Catecholamines, the hypothalamus and neuroendocrinology — applications of electrochemical methods

60 reach their regulatory centres in the CNS, and thus this would not be an efficient route for the system to use. With the recognition of the structure and angioarchitecture of the median eminence, the topographical relations of the pituitary gland and the surrounding extracellular space reveal a system for n e u r o h u m o r a l regulation built up in an outstanding way. What is most outstanding is its simplicity. Nature avoids being complicated.

Reading list 1. Ambach, G., Palkovits, M. and Szent~gothai,J. (1978) Acta Morph. Acad. Sci. Hung. 24, 93-119. 2. Berg,land, R. M. and Page, R. B. (1978) . Endocrinology 102, 1325-1338. 3. Bloom, F., Battenberg, E., Rossier, J., Ling, N. and Guinemin, R. (1978) Proc. Nat. Acad. Sci. U.S.A. 75, 1591-1594. 4. Broadwell, R.D. and Brightman, M.W. (1976) J. Comp. Neurol. 166, 257-284. 5. Dub6, D., Lissitzky, J.C., LeClerc, R. and Pelletier, G. (1978) Endocrinology 102, 1283-1291. 6. Fischer, A. W. F. and Price, P. G. (1976) Anat. Rec. 184, 403. 7. Green, D. and Harris, G. W. (1949) J. Physiol. (Lond.) 108, 359-361. 8. Harris, G. W. (1955) Neural Control of the Pituitary Gland, Edward Arnold, London. 9. Krieger, D.T.,Liotta, A. andBrownstein, M. J. (1977) Brain Res. 128, 575-579. 10. Mezey, I~., Palkovits, M., De Kloet, E.R., Verhoef, J. and de Wied, D. (1978) Life Sci. 22, 831-838. 11. Moldow, R. and Yalow, R. S. (1978) Proc. Nat. Acad. Sci. U.S.A. 75, 994--998. 12. Nakai, Y. and Naito, N. (1975) In: K.M. Knigge, D. E. Scott, H. Kobayashi and S. lshii (eds) Brain-Endocrine Interaction. 1l. The Ventricular System. Karger, Basel, pp. 94--108. 13. Oliver, D., Mical, R. S. and Porter, J. C. (1977) Endocrinology 101, 598-604. 14. Page, R. B. and Bergland, R. M. (1976) Vth Int. Congr. Endocrinol., Hamburg, Abst. 502. 15. Page, R. B. and Bergland, R. M. (1977)Am. J. Anat. 148, 345-358. 16. Popa, G. and Fielding, V. (1930) Lancet ii, 238-240. 17. Rodriguez, E. M. (1969) Z. Zellforsch. Mikrosk. Anat. 102, 153-171. 18. Szentagothai, J., Flerk6, B., Mess, B. and Hal6sz, B. (1968) Hypothalamic Control of the Anterior Pituitary, Akad6miai Kiad6, Budapest. 19. T6r6k, B. (1962)Anat. Anz. 109, 622-629. 20. Tamu, G., Leonardelli, J. and Dubois, M.P. (1977) Neurosci. Lett. 6, 305-309. 21. Wislocki, G. B. (1937) Am. J. Anat. 61, 95-129. 22. Wislocki, G. B. and King, L. S. (1936) Am. J. Anat. 58, 421--472. 23. Worthington, W. C. (1960) Endocrinology 66, 19-31. 24. Zimmerman, E.A., Liotta, A. and Krieger, D. T. (1978) Cell Tiss. Res. 186, 393-398.

