150

Brain Research, 86 (1975) 150-154 ,~Z.Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Thyrotropin releasing hormone (TRH): depressant action on central neuronal activity

L. P. RENAUD AND J. B. MARTIN Division of Neurology, Montreal General Hospital and McGill University, Montreal (Canada)

(Accepted December 3rd, 1974).

Available evidence from physiological and anatomical studies indicates that neural regulation of adenohypophyseal secretion is mediated by 'releasing' or 'inhibiting' peptide hormonesZ, tS. The identification and structural elucidation of 3 of these peptides, i.e., thyrotropin releasing hormone (TRH) 4,13, luteinizing hormone releasing hormone (LH-RH)I, 17 and growth hormone release-inhibiting hormone (SRIF or somatostatin)Z has facilitated their localization in brain. With respect to TRH, the use of a specific sensitive radioimmunoassay has revealed that TRH, although in highest concentration in the hypothalamus, is widespread in the central nervous system of both mammals t9 and submammalian chordates 9. It appears that behavioral changes can follow systemic administration of both T R H 14-16 and L H - R H 12, which in the case of T R H are independent of any action on the pituitary 15 or thyroid glands 14. These findings have heightened the suspicion that these, and other polypeptides may have a role in brain function, possibly as neurotransmitters. We wish to report some preliminary findings which indicate that T R H has a depressant action on the activity of central neurons in several areas of the central nervous system, including the cerebellar cortex where there is no appreciable concentration of T R H according to radioimmunoassay 19 nor detectable high affinity T R H binding sites 5. Experiments were performed on male Sprague-Dawley rats anesthetized with intraperitoneat pentobarbital (35 mg/kg with supplemental i.v. doses) or urethane (1.25 mg/kg in 2 5 ~ w/v solution). Action potentials were recorded from single neurons located in 4 areas of the central nervous system: the parietal cortex, the cerebellar cortex in the region of the posterior lobe vermis, a dorsal column (cuneate) nucleus, and the ventromedial hypothalamus. The first 3 areas were exposed by removal of the overlying bone, and pulsations minimized by gentle pressure of a small perspex plate against the surface of the brain. All exposed surfaces were perfused continuously with a mammalian Ringer's solution maintained at body temperature. The ventral surface of the hypothalamus was exposed by a direct retropharyngeal approach, and unit activity recorded from neurons located on either side of the median eminence. Subsequent histological studies verified that many of these hypothalamic units were located within the ventromedial and arcuate nucleus.

151

TABLE I RESPONSE OF THE SPIKE DISCHARGE FREQUENCY OF CENTRAL NEURONS TO

TRH

APPLIED BY MICROION-

TOPHORE~IS

Recording site

No. of cells tested

Cuneate nucleus Cerebellarcortex Parietal cortex Ventromedial hypothalamus

43 56 36 41

Change in firing frequency Depression

No effect

12 19 17 21

31 37 19 20

(28~) (34~) (47~) (51~)

(72~) (66~) (53~) (49~)

Single neuronal activity was studied using 2 types of glass micropipettes. For areas with relatively large neurons, i.e. cerebral and cerebellar cortices and the cuneate nucleus, extracellular action potentials of suitable amplitude could be recorded through 3.0 M NaC1 contained in the center channel o f multibarrelled glass micropipettes (tip outer diameter < 5 #m), while the other channels were filled with compounds for microiontophoresis. Action potentials from smaller units in these areas, and from all cells studied in the hypothalamus, were recorded through single 3.0 M NaC1 filled micropipettes (tip outer diameter < 1/~m) rigidly fixed to the shaft of multibarrelled micropipettes s o that the tip of the single electrode protruded 10-20 # m beyond the tip of the multibarrelled electrode. After amplification, action potentials were fed through an adjustable voltage gate to a frequency counter whose output was connected to a polygraph. Micropipette channels for iontophoresis were filled with sodium L-glutamate (1.0 M, p H 7.0), T R H (10 m M in distilled water, p H 6.5-7.0), bicuculline hydrochloride (5 m M in 165 m M NaC1, p H 5.0), strychnine

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Fig. 1. Depressant action of TRH in the cerebellar cortex. Continuous traces of the spontaneous activity of a cerebellar Purkinje neuron indicate a decrease in firing frequency and increase in spike height during iontophoretic application of 2 doses of TRH ( ). Note the brief delay in onset and abrupt termination of action of TRH.

