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Catecholamine mechanisms in medio-basal hypothalamus influence prolactin but not growth hormone secretion
Brain Research, 253 (1982) 213-219 Elsevier Biomedical Press
213
Catecholamine Mechanisms in Medio-Basal Hypothalamus Influence Prolactin but not Growth Hormone Secretion TREVOR A. DAY*, PETA M. JERVOIS, MARGARET F. MENADUE and JOHN O. WILLOUGHBY Centre for Neuroscience and Department of Medicine, The Flinders University of South Australia, Bed]brd Park, South Australia, 5042 (Australia) (Accepted March 18th, 1982) Key words: growth hormone - - prolactin - - catecholamine - - medio-basal hypothalamus
Medio-basal hypothalamic (MBH) catecholamine mechanisms in the regulation of prolactin and growth hormone (GH) secretion were investigated in unanesthetized rats with chronic indwelling venous cannulae and bilateral MBH directed intracerebral guide cannulae. MBH injections of the catecholamine-specificneurotoxin 6-hydroxydopamine (6-OHDA; 2 #g base in 0.5/~10.9 ~ saline) had no effect upon basal prolactin or GH secretion. Examination of catecholamine fluorescence indicated that MBH 6-OHDA treatment produced widespread disruption of MBH catecholamine afferents but did not destroy tuberoinfundibular dopamine neurons of the arcuate nucleus, nor median eminence catecholamine structures. MBH injections (0.5 #1, 0.032 M solutions) of dopamine, noradrenaline or adrenaline all produced statistically significant increases in plasma prolactin levels. The potency of these 3 catecholamines in evoking prolactin release differed markedly, adrenaline having the greatest effect. MBH catecholamine injections had no effect upon plasma GH levels compared to saline injected controls. The present data suggest that MBH catecholamine afferents are unimportant in the regulation of basal patterns of GH or prolactin secretion. As MBH catecholamine injections stimulate prolactin release this region may contain a prolactin-facilitatory catecholamine mechanism which is capable of generating prolactin surges in response to certain environmental or endogenous stimuli. INTRODUCTION Central c a t e c h o l a m i n e structures are p r e d o m i n a n t l y s t i m u l a t o r y to G H secretiong,24, 3s, a l t h o u g h evidence has also been o b t a i n e d for a G H - i n h i b i t o r y n o r a d r e n e r g i c p r o j e c t i o n to the m e d i a n eminence 6. It seems p r o b a b l e , therefore, t h a t the s t i m u l a t o r y role o f c a t e c h o l a m i n e in G H regulation involves an i n h i b i t o r y action on periventricular p r e o p t i c - a n t e r ior h y p o t h a l a m i c a r e a ( P O / A H A ) G H release-inhibiting factor ( S R I F ) p e r i k a r y a ~9 or a s t i m u l a t o r y a c t i o n on G H releasing factor ( G R F ) neurons1, ~7. H y p o t h a l a m i c ablation12, 25, deafferentation~9,80, 40 a n d stimulationaS,23, 26 studies indicate t h a t G R F n e u r o n s reside in the m e d i o b a s a l h y p o t h a l a m u s ( M B H ) , p r o b a b l y within either the v e n t r o m e d i a l
( V M N ) or a r c u a t e nuclei. It is now widely accepted that d o p a m i n e released b y t u b e r o i n f u n d i b u l a r a r c u a t e n e u r o n s acts as a physiological p r o l a c t i n release-inhibiting factor15,16, 81,33. T h e r e is also evidence to indicate that catec h o l a m i n e m a y have a s t i m u l a t o r y role in p r o lactin regulation27, ~2. A positive c o r r e l a t i o n between M B H c a t e c h o l a m i n e t u r n o v e r a n d p r o l a c t i n levels in rats subjected to a variety o f e n d o c r i n o l o g i c a l m a n i p u l a t i o n s has also been r e p o r t e d 17. The present study sought to d e t e r m i n e whether M B H c a t e c h o l a m i n e m e c h a n i s m s (distinct f r o m t u b e r o i n f u n d i b u l a r d o p a m i n e neurons) c o n t r i b u t e to the regulation o f either G H or p r o l a c t i n secretion. Thus, the effects on G H a n d p r o l a c t i n secretion o f d i s r u p t i o n o f M B H c a t e c h o l a m i n e afferents (Expt.