The authors are members of the 1st Department of Anatomy, Semmelweis University Medical School, 1450 Tiizolt6 utca 58, Budapest IX, Hungary. El~vter/North-HoUandBiomedicalPr~a 1979

TINS - March 1979

Catecholamines, the hypothalamus and neuroendocrinology applicati s of electrochemical methods -

Jimmy D. Neill, Paul M. Plotsky and Wim J. de Greef

Hormone secretion from the anterior pituitary gland is regulated by the hypothalamus. This regulation is accomplished by secretion o f neurally-produced hypophysiotropic hormones into the vascular portal system connecting the hypothalamus with the anterior piadZary, thus functionally linking the nervous and endocrine systems. The three hypophysiotropic hormones isolated and synthesized to date have proven to be small peptides. They are thyroid stimulating hormone-releasing hormone (TRH), luteinizing hormone-releasing hormone ( L H R H ) and growth hormone release-inhibiting hormone (somatostatin). Schally and Guillemin shared the 1 977 Nobel Prize in Physiology and Medicine for their work in elucidating the nature o f these substances. This article describes electrochemical methods which have been used to establish dopamine as the fourth hypophysiotropic hormone, one which is involved in the inhibition o f prolactin secretion from the anterior pituitary gland. These methods were originally developed in the laboratory of Ralph Adams at the University o f Kansas ~ and have been adapted and successfully applied in our laboratory to some problems confronting neuroendocrinology. The science of neuroendocrinology experienced a rebirth in 1955 with the publication of Geoffrey Harris' m o n o graph 5, 'Neural Control of the Pituitary Gland'. This work illustrated the dependence of pituitary h o r m o n e secretion upon proximity to the brain. Since no neuronal connections exist between the brain and the anterior pituitary, he proposed that regulation occurred v/a the short vascular pathway which linked these two organs. According to his chemon e u r o h u m o r a l hypothesis, hypothalamic substances (hypophysiotropic h o r m o n e s ) were secreted by nerve endings of the hypothalamic median eminence into this specialized vascular communication system. While catecholamines were considered as h o r m o n e candidates, work in this direction was overshadowed by efforts to isolate and identity peptide h o r m o n e s concerned with regulation of pituitary function. Catecholamines were relegated to the role of classical neurotransmitters involved in the release of various hypothalamic peptide hormones.

catecholamine distribution within nervous tissue, renewed speculation as to the role of these monoamines, specifically dopamine, in neuroendocrine control. Dopaminergic cell bodies were found in the arcuate and periventricular nuclei of the hypothalamus. Their processes extended ventrally to the median eminence, ending in close proximity to the portal capillary network. T h u s a morphological basis for dopamine participation in pituitary regulation was provided. Early physiological studies showed that removal of the pituitary from hypothalamic influence, such as autotransplanting the gland under the kidney capsule, resulted in greatly e n h a n c e d prolactin secretion suggesting that the hypothalamus provided a negative influence to the pituitary as far as prolactin secretion was concerned 6. Pharmacological evidence supported the view of an inverse relationship between hypothalamic catecholamine content or turnover and prolactin secretion. Agents depleting brain catecholamines (e.g. reserpine, a-methyl-p-tyrosine) resulted in Prolactin secretion control by doimmine increased plasma prolactin levelsL FurT h e observations of Fuxe =, utilizing the thermore, Shaar and Clemens 1° showed histofluorescent m e t h o d for visualizing that, in vitro, dopamine could act at the

61

TINS - March 1979

level of the pituitary to inhibit prolactin secretion. Subsequently, specific dopamine receptors were identified in the pituitary. These diverse lines of evidence led to the much-debated question: Is dopamine acting directly on the pituitary gland as the long sought-after prolactin inhibiting factor (PIF) or does it regulate the secretion of a putative peptide PIF at the hypothalamic level? Although this issue remains incompletely resolved, we will present evidence suggesting that dopamine acts in both capacities. When our studies began several years ago the crucial evidence missing to establish dopamine as a hypophysiotropic hormone was two-fold: (1) demonstration that'dopamine was secreted by the median eminence into the portal vessels linking the brain and pituitary, and (2) demonstration that the quantity of dopamine present in hypophysial portal blood was sufficient to account for inhibition of prolactin secretion by the pituitary gland in v i v o . While the technique of collecting this portal blood had been developed by John Porter in Dallas9, analytical methods for determining low levels of catecholamines in small plasma samples were not readily available. Thus, before these critical studies could be undertaken, a suitable catecholamine assay had to be developed. Dopamine assay The liquid chromatographic-electrochemical (LCEC) method ~ developed by Ralph Adams for tissue levels of catecholamines was chosen due to its simplicity, rapidity, and. sensitivityL Briefly, catecholamines from the plasma sample are adsorbed onto activated alumina, eluted with perchioric acid, and then injected into a high performance liquid chromatographic column (2 × 1000mm) packed with a strong cation exchange resin. This effects the separation of dopamine, adrenaline and noradrenaline (Fig. 1). Detection results from the oxidation of the catecholamine molecules at the surface of a carbon paste electrode attached to the effluent end of the chromatographic system. The electrochemical current generated by this oxidation is linearly related to catecholamine content from 20 pg to at least 10 ng. Specificity is provided by: the limited number of compounds having oxidation potentials similar to that of dopamine; the preferential adsorption of catechol molecules onto alumina; and the retention volume of dopamine on the ion exchange column.