152

A HYPOTHALM AUS 30

20

10

40

30

30

20

B CEREBRALCORTEX 40

30sec

C CUNEATE No 40

TRH 6

7

10

12

30 see

Fig. 2. Action of TRH on glutamate-evoked activity. The polygraph records taken from individual neurons in the ventromedial hypothalamus (A), cerebral cortex (B) and cuneate nucleus (C) indicate a dose related depression of firing frequency of glutamate-evoked activity (---) during administration of TRH ( ). The upper 2 records indicate some prolongation in recovery to control excitability levels after larger or longer TRH applications. The vertical bar on the right indicates number of spike counts per single bin. Data from the lower 2 traces are included in the log current-response curves in Fig. 3.

sulfate (5 m M in 165 m M NaCI, pH 5.5) and sodium chloride (165 mM), and connected to constant current sources. A total of 176 neurons were studied in detail and distributed as shown in Table I. In the population of cells whose spontaneous or glutamate-evoked activity was influenced by TRH the response was consistently a depression of firing frequency which in more than 70 70 of cells was accompanied by an increase (up to 30 70) in the height of the spike (cf Fig. 1). Characteristic of the action of TRH on the majority of TRH-responsive neurons was its relatively brief onset of action and ready reversibility. A detectable depression in firing frequency could usually be achieved with a relatively low range (5-20 nA) of iontophoretic currents (Fig. 2). After cessation of brief (10-20 sec) TRH applications with ejection currents sufficient to cause greater than 50 ~ reduction in firing frequency, a rapid recovery of firing frequency was observed for most neurons. A small number of neurons (10 cells) whose activity was evoked b y periodic 10-20 sec applications of L-glutamate repeatedly exhibited a prolonged recovery period (Fig. 2A, B) which could be overcome by increasing the amount of current used to release glutamate. Control ejections of positive ions (sodium) had no similar effect on cell excitability. The observation that depressions of activity were often evident at lower ranges of ejection currents during experiments where multibarrelled micropipettes (as op-

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Fig. 3. TRH log current-response curves. Depression of control glutamate-evoked spike discharge frequency is plotted as a function of the log of the currents (in nA) used to release TRH (cfi Kelly and Renaud, ref. 10). Data were obtained from 3 cuneate neurons recorded with the same multibarrelled electrode, and 2 cortical neurons recorded with another similar electrode. The lines through the points for each cuneate (.... ) and cortical ( ) neuron were drawn by eye.

posed to combination single plus multibarrelled micropipettes) were used might be partly explained by the closer apposition of the orifice o f iontophoretic channels to the recorded neuron, and, therefore, shorter diffusion distances. However, adjacent TRH-responsive neurons recorded and tested with the same microelectrode did display different log current-response curves for T R H (Fig. 3), which might reflect differences in the sensitivity o f individual neurons to TRH. The brevity of the onset and termination of TRH-induced depression of activity is similar to the depressive effects associated with electrophoretic administration of the short-chain monocarboxylic omega amino acids, gamma-aminobutyric acid and glycine. Simultaneous applications o f their respective antagonist compounds, i.e. bicuculline 6 or strychnine 7, for several minutes with iontophoretic currents of 40 nA and 160 nA respectively (sufficient to block the responses to iontophoretic gamma-aminobutyric acid 10and glycine 11 in the cuneate nucleus) had no significant effect on either neuronal firing frequency or TRH-evoked depression in 8 TRH-responsive neurons tested with both compounds. This would suggest that T R H exerts its effect through other specific membrane receptors. In this respect it is interesting that Burt and Snyder 5 have recently reported specific high affinity T R H binding sites in rat brain. Their failure to detect high affinity binding in the cerebellum 5 might have some explanation in the fact that both cerebellum and brain stem appear to contain lower numbers of TRH-responsive neurons (Table I) and lowest values of endogenous T R H xg. The postulate of an important role for T R H in the mammalian brain has received support with the reports of TRH-induced behavioral activity x4-16 (however, cf ref. 8) and evidence o f widespread distribution o f T R H in neural tissueg, tg. The argument is now considerably strengthened by data for high affinity T R H binding sites in both pituitary and in brain 5 and by the detection of a potent depressant action of T R H on the activity of a certain population o f neurons in several areas of the

154 central nervous system. Since T R H is also f o u n d in the b r a i n of some s u b m a m m a l i a n chordates 9 further studies m a y indicate that its role on the p i t u i t a r y - t h y r o i d axis is b u t one facet of a more general rcle of T R H in b r a i n function. We t h a n k A. O. Geiszler a n d W. F. White ( A b b o t t Laboratories, Chicago) for supplies of T R H , Mr. Brian M a c K e n z i e for technical assistance, a n d the Medical Research C o u n c i l for financial support.