* Present address: Division of Neurology, Montreal General Hospital and McGill University, 1650 Cedar Ave., Montreal, Quebec, Canada. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
214 I), and of localized MBH injections of catecholamine (Expt. ll), were examined in the unanesthetized male rat. MATERIALS AND METHODS
Male albino Porton rats (250-350 g), permitted free access to food and water and maintained on a 12 h light-dark cycle (lights on 09.00 h) at an ambient temperature of 23 °C, were used. Pentobarbitone anesthetized animals were prepared with chronic indwelling right atrial cannulae 4. At the same time an MBH directed bilateral intracerebral guide cannulae assembly, fashioned from 22-gauge stainless steel tubing and fitted with indwelling stylets, was secured to the skull of each animal with stainless steel screws and dental cement. Coordinates of placement (skull level between lambda and bregma) were: 2.4 mm caudal and 5_0.5 mm lateral to bregma, 2.0 mm ventral to brain surface. MBH injections were made using 30-gauge injection needles which extended 6.3 mm beyond the tips of the intracerebral cannulae. This injection site was centered at the common border of the VMN and arcuate nucleus approximately 4.3 mm anterior to the interaural line in the stereotaxic atlas of K6nig and Klippe119. Four days after surgery animals were housed in isolation boxes such that blood samples could be withdrawn without disturbing the animals, via tubing leading to the outside of the box. Animals were allowed 3 days to adapt to these cages before sampling was undertaken. Blood samples (0.4 ml) were taken every 15 (Expt. I) or 10 (Expt. II) min during sampling sessions, plasma and red blood cells were immediately separated by centrifugation. Plasma required for subsequent assay (0.2 ml) was then frozen and the red blood cells resuspended in 0.2 ml physiological saline and reinjected into the rat after collection of the next sample. Plasma GI-! and prolactin concentrations were determined by radioimmunoassay using a double antibody separation technique. Materials were provided by N I A M D D and values are expressed in terms of the respective N I A M D D reference preparations. The range of sensitivities of the assays were 12.5-800 ng/ml for G H and 1.25-320 ng/ml for prolactin.
Experiment 1 Animals were sampled the day before (baseline) and 1 and 7 days after bilateral MBH injection of the catecholamine-specific neurotoxin 2° 6-hydroxydopamine (6-OHDA, 2 /zg base in 0.5 /d 0.9'!, saline). Preliminary studies indicated that MBH injection of saline had no effect upon hormone secretory patterns. Additional animals were treated with 6-OHDA and one day later were anesthetized and perfused with a mixture of formaldehyde and glutaraldehyde which has been shown both to fix CNS tissue and to convert noradrenaline and dopamine to fluorescent derivatives 14. Brains were then removed, 40 tzm sections cut on an Oxford Vibratome and the sections mounted, dried and cover-slipped with liquid paraffin. Sections were examined with a Leitz Ortholux fluorescence microscope and the extent of 6OHDA induced damage to CNS catecholamine fiber systems determined. Experiment 1I Animals underwent 4 sampling sessions, each of 90 rain duration and each on separate days. Immediately prior to each session animals received bilateral MBH injections of either noradrenaline, dopamine, adrenaline or physiological saline. Injections were 0.5 #1 in volume; catecholamine solutions were 0.032 M, pH 6.0-7.0, a dose found in previous work to be sufficient to elicit marked but submaximal physiological changes when injected into the hypothalamus 8. Animals received all 4 treatments in random order. At the completion of the final sampling session animals were sacrificed and CNS tissue sections prepared as in Expt. I. Only data from animals in which intracerebral injections were found to have been accurately placed were used. RESULTS
Experiment 1 Examples of baseline, day 1 and day 7 G H and prolactin secretory profiles from 3 animals are illustrated in Fig. i. Mean G H and prolactin plasma concentrations are shown in Table I. Baseline patterns of G H secretion consisted of polyphasic bursts of release occurring approximately every 3 h. Base-
215
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Fig. 1. Three examples of 6 h GH and prolactin profiles obtained from animals sampled the day before (baseline) and 1 and 7 days after bilateral MBH 6-OHDA injections (2/,g base in 0.5 td 0.9 ~ saline).