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Portal vessel cannulatlon The initial experiments were designed to determine whether dopamine was secreted into the portal vessels. The pituitary gland was exposed in pentobarbital- or urethane-anaesthetized female rats using the microsurgical parapharyngeal approach. Dissection was used to expose the basi-sphenoid bone overlying the.pituitary (Fig. 2), and then a hole was drilled through the bone to expose the pituitary. The infundibulum (pituitary stalk), upon which the portal vessels were located, was cut and the flared tip of a polyethylene cannula was placed over the distal end of the stalk. Blood was withdrawn for 1 h at 6-8/~l/min via a syringe withdrawal pump. Plasma samples were then prepared for dopamine measurement as described above. The dopamine concen-

tration in hypophyseal stalk plasma of ten female rats was found to be 6.0 - 1.1 ng/ml(mean - standard error). Dopamine concentrations in peripheral plasma collected from the same rats were undetectable (<0.6 ng/ml). The higher concentration of dopamine in hypophyseal stalk plasma compared with that in peripheral plasma strongly supports the view that dopamine is a secretory product of the median eminence. In contrast, adrenaline and noradrenaline levels were identical in peripheral and stalk plasma - thus they cannot be considered on these criteria to be secretory products of the median eminence, although they, like dopamine, are found in high concentration there. In order to determine the physiological significance of these stalk blood dopamine levels in the tonic inhibition of pituitary

62

T I N S - March 1979

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portal system does not appear to be involved in the dynamic regulation of prolactin secretion. However, the possible importance of patterning of dopamine secretion cannot be overlooked. Changes in the rate of dopamine secretion occurring with a duration significantly less than the 15-rain sampling period could not be detected. More frequent sampling is not feasible at present due to limitations in the sensitivity of the available assays. Preliminary work in our laboratory suggests that optimization of various aspects of the LCEC (e.g. redesign of the carbon electrode detector) may increase the sensitivity of the method 5-10-fold.

',;S /n situ flopamine measurement technique

Fig. 2. Dia•rammatic representati•n •f the surgica• pr•cedure used t• exp•se the pituitary gland and t• c••lect hypophyseal stalk blood. Dura, dura mater: ME, median eminence o[ the hypothalamus; OC, optic chiasm; Stalk, hypophyseal stalk; Pit, pituitary gland.

prolactin secretion, the classical removal and replacement paradigm was employeds. Rats were pretreated with a-methyl-p-tyrosine, a competitive inhibitor of catecholamine synthesis. This treatment results in a rapid 100-fold increase in plasma prolactin levels, while reducing stalk blood dopamine levels to undetectable levels. In another group of t~-methyl-p-tyrosine treated rats, dopamine was infused peripherally at a rate experimentally determined to produce normal levels of stalk blood dopamine. These infusions suppressed prolactin secretion by 70% within 30 min (Fig. 3). Similar results were obtained when the median eminence was electrolytically lesioned, a manipulation which leads to elevation of prolactin secretion quantitatively similar to that caused by ,~-methyl-p-tyrosine treatment. These experiments establish dopamine as a physiological prolactin inhibiting factor (PIF), acting directly on the pituitary gland. However, since the infusion of dopamine did not reduce prolactin secretion to baseline values, the existence of other dopamine-dependent or independent PIFs cannot be ruled out.

