyrotrophin releasing hormone-induced prolactin secretion in ovariectomized, oestrogen-treated rats, J. Endocr., 66 (1975) 151-157. 12 Malven, P. V., Prolactin release induced by electrical stimulation of the hypothalamic preoptic area in unanesthetize sheep, Neuroendocrinology, 18 (1975) 65-71. 13 Martin, J. B., Kontor, J. and Mead, P., Plasma GH responses to hypothalamic, hippocarnpal and amygdaloid electrical stimulation: effects of variation in stimulus parameters and treatment with a-methyl-p-tryosine (a-MT), Endocrinology, 92 (1973) 1354-1361.
395 14 McCann, S. M. and Friedman, H. M., The effect of hypothalamic lesions on the secretion of luteotrophin, Endoerinology, 67 (1960) 597-608. 15 Pe••egrin•• L J. and Cushman•A. J.• A Stere•taxic At•as •f the Rat Brain• App•et•n-Century-Cr•fts• New York, 1967. 16 Renaud, L. P. and Martin, J. B., Electrophysiologicai studies of connections of hypothalamic ventromedial nucleus neurons in the rat: evidence for a role in neuroendocrine regulation, Brain Research, 93 (1975) 145-151. 17 Seggie, J. A. and Brown, G. M., Stress response patterns of plasma corticosterone, prolactin and growth hormone in the rat following handling or exposure to novel environments, Canad. J. Physiol. Pharmacol., 53 (1975) 629-637. 18 Selye, H., On the nervous control of lactation, Amer. J. PhysioL, 107 (1934) 535-538. 19 Smith, S. W. and Gala, R. R., Influence of restraint on plasma prolactin and corticosterone in female rats, J. Endocr., 74 (1977) 303-314. 20 Smith, V. G. and Convey, E. M., TRH-stimulation of prolactin release from bovine pituitary cells Proc. Soc. exp. BioL (N. Y.), 149 (1975) 70-74.
Brain Research, 147 (1978) 390-395 © Elsevier/North-Holland Biomedical Press
Rat plasma prolactin levels in response to electrical stimulation of the ventromedial nucleus of the hypothalamus: dependence upon stimulation parameters
STUART W. SMITH* and RICHARD R. GALA** Department of Physiology, Wayne State University, School of Medicine, Detroit, Mich. 48201 (U.S.A.)
(Accepted December 1st, 1977)
A neural role for prolactin homeostasis was initially suspected over 40 years ago is. Subsequent experiments demonstrated that hypothalamic lesions6,14 or electrical stimulation s resulted in increased prolactin release as determined by biological parameters. With the introduction of specific prolactin radioimmunoassays a number of studies were initiated to evaluate the effect of electrical stimulation of the brain on prolactin release1,4, s,7. A question inherent in the above studies was the influence of the anesthetic and/or the surgical procedures on the induced prolactin release. The purpose of the present experiment was to compare the effect of several electrical stimulation parameters on the plasma concentration of prolactin in conscious non-stressed rats bearing chronically implanted electrodes in the ventromedial hypothalamic nucleus. All animals were Sprague-Dawley derived female rats (Spartan Research Animals, Inc., Haslett, Mich.) and weighed 200-250 g upon arrival at the laboratory. They were housed in a constant temperature (23 °C), light controlled room (14 h ligbt: 10 h dark, lights on at 06.00 h) and given tap water and standard rat chow ad libitum. Bilateral ovariectomies were performed 3-5 days after arrival. Fourteen days later an indwelling catheter was inserted into the aorta via the common carotid by a method previously described 10. Three to seven days after catheterization, bipolar Nichrome electrodes were implanted in the hypothalamus and each rat was injected s.c. with 1 mg of Estradurin (Ayerst Laboratories; 0.5 mg polyestradiol phosphate). Most experiments were performed 6-8 days after electrode implants; however, some animals were initially stimulated after 4 or 12 days. Some animals were stimulated again 7 days after the initial experiment. All animals were conditioned to experimental procedures prior to the initiation of stimulation and blood sampling. During preliminary experiments we found that animals could give overt 'agitated' motor behavior reactions when 60 Hz stimuli were used at higher current levels. The current level was, therefore, increased from 0 / t A to * Present address: Nuclear Medicine Service, Allen Park Veterans Administration Hospital, Allen Park, Mich., 48101, U.S.A. ** To whom reprint requests should be addressed.