1 AMOSS,M., BURGUS,R., BLACKWELL,R., VALE, W., FELLOWS,R., AND GUILLEMIN,R., Purification, amino acid composition and N-terminus of the hypothalamic luteinizinghormone releasing factor (LRF) of ovine origin, Biochem. biophys. Res. Comman., 44 (1971) 205-210. 2 BLACKWELL,R. E., AND GUILLEMIN,R., Hypothalamic control of adenohypophyseal secretions, Ann. Rev. Physiol., 35 (1973) 357-390. 3 BRAZEAU,P., VALE, W., BURGUS,R., LING,N., BUTCHER, M., RIVIER, J., AND GUILLEMIN,R., Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science, 179 (1973) 77-79. 4 BURGUS,R., DUNS, T. F., DESIDERIO,D., WARD, D. N., VALE, W., AND GUILLEMIN,R., Characterization of ovine hypothalamic hypophysiotropic TSH-releasing factor, Nature (Lond.), 226 (1970) 321-325. 5 BURT,D. R., AND SNYDER, S. H., A second site for binding of thyrotropin releasing hormone to rat brain, Proc. Soc. Neuroscience, Fourth Annual Meeting, St. Louis, Mo., 1974, Abstract 101. 6 CURTIS, D. R., DUGGAN,A. W., FELIX, D., JOHNSTON,G. A. R., AND MCLENNAN,H., Antagonism between bicuculline and GABA in the cat brain, Brain Research, 33 (1971) 57-73. 7 CURTIS, D. R., HOSLI,L., ANDJOHNSTON,G. A. R., A pharmacological study of the depression of spinal cord neurones by glycine and, related amino acids, Exp. Brain Res., 6 (1968) 1-18. 8 HOLLISTER,L. E., BERGER,P., OGLE, F. L., ARNOLD,R. C., AND JOHNSON,A., Protirelin (TRH) in depression, Arch. sen. Psychiat., 31 (1974) 468-470. 9 JACKSON, I. M. D., AND REICHLIN, S., Thyrotropin-releasing hormone (TRH): distribution in hypothalamic and extrahypothalamic brain tissues of mammalian and submammalian chordates, Endocrinology, 95 (1974) 854-862. 10 KELLY,J. S., ANDRENAUD,L. P., On the pharmacology of the GABA receptors on the cuneothalamic relay cells of the cat, Brit. J. Pharmacol., 48 (1973) 369-386. ll KELLY, J. S., AND RENAUD.,L. P., On the pharmacology of the glycine receptors on the cuneothalamic relay cells of the cat, Brit. J. Pharmacol., 48 (1973) 387-395. 12 Moss, R. L., ANDMCCANN,S. M., Induction of mating behaviour in rats by luteinizing hormonereleasing factor, Science, 181 (1973) 177-179. 13 NAIR,R. M. G., BARRETT,J. F., BOWERS,C. Y., ANDSCHALLY,A. V., Structure of porcine thyrotropin releasing hormone, Biochemistry, 9 (1970) 1103-1106. 14 PLOTNIKOFE,N. P., PRANGE,JR., A. J., BREESE,G. R., AND WILSON, I. C., Thyrotropin releasing hormone: enhancement of DOPA activity in thyroidectomized rats, Life Sci., 14 (1974) 1271-1278. 15 PLOTNIKOFF,N.P., PRANGE,JR., A.J., BREESE, G.R., ANDERSON, M.S., AND WILSON, 1. C., Thyrotropin releasing hormone: enhancement of dopa activity by a hypothalamic hormone, Science, 178 (1972) 417-418. 16 PRANGE,A. J., The Thyroid Axis, Drugs, and Behaviour, Raven Press, New York, 1974. 17 SCHALLY,A. V., ARIMURA,A., BABA,Y., NAIR,R. M. G., MATSUO,J., REDDING,T. W., DEBELJUK, L., AND WHITE, W. F., Isolation and properties of the FSH- and LH-releasing hormone, Biochem. biophys. Res. Commun., 43 (1971) 393-399. 18 SZENT.~GOTHAI,J,, FLERKO,B., MESS,B., AND HALASZ,B., Hypothalamic Control of the Anterior Pituitary, Akademiai Kiado, Budapest, 1968. 19 WINOKUR,A., AND UTIGER, R. D., Thyrotropin-releasing hormone: regional distribution in rat brain, Science, 185 (1974) 265-267.

Thyrotropin releasing hormone (TRH): depressant action on central neuronal activity.

150 Brain Research, 86 (1975) 150-154 ,~Z.Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands Thyrotropin releasing hormo...
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