I ) o ¢ - , , ~ aed pro~-t~ rek~e d . , , ~ meUlae Having fulfilled the criteria necessary to establish D A as a physiological PIF, we designed a series of experiments to test the simplest hypothesis for neuroendo-

crinological control: Is dopamine secretion into the portal blood inversely correlated with pituitary prolactin secretion? Is dopamine the dynamic regulator of prolactin secretion? Among the physiological stimuli which evoke prolactin secretion, suckling of the nipples by the young is considered most significant. During suckling stimulation, plasma prolactin levels rise within 3 min and remain substantially elevated during the suckling episode. Lactating rats were anaesthetized with urethane and the mammary nerve was surgically isolated. Electrical stimulation of this nerve for 15 min produced a pattern of prolactin secretion (Fig. 4) similar to that observed during normal suckling of the unanaesthetized lactating rat. In a second group of lactating rats, the pituitary was exposed and hypophyseal portal blood was collected before, during, and after the stimulation period, which lasted for 15 rain. Preliminary results from two animals are illustrated in Fig. 4, showing that no alteration of dopamine secretion into portal blood occurred during this major alteration in the rate of prolactin secretion. A similar lack of correlation between the secretion of the two hormones has been observed during prolactin surges evoked by pharmacological stimulation (histamine and acetylcholine) and by mating'. Thus, within the constraints of this experimental approach, dopamine secretion into the hypophyseal

The possibility of a dynamic role for dopamine in the regulation of prolactin secretion is currently being explored with another electrochemical approach -in situ catecholamine-sensitive microelectrodes. These electrodes (tip diameter 30-80 p.m) are fabricated from carbonfilled glass micropipettes and are implanted directly into the surgicallyexposed median eminence. The principles governing these electrodes are quantitatively similar to those for the LCEC detector electrode with three major exceptions: (1) the microelectrode potential is applied as a square-wave pulse while the LCEC detector potential is constant, (2) the potential applied to the microelectrode (+0.40V v. Ag/AgCl) is much lower than that applied to the LCEC detector (+0.65 V), and (3) a steadystate, diffusion-limited condition is assumed to occur at the microelectrode surface, whereas hydrodynamic flow (non-diffusion limited) conditions exist at the LCEC detector surface. Oxidation of electroactive molecules diffusing to the electrode surface generates a current

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63

T I N S - March 1 9 7 9

proportional to the concentration of the electroactive species in solution. The specificity of the microelectrode is determined by the applied potential. Large molecules (peptides, proteins) are usually not detectable due to their slow diffusion and unavailability of their electroactive groups for electron transfer. At the potential chosen, + 0 . 4 0 V v. Ag/AgC1, the following molecules present in the tissue matrix may contribute to the signal: dopamine, noradrenaline, adrenaline, dihydroxyphenylacetic acid (DOPAC), and ascorbic acid. In vivo experiments with the microelectrodes implanted in the median eminence confirmed that the measured current responded in expected fashion to various pharmacological agents. Administration of tx-methyl-p-tyrosine (200 mg/kg, i.p.), a catecholamine synthesis blocker, caused a 43% _ 7.3 decrease in the signal within 15-30 min. Direct application of 10-'M acetylcholine or electrical field stimulation of the median eminence caused a transient 2-3-fold increase in the current signal. In an attempt to define which molecular species were responsible for these changes in electrochemical current, in vitro, median eminence incubation experiments were undertaken'. After isolation, the median eminences were individually maintained in a static incubation of physiological salt solution and supplied with glucose and oxygen. Aliquots of this medium were withdrawn at 10rain intervals before, during, and after experimental manipulation (acetylcholine application or electrical field stimulation). a-methyl-p-tyrosine treated 140 t20