391 the point where increased motor activity was just noticeable. The current was then reduced slightly ( ~ 5/~A) and that level was noted as the animals' behavior limit (BL) for subsequent experiments. This testing procedure never took more than 5-10 sec. On the day of the experiment this BL was again verified, since some animals demonstrated increased sensitivity over time as indicated by overt motor behavior to the previously established electrical parameters. This verified behavioral limit was the one used for the immediate experiment. Preparations for sampling and stimulation were completed before 08.00 h after which all animals were allowed to acclimate at least 2 h before taking the first blood sample. An initial 0.4 ml blood sample was taken at 10.00 h and mixed with 0.4 ml of sterile saline containing 20--50 U/ml heparin. Subsequent samples were obtained at 10, 30, 50 and 110 min after the start of electrical stimulation. Immediately after taking the 30-min blood sample the current was turned off. The blood volume removed at each sampling was immediately replaced with warm (37 °C) sterile saline. The plasma was recovered by centrifugation and stored frozen (--20 °C) until assayed for prolactin. Electrodes were supported on a 1.0-1.3 mm length of 23 g stainless steel tubing that had parallel flat surfaces milled onto it. Two 32-g Nichrome wires, one each against the flat surfaces, extended 2-3 mm from the tubing and were adjusted to be 1 mm apart. Epoxylite (6001-M) coated wires were held in place with a 1 mm segment of silicone tubing (Silastic Tubing, Dow-Corning, 0.50 × 0.93) at the distal end and the unit double coated with Epoxylite. The distal ends of the wires were soldered to male microconnector pins that were subsequently secured in a microconnector manifold. The solder connections and exposed wire at the distal end of the electrode was coated with a thin layer of silicone glue (Dow-Corning 891, Type A medical adhesive). Prior to implanting, 0.5 mm of insulation was scraped from the tip of each wire and the electrode tested for isolation of each pole and for conduction leaks. Electrically faulty electrodes were implanted into control animals. Coordinates for the ventromedial nucleus of the hypothalamus (VMH) were derived from the rat brain atlas of Pellegrino and Cushman 15. To reduce the possibility of rupturing the sagittal sinus, the electrodes were implantea at a 10° angle. Bidirectional constant current square-wave pulses were delivered to the bipolar electrodes with a Grass model $88 stimulator and a floating ground was provided by two photoelectric stimulus isolation units (Grass Inst. Co. PSIU6B). The current was continuously monitored with an oscilloscope. Stimulation parameters are presented in Table I. The BL current was found to range from 30 #A to 150 #A. The duration of the current pulse was 1 msec in one polarity, then instantly reversed to the opposite polarity for another 1 msec. Stimulation for each electrical pattern was continued for 30 min. Brains were fixed in a formalin-acetic acid-alcohol-water solution (0.54).5-4.54.5) for 5-14 days, imbedded in paraffin, serially sectioned at 15-20/~m and stained with crystal violet to verify the placement of the electrodes. Plasma prolactin levels were determined at two dilutions in duplicate by a double antibody radioimmunoassayL All plasma samples from a given rat were run in the same assay. The prolactin standard was NIAMDD-RP-1 which had 11 IU/mg. Dunnetts' test 2 was used to estimate the statistical significance of the data.
392 Animals were included in the study on the basis of the following criteria: (1) maintenance of body weight, (2) true square wave stimulus during the experiment, and (3) proper electrode placement as revealed by histological brain examination with at least one of the poles imbedded in the VMH. When the 60 Hz stimulus was used, no animal was observed to have agitated motor activity when the current level was below 30/,A; therefore, the low current level was routinely 20-25 # A when 60 Hz frequency was used. No behavioral reactions were evident when the 5 Hz frequency was used, thus, low and high current levels were defined in absolute terms of 20-25/.,A and 100 #A, respectively. Table I summarizes the data from this study. Control animals showed no responses to the caging or blood sampling procedures. When 5 Hz was used neither the 25 # A nor 100 #A current level induced a change in the plasma prolactin concentration. Also, no change in plasma prolactin was observed when either 60 Hz-continuous or 60 Hzintermittent stimuli were used at 25/zA; however, when the BL current was used, the plasma prolactin level was significantly elevated at 10 and 30 min after the start of the stimulus. The 50 and 110 min (20 and 80 min after terminating the stimulus) plasma prolactin levels were not significantly different from those of prestimulation. We were able to perform a second experiment with some of the animals one week after initial stimulation. This was done to determine whether our animal model could be used in sequential experiments. Some animals were stimulated a second time while others served as controls. The stimulated animals received the same electrical parameters during the second experiment as they did the first. Of the animals that served as controls for the first experiment, 3 died before the second experiment, 5 had elevated TABLE I Influence o f the stimulation o f the ventromedial hypothalamus by various electricalparameters on prolactin release Stimulation parameters Fre-
Train
Current
No. Plasma prolactin levels (ng/ml) * animals Time after initiation o f stimulation
quency
sham control - 60 Continuous Continuous 5 sec on 10 sec off 5 sec on 10 sec off Continuous Continuous
Pre-stim
+10 min
-11 < 25/~A 9 B.L.** 8 < 25/*A 7
37±4 50±7 50&8 41 ±4
33±3 32±3 40±3 524-4 404-2 514-7 1114-13§§1054-18§§ 594-7 47±5 5 2 4 - 1 0 49±9
B.L.