TABLE I
Mean plasma GH and prolactin concentrations (ng/ml :k S.E.M.) over 6 h the daybefore (baseline), 1 and 7 days after M B H 6-OHDA Student's t-tests show no significant differences.
Baseline Day 1 Day 7
n
GH
Prolactin
7 7 6
153.2 ± 22.0 183.7 d- 31.3 184.0 d: 24.0
17.7 ~: 2.9 17.3 :k 3.8 20.4 :E 4.3
line prolactin profiles were characterized by long troughs and sporadic bursts of secretion. M B H 6O H D A treatment had no effect upon either G H or prolactin secretion at days 1 or 7. Histofluorescence examination of tissue sections one day after the intracerebral injection of 6 - O H D A indicated severe disruption of M B H catecholamine structures. The density o f catecholamine termina[ fibers in the arcuate nucleus was markedly reduced although A12 dopamine neurons were still visible (Fig. 2). Few catecholamine processes are normally
observed in the V M N of intact animalslS; none were visible in M B H 6 - O H D A treated animals. A noticeable reduction was observed in the number of catecholamine processes within the M B H though dorsolaterally large numbers of swollen, presumably degenerating catecholamine axons could be seen in the region of the ventral ascending catecholamine bundle. No alteration was observed in the appearance of median eminence nor P O / A H A catecholamine processes.
Experiment H Mean group G H and prolactin profiles following M B H injections of noradrenaline, dopamine, adrenaline and saline are illustrated in Fig. 3. Mean plasma hormone concentrations are shown in Table II. Catecholamine injections failed to affect G H secretion. All 3 catecholamine stimulated prolactin secretion but there were noticeable differences in the amplitude of the effect. The ascending order of potency was: dopamine, noradrenaline, adrenaline.
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Fig. 2. Catecholamine-fluorescence micrographs of control (a) and 6-OHDA (b) injected sides of a hypothalamus from an animal 1 day after treatment. The PO/AHA (I a and I b) was not affected by M BH injections whereas in tile MBH anteriorly (2a and 2b) and posteriorly (3a and 3b) pronounced depletion of catecholamine fluorescent fibers is evident. In 3b, dopamine neurons of the A12 group appear a little more prominent than on the control side, because of the paucity of brightly fluorescent catecholamine fibers in the adjacent neuropil. A damaged swollen catecholamine-containing fiber is evident in 3b (center) as weft as a few apparently intact fibers. 800
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Fig. 3. Mean (~: S.E.M.) group GH and prolactin secretory profiles over 90 min following bilateral MBH injection of dopamine, noradrenaline, adrenaline (0.5/d, 0.032 M solutions) or saline; n = 8 per point.