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Only a transient decrease in the concentration of electroactive species occurred during the period of stimulation. The physiological significance'of this decline is unknown, but its occurrence would be completely missed using the stalk blood collection approach. Striking alterations in the pattern of electroactive species concentration were observed within 6 ~ n o . 1 20 rain of cessation of nerve stimulation. T~o41 ~i ~ 1 .__._.. Rat The periodicity of these changes is of the order of a few minutes. The average .~o 0 | . . . . . . . . . concentration of electroactive species appeared to be elevated at this time. Again, physiological significance cannot .~ 220 yet be assigned to these observations. 200 Ii ", e~ .~100 ]l ~ill'" t n l ~ o t " 3 However, these data highlight the importance of achieving adequate time resolution in studies of this complex system ~ 60 ~ ' ~ . ~ t ~'~i ..~p Rat resolution which is presently afforded ° i ~ ',.~ 'N no 4 [] ,0 ~.~: solely by electrochemical monitoring ,, 20 xw~,,""l techniques. Major questions remain to be addressed 0 -30-20-10 | . . . 0. .I0 . 20. 30 . . 40. 50 60 with regard to the meaning of these Time (rain) Fig. 4. Top Panel. Effect of electrical stimulation o f measurements. The origin and function of the dopamine component of the elecan isolated mammary nerve trunk on prolactin secretion in urethane-annesthetized rats. Middle trochemical signal is unclear. This Panel. Stalk plasma dopamine concentrations dopamine may be neurotransmitter overraeusured with the LCEC method in two rats before, flow or ma~ represent a special pool during, and after mammary nerve stimulation. Bottom Panel. Electrochemical recordings from two secreted into stalk blood. Resolution of rats with catechoiamine-sensitive electrodes inserted this question is necessary before full and into the median eminence of the hypothaiamus before, meaningful interpretation of our results is during, and after mammary nerve stimulation. The possible.

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These samples were analysed by LCEC for dopamine, adrenaline, noradrenaline, DOPAC, and ascorbic acid. The results clearly indicated that the species undergoing changes were dopamine and DOPAC. While ascorbic acid was present in all samples, it maintained a stable concentration, and thus provided a steady-state background current. These studies and those undertaken by Adams' group in Kansas ~ strongly support the assumption that changes in the levels of catecholamines and their metabolites are reflected in the electrochemical signal.

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Reading list 1. Adams, R. N. (1976) Anal. Chem. 48, l126A-1137A; (1978) Trends NeuroSci. 1, 160-164. 2. Fuxf, K. (1964) Z. Zellforsch. Mikrosk. Anat. 61,710-724. 3. Gibbs, D. M. and Neill, J. D. (1978) Endocrinology 102, 1895-1900. 4. de Greef, W. J. and Neill, J. D. (1978) Society for Neuroscience Abstracts 4, 343. 5. Harris, G. W. (1955) Neural Control o f the Pituitary Gland Arnold, London. 6. Neill, J. D. (1974) In: E. Knobil and W.H. Sawyer (eds), Handbook of Physiology Section 7, Vol. IV, Part 2, American Physiological • Society, Washington, D.C., pp. 469-488. 7. Plotsky, P. M., Gibbs, D. M. and Neill, J. D. (1978) Endocrinology 102, 1887-1894. 8. Plotsky, P. M. and Neill, J. D. (1978) Endocrine Society Abstracts, p. 339. 9. Porter, J. C., Kamberi, 1. A. and Grazla, Y. R. (1971) In: L. Martini and W. F. Ganong (eds), Frontiers in Neuroendocrinology, 1971 Oxford University Press, London, pp. 145-175. 10. Shaar, C. J. and Clemens, J. A. (1974) Endocrinology 95, 1202-1212.

the median eminence With this assurance, we proceeded to repeat the mammary nerve stimulation experiments described earlier - this time with microelectrodes in the median eminence. Electrochemical measure- J. D. Neill is the William Timmie Patterson Professor ments were taken at 1 min intervals of Physiology and P. M. PIotsky a PhD candida~ in throughout the experiment, and the Department of Physiology, Emory University, peripheral blood samples for prolactin Atlanta, GA 30322, U . S . A . W . J . de Greef is a measurements were obtained at 15 min Visiting Assistant Professor; his permanent address is Department of Endocrinology, Growth, and Reintervals via a femoral cannula. The production, Erasmus University, Rotterdam, The results obtained are displayed in Fig. 4. Netherlands.