7
49±3
63±5
81 4-15§ 61i6
594-7
_< 25/*A 100/~A
6 8
364-1 57±7
374-2 62±10
38±3 75±11
454-4 60±13
* Mean + S.E.M. ** B.L. = behavioral limits. § Significantlydifferent from pre-stimulation values, P < 0.05. §§ Significantlydifferent from pre-stimulation values, P < 0.01.
+30 min
+50 min
36±2 684-18
+110 min
37±4 454-4 464-4 454-4
393 initial prolactin values that were maintained throughout the second experiment and 5 animals maintained normal low prolactin levels throughout the experiment. Animals that had been stimulated the previous week and served as sham controls in the second experiment had a similar distribution; most had elevated initial prolactin levels but some developed and sustained high prolactin levels after the first blood sample was obtained. Three animals that were subjected to continuous 5 Hz 100 # A stimuli for a second time each maintained their initial low prolactin levels. Three out of 4 animals subjected to continuous 60 Hz behavioral limit current for the second time gave clear elevations in plasma prolactin while the fourth gave a mild rise. It became apparent that animals could not be uniformly utilized more than once in an experiment. Several initial experiments with a 60 Hz stimulus had to be terminated because animal behavioral reactions were so intense that they either tangled the electrode cable and sampling catheter extension or jumped out of the sampling cage. The stimulus to several animals exhibiting these behavioral conditions was immediately stopped; however, sampling was continued. If an increase in plasma prolactin had occurred in these animals it could be attributed to a stress condition17,19. The results in these cases did not reveal an elevated plasma prolactin, suggesting that the behavioral motor responses were not analogous to classical stress conditions 17,19. We concluded from this experience that the absolute current level may not represent the most reliable stimulus and may in fact stimulate inhibitory components to prolactin release. Thus, BL current was used routinely in experiments using 60 Hz stimuli. This definition was not needed with 5 Hz stimuli because a behavioral response with 5 Hz could not be elicited with current levels up to 250 keA, the practical range of our equipment. When a pulsed (5 sec on, 10 sec off) 60 Hz stimulus was used, animals did not develop the pronounced behavioral agitation demonstrated with continuous stimulus. To provide a means of comparison between 60 Hz stimulus groups, the high current level delivered to the pulsed stimulus group was the BL current defined by a continuous 60 Hz stimulus. Although the 60 Hz stimuli provoked an elevation of plasma prolactin when behavioral limit current was used (Table I), the mechanism for the elevation is not clear. We believe, however, that the elevation of plasma prolactin was not due to a stress reaction because animals that received a short 60 Hz continuous stimulus well over the behavioral limit and were highly agitated, were not observed to have elevated plasma prolactin levels. Further, animals that received pulsed 60 Hz behavioral limit current had elevated plasma prolactin with no evidence of behavioral motor arousal. There is evidence that both a prolactin releasing factor (PRF) and a prolactin inhibiting factor (PIF) is concentrated in the median eminence region s. Renaud and Martin 16 have demonstrated that there are direct neural connections to the median eminence (ME) originating in the VMH. It is possible, then, that some of these connections facilitate the release of a P R F from the ME and were activated by our stimulus conditions. Alternatively, the primary effect of our electrical stimulations may have activated internuncial neurons associated with the V M H that predominantly inhibited P I F associated neurons. A third mechanism may involve the release of thyrotrophin releasing hormone ( T R H ) in large enough quantities to induce prolactin release 2°. Our animal model does respond to exogenous T R H 11.
394 In previous experiments on unanesthetized sheep, electrical s t i m u l a t i o n o f the p r e o p t i c a r e a resulted in an increase in p l a s m a p r o l a c t i n 12 a n d i n d u c e d b e h a v i o r a l activity were e n c o u n t e r e d with certain stimulus currents similar to t h a t r e p o r t e d here in the rat for V M H stimulation. O t h e r a u t h o r s have also r e p o r t e d t h a t some stimulus p a r a m e t e r s are m o r e effective t h a n others lz in inducing the release o f p i t u i t a r y h o r m o nes; the physiological i n t e r p r e t a t i o n o f their results, however, m a y be c o n f o u n d e d by the presence o f anesthesia a n d high stimulus currents. The different efficiency o f various stimulation p a r a m e t e r s to elicit a change o f p l a s m a p r o l a c t i n in conscious animals underscores the i m p o r t a n c e o t careful e x a m i n a tion o f stimulating p a r a m e t e r s when studying the d i s t r i b u t i o n o f neural influences over p i t u i t a r y h o r m o n e levels in plasma. W e w o u l d like to express our a p p r e c i a t i o n to Mrs. C y n t h i a Van D e W a l l e for her excellent technical assistance in the p e r f o r m a n c e o f the p r o l a c t i n r a d i o i m m u n o a s s a y . The a u t h o r s also a p p r e c i a t e receiving as a gift from the R a t Pituitary D i s t r i b u t i o n A g e n c y o f N I A M D D the r a t p r o l a c t i n used for i o d i n a t i o n and standards. S u p p o r t e d in p a r t by N S F Research G r a n t No. 74-17332.
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