217 TABLE II Effect of bilateral MBH injections (0.5 td) of dopamine, noradrenaline and adrenaline (0.032M solutions) on mean plasma GH and prolactin concentrations (ng/ml d_ S.E.M.) n
Saline 8 Dopamine 8 Noradrenaline 8 Adrenaline 8
GH
Prolactin
124.5 4- 39.5 142.9 ~ 42.9 90.3 ± 27.9 78.1 ~ 16.1
21.3 -I- 2.7 35.8 i 4.5* 68.7 4- 6.6** 158.6 ± 19.7"*
Significantlydifferent from saline control, * P < 0.01, ** P < 0.001. Paired Student's t-test. DISCUSSION Prolactin secretion
In addition to the inhibitory influence of tuberoinfundibular dopamine neurons~S,16,31,3z, central catecholamine structures also appear to play a facilitatory role in the regulation of prolactin secretionZT,32. Although our knowledge of the location of prolactin-regulating catecholamine structures is incomplete, it has been reported that elevated plasma prolactin levels in proestrous and ovariectomized steroid-primed animals coincide with increased noradrenaline turnover in the MBH ~7. Electrical stimulation studies have also implicated MBH structures, apart from the arcuate, as being significant in the regulation of prolactin: stimulation of the VMN has been reported to induce acute prolactin release a4 and to trigger the onset of pseudopregnancy-associated prolactin surges 11. In the present study MBH injections of all 3 catecholamine (Expt. II) produced statistically significant increases in plasma prolactin levels. In contrast to this observation, MBH 6-OHDA treatment was found to have no effect upon basal hormone profiles, despite producing a severe disruption of local catecholamine afferents. This suggests that although 6-OHDA-sensitive MBH catecholamine afferents are not important to the regulation of basal patterns of prolactin secretion, this region may contain a catecholamine mechanism which subserves the generation of prolactin surges in response to certain environmental or endogenous stimuli. In accord with this proposal it has been reported that depletion of central catecholamine levels prevents
stress induced prolactin rises 1° and the afternoon prolactin surge observed in ovariectomized steroidprimed rats 35. One explanation of the differing potencies of dopamine, noradrenaline and adrenaline in stimulating p~ olactin secretion (see Fig. 3) may be that the MBH prolactin-facilitatory catecholamine mechanism involves receptors for which adrenaline is the normal ligand. The quantities of dopamine and noradrenaline injected into the MBH were conceivably sufficient to cause some degree of stimulation of these receptors despite a lower receptor affinity. Furthermore the order of agonist potency reported for az-adrenergic receptors is adrenaline > noradrenaline > > dopamine 3. Although MBH 6-OHDA injections (Expt. I) destroyed local catecholamine afferents, they had no apparent effect upon arcuate dopamine neurons. The resistance of these neurons to the toxic effects of 6-OHDA has been noted previouslyS, 6,36 and is attributed to the lack of a high affinity uptake system for catecholamine 8. Given the considerable evidence that tuberoinfundibular arcuate dopamine neurons are tonically inhibitory to prolactin secretion tS,16,22,at,3a, the observation that prolactin secretion was normal at days 1 and 7 may be viewed as further evidence of the insensitivity of these neurons to 6-OHDA. G H secretion
As catecholamines do not act directly upon the pituitary to influence G H secretion 2s their role in the regulation of this hormone presumably stems from their ability to affect the activity of SRIF and/or G R F neurons. We have previously reported that, in the rat, median eminence catecholamine afferents are inhibitory to G H secretion, probably as a result of a facilitatory effect upon SRIF terminals 6. Although there is some evidence that the facilitatory role of catecholamine in G H regulation involves a stimulatory influence upon G R F neurons as well as an inhibitory input to SRIF neuronsl, aT, the present findings indicate that it is unlikely that MBH G R F perikarya receive a direct input from central catecholamine neurons. The possibility remains, however, that CNS catecholamine structures facilitate G R F activity via interneurons whose perikarya lie outside of the
218 M BH. For example, periventricular P O / A H A S R I F n e u r o n s may be the inhibitory interneurons by
cholamine antagonists suppress G H secretion in animals passively i m m u n i z e d with S RI F antiserum ~,
which catecholamine influence G R F release. It is
:% in these studies, S R I F antiserum was administered into the peripheral circulation, and thus
k n o w n that S R I F n e u r o n s project to the V M N arcuate region e a n d that periventricular P O / A H A n e u r o n s receive a catecholamine i n n e r v a t i o n 2~. Such
would be likely to leave any central S R I F mechanisms unaffected.
an a r r a n g e m e n t would provide a simple means of regulating the rhythmic pattern which characterizes
ACKNOWLEDGEMENTS
G H secretion. W h e n P O / A H A catecholamine afferents are active, median eminence S R I F release and the proposed inhibitory S R I F i n p u t on G R F n e u r o n s would both be blocked, thus permitting a G H surge. W h e n P O / A H A catecholamine afferents are inactive median eminence S R I F release and S R I F i n h i b i t i o n of G R F n e u r o n s would be maximal and a G H trough would result. Even though cate-
R a d i o i m m u n o a s s a y materials were kindly provided by Dr. A. Parlow, N I A M D D . This work was supported by grants to J.O.W. from the N a t i o n a l Health a n d Medical Research C o u n c i l o f Australia, T.A.D. was a Postdoctoral Fellow of the Neurosurgical Research F o u n d a t i o n of South Australia.
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213
Catecholamine Mechanisms in Medio-Basal Hypothalamus Influence Prolactin but not Growth Hormone Secretion TREVOR A. DAY*, PETA M. JERVOIS, MARGARET F. MENADUE and JOHN O. WILLOUGHBY Centre for Neuroscience and Department of Medicine, The Flinders University of South Australia, Bed]brd Park, South Australia, 5042 (Australia) (Accepted March 18th, 1982) Key words: growth hormone - - prolactin - - catecholamine - - medio-basal hypothalamus
Medio-basal hypothalamic (MBH) catecholamine mechanisms in the regulation of prolactin and growth hormone (GH) secretion were investigated in unanesthetized rats with chronic indwelling venous cannulae and bilateral MBH directed intracerebral guide cannulae. MBH injections of the catecholamine-specificneurotoxin 6-hydroxydopamine (6-OHDA; 2 #g base in 0.5/~10.9 ~ saline) had no effect upon basal prolactin or GH secretion. Examination of catecholamine fluorescence indicated that MBH 6-OHDA treatment produced widespread disruption of MBH catecholamine afferents but did not destroy tuberoinfundibular dopamine neurons of the arcuate nucleus, nor median eminence catecholamine structures. MBH injections (0.5 #1, 0.032 M solutions) of dopamine, noradrenaline or adrenaline all produced statistically significant increases in plasma prolactin levels. The potency of these 3 catecholamines in evoking prolactin release differed markedly, adrenaline having the greatest effect. MBH catecholamine injections had no effect upon plasma GH levels compared to saline injected controls. The present data suggest that MBH catecholamine afferents are unimportant in the regulation of basal patterns of GH or prolactin secretion. As MBH catecholamine injections stimulate prolactin release this region may contain a prolactin-facilitatory catecholamine mechanism which is capable of generating prolactin surges in response to certain environmental or endogenous stimuli. INTRODUCTION Central c a t e c h o l a m i n e structures are p r e d o m i n a n t l y s t i m u l a t o r y to G H secretiong,24, 3s, a l t h o u g h evidence has also been o b t a i n e d for a G H - i n h i b i t o r y n o r a d r e n e r g i c p r o j e c t i o n to the m e d i a n eminence 6. It seems p r o b a b l e , therefore, t h a t the s t i m u l a t o r y role o f c a t e c h o l a m i n e in G H regulation involves an i n h i b i t o r y action on periventricular p r e o p t i c - a n t e r ior h y p o t h a l a m i c a r e a ( P O / A H A ) G H release-inhibiting factor ( S R I F ) p e r i k a r y a ~9 or a s t i m u l a t o r y a c t i o n on G H releasing factor ( G R F ) neurons1, ~7. H y p o t h a l a m i c ablation12, 25, deafferentation~9,80, 40 a n d stimulationaS,23, 26 studies indicate t h a t G R F n e u r o n s reside in the m e d i o b a s a l h y p o t h a l a m u s ( M B H ) , p r o b a b l y within either the v e n t r o m e d i a l
( V M N ) or a r c u a t e nuclei. It is now widely accepted that d o p a m i n e released b y t u b e r o i n f u n d i b u l a r a r c u a t e n e u r o n s acts as a physiological p r o l a c t i n release-inhibiting factor15,16, 81,33. T h e r e is also evidence to indicate that catec h o l a m i n e m a y have a s t i m u l a t o r y role in p r o lactin regulation27, ~2. A positive c o r r e l a t i o n between M B H c a t e c h o l a m i n e t u r n o v e r a n d p r o l a c t i n levels in rats subjected to a variety o f e n d o c r i n o l o g i c a l m a n i p u l a t i o n s has also been r e p o r t e d 17. The present study sought to d e t e r m i n e whether M B H c a t e c h o l a m i n e m e c h a n i s m s (distinct f r o m t u b e r o i n f u n d i b u l a r d o p a m i n e neurons) c o n t r i b u t e to the regulation o f either G H or p r o l a c t i n secretion. Thus, the effects on G H a n d p r o l a c t i n secretion o f d i s r u p t i o n o f M B H c a t e c h o l a m i n e afferents (Expt.
* Present address: Division of Neurology, Montreal General Hospital and McGill University, 1650 Cedar Ave., Montreal, Quebec, Canada. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
214 I), and of localized MBH injections of catecholamine (Expt. ll), were examined in the unanesthetized male rat. MATERIALS AND METHODS
Male albino Porton rats (250-350 g), permitted free access to food and water and maintained on a 12 h light-dark cycle (lights on 09.00 h) at an ambient temperature of 23 °C, were used. Pentobarbitone anesthetized animals were prepared with chronic indwelling right atrial cannulae 4. At the same time an MBH directed bilateral intracerebral guide cannulae assembly, fashioned from 22-gauge stainless steel tubing and fitted with indwelling stylets, was secured to the skull of each animal with stainless steel screws and dental cement. Coordinates of placement (skull level between lambda and bregma) were: 2.4 mm caudal and 5_0.5 mm lateral to bregma, 2.0 mm ventral to brain surface. MBH injections were made using 30-gauge injection needles which extended 6.3 mm beyond the tips of the intracerebral cannulae. This injection site was centered at the common border of the VMN and arcuate nucleus approximately 4.3 mm anterior to the interaural line in the stereotaxic atlas of K6nig and Klippe119. Four days after surgery animals were housed in isolation boxes such that blood samples could be withdrawn without disturbing the animals, via tubing leading to the outside of the box. Animals were allowed 3 days to adapt to these cages before sampling was undertaken. Blood samples (0.4 ml) were taken every 15 (Expt. I) or 10 (Expt. II) min during sampling sessions, plasma and red blood cells were immediately separated by centrifugation. Plasma required for subsequent assay (0.2 ml) was then frozen and the red blood cells resuspended in 0.2 ml physiological saline and reinjected into the rat after collection of the next sample. Plasma GI-! and prolactin concentrations were determined by radioimmunoassay using a double antibody separation technique. Materials were provided by N I A M D D and values are expressed in terms of the respective N I A M D D reference preparations. The range of sensitivities of the assays were 12.5-800 ng/ml for G H and 1.25-320 ng/ml for prolactin.
Experiment 1 Animals were sampled the day before (baseline) and 1 and 7 days after bilateral MBH injection of the catecholamine-specific neurotoxin 2° 6-hydroxydopamine (6-OHDA, 2 /zg base in 0.5 /d 0.9'!, saline). Preliminary studies indicated that MBH injection of saline had no effect upon hormone secretory patterns. Additional animals were treated with 6-OHDA and one day later were anesthetized and perfused with a mixture of formaldehyde and glutaraldehyde which has been shown both to fix CNS tissue and to convert noradrenaline and dopamine to fluorescent derivatives 14. Brains were then removed, 40 tzm sections cut on an Oxford Vibratome and the sections mounted, dried and cover-slipped with liquid paraffin. Sections were examined with a Leitz Ortholux fluorescence microscope and the extent of 6OHDA induced damage to CNS catecholamine fiber systems determined. Experiment 1I Animals underwent 4 sampling sessions, each of 90 rain duration and each on separate days. Immediately prior to each session animals received bilateral MBH injections of either noradrenaline, dopamine, adrenaline or physiological saline. Injections were 0.5 #1 in volume; catecholamine solutions were 0.032 M, pH 6.0-7.0, a dose found in previous work to be sufficient to elicit marked but submaximal physiological changes when injected into the hypothalamus 8. Animals received all 4 treatments in random order. At the completion of the final sampling session animals were sacrificed and CNS tissue sections prepared as in Expt. I. Only data from animals in which intracerebral injections were found to have been accurately placed were used. RESULTS
Experiment 1 Examples of baseline, day 1 and day 7 G H and prolactin secretory profiles from 3 animals are illustrated in Fig. i. Mean G H and prolactin plasma concentrations are shown in Table I. Baseline patterns of G H secretion consisted of polyphasic bursts of release occurring approximately every 3 h. Base-
215
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Fig. 1. Three examples of 6 h GH and prolactin profiles obtained from animals sampled the day before (baseline) and 1 and 7 days after bilateral MBH 6-OHDA injections (2/,g base in 0.5 td 0.9 ~ saline).
TABLE I
Mean plasma GH and prolactin concentrations (ng/ml :k S.E.M.) over 6 h the daybefore (baseline), 1 and 7 days after M B H 6-OHDA Student's t-tests show no significant differences.
Baseline Day 1 Day 7
n
GH
Prolactin
7 7 6
153.2 ± 22.0 183.7 d- 31.3 184.0 d: 24.0
17.7 ~: 2.9 17.3 :k 3.8 20.4 :E 4.3
line prolactin profiles were characterized by long troughs and sporadic bursts of secretion. M B H 6O H D A treatment had no effect upon either G H or prolactin secretion at days 1 or 7. Histofluorescence examination of tissue sections one day after the intracerebral injection of 6 - O H D A indicated severe disruption of M B H catecholamine structures. The density o f catecholamine termina[ fibers in the arcuate nucleus was markedly reduced although A12 dopamine neurons were still visible (Fig. 2). Few catecholamine processes are normally
observed in the V M N of intact animalslS; none were visible in M B H 6 - O H D A treated animals. A noticeable reduction was observed in the number of catecholamine processes within the M B H though dorsolaterally large numbers of swollen, presumably degenerating catecholamine axons could be seen in the region of the ventral ascending catecholamine bundle. No alteration was observed in the appearance of median eminence nor P O / A H A catecholamine processes.
Experiment H Mean group G H and prolactin profiles following M B H injections of noradrenaline, dopamine, adrenaline and saline are illustrated in Fig. 3. Mean plasma hormone concentrations are shown in Table II. Catecholamine injections failed to affect G H secretion. All 3 catecholamine stimulated prolactin secretion but there were noticeable differences in the amplitude of the effect. The ascending order of potency was: dopamine, noradrenaline, adrenaline.
1 L~
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.
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Fig. 2. Catecholamine-fluorescence micrographs of control (a) and 6-OHDA (b) injected sides of a hypothalamus from an animal 1 day after treatment. The PO/AHA (I a and I b) was not affected by M BH injections whereas in tile MBH anteriorly (2a and 2b) and posteriorly (3a and 3b) pronounced depletion of catecholamine fluorescent fibers is evident. In 3b, dopamine neurons of the A12 group appear a little more prominent than on the control side, because of the paucity of brightly fluorescent catecholamine fibers in the adjacent neuropil. A damaged swollen catecholamine-containing fiber is evident in 3b (center) as weft as a few apparently intact fibers. 800
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Fig. 3. Mean (~: S.E.M.) group GH and prolactin secretory profiles over 90 min following bilateral MBH injection of dopamine, noradrenaline, adrenaline (0.5/d, 0.032 M solutions) or saline; n = 8 per point.
217 TABLE II Effect of bilateral MBH injections (0.5 td) of dopamine, noradrenaline and adrenaline (0.032M solutions) on mean plasma GH and prolactin concentrations (ng/ml d_ S.E.M.) n
Saline 8 Dopamine 8 Noradrenaline 8 Adrenaline 8
GH
Prolactin
124.5 4- 39.5 142.9 ~ 42.9 90.3 ± 27.9 78.1 ~ 16.1
21.3 -I- 2.7 35.8 i 4.5* 68.7 4- 6.6** 158.6 ± 19.7"*
Significantlydifferent from saline control, * P < 0.01, ** P < 0.001. Paired Student's t-test. DISCUSSION Prolactin secretion
In addition to the inhibitory influence of tuberoinfundibular dopamine neurons~S,16,31,3z, central catecholamine structures also appear to play a facilitatory role in the regulation of prolactin secretionZT,32. Although our knowledge of the location of prolactin-regulating catecholamine structures is incomplete, it has been reported that elevated plasma prolactin levels in proestrous and ovariectomized steroid-primed animals coincide with increased noradrenaline turnover in the MBH ~7. Electrical stimulation studies have also implicated MBH structures, apart from the arcuate, as being significant in the regulation of prolactin: stimulation of the VMN has been reported to induce acute prolactin release a4 and to trigger the onset of pseudopregnancy-associated prolactin surges 11. In the present study MBH injections of all 3 catecholamine (Expt. II) produced statistically significant increases in plasma prolactin levels. In contrast to this observation, MBH 6-OHDA treatment was found to have no effect upon basal hormone profiles, despite producing a severe disruption of local catecholamine afferents. This suggests that although 6-OHDA-sensitive MBH catecholamine afferents are not important to the regulation of basal patterns of prolactin secretion, this region may contain a catecholamine mechanism which subserves the generation of prolactin surges in response to certain environmental or endogenous stimuli. In accord with this proposal it has been reported that depletion of central catecholamine levels prevents
stress induced prolactin rises 1° and the afternoon prolactin surge observed in ovariectomized steroidprimed rats 35. One explanation of the differing potencies of dopamine, noradrenaline and adrenaline in stimulating p~ olactin secretion (see Fig. 3) may be that the MBH prolactin-facilitatory catecholamine mechanism involves receptors for which adrenaline is the normal ligand. The quantities of dopamine and noradrenaline injected into the MBH were conceivably sufficient to cause some degree of stimulation of these receptors despite a lower receptor affinity. Furthermore the order of agonist potency reported for az-adrenergic receptors is adrenaline > noradrenaline > > dopamine 3. Although MBH 6-OHDA injections (Expt. I) destroyed local catecholamine afferents, they had no apparent effect upon arcuate dopamine neurons. The resistance of these neurons to the toxic effects of 6-OHDA has been noted previouslyS, 6,36 and is attributed to the lack of a high affinity uptake system for catecholamine 8. Given the considerable evidence that tuberoinfundibular arcuate dopamine neurons are tonically inhibitory to prolactin secretion tS,16,22,at,3a, the observation that prolactin secretion was normal at days 1 and 7 may be viewed as further evidence of the insensitivity of these neurons to 6-OHDA. G H secretion
As catecholamines do not act directly upon the pituitary to influence G H secretion 2s their role in the regulation of this hormone presumably stems from their ability to affect the activity of SRIF and/or G R F neurons. We have previously reported that, in the rat, median eminence catecholamine afferents are inhibitory to G H secretion, probably as a result of a facilitatory effect upon SRIF terminals 6. Although there is some evidence that the facilitatory role of catecholamine in G H regulation involves a stimulatory influence upon G R F neurons as well as an inhibitory input to SRIF neuronsl, aT, the present findings indicate that it is unlikely that MBH G R F perikarya receive a direct input from central catecholamine neurons. The possibility remains, however, that CNS catecholamine structures facilitate G R F activity via interneurons whose perikarya lie outside of the
218 M BH. For example, periventricular P O / A H A S R I F n e u r o n s may be the inhibitory interneurons by
cholamine antagonists suppress G H secretion in animals passively i m m u n i z e d with S RI F antiserum ~,
which catecholamine influence G R F release. It is
:% in these studies, S R I F antiserum was administered into the peripheral circulation, and thus
k n o w n that S R I F n e u r o n s project to the V M N arcuate region e a n d that periventricular P O / A H A n e u r o n s receive a catecholamine i n n e r v a t i o n 2~. Such
would be likely to leave any central S R I F mechanisms unaffected.
an a r r a n g e m e n t would provide a simple means of regulating the rhythmic pattern which characterizes
ACKNOWLEDGEMENTS
G H secretion. W h e n P O / A H A catecholamine afferents are active, median eminence S R I F release and the proposed inhibitory S R I F i n p u t on G R F n e u r o n s would both be blocked, thus permitting a G H surge. W h e n P O / A H A catecholamine afferents are inactive median eminence S R I F release and S R I F i n h i b i t i o n of G R F n e u r o n s would be maximal and a G H trough would result. Even though cate-
R a d i o i m m u n o a s s a y materials were kindly provided by Dr. A. Parlow, N I A M D D . This work was supported by grants to J.O.W. from the N a t i o n a l Health a n d Medical Research C o u n c i l o f Australia, T.A.D. was a Postdoctoral Fellow of the Neurosurgical Research F o u n d a t i o n of South Australia.
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