Behavioural, electrocortical and body temperature effects after intracerebral infusion of TRH in fowls

European Journal of Pharmacology, 50 (1978) 253--260

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© Elsevier/North-Holland Biomedical Press

BEHAVIOURAL, ELECTROCORTICAL AND BODY TEMPERATURE EFFECTS AFTER INTRACEREBRAL INFUSION OF TRH IN FOWLS GIUSEPPE NISTIC(~ *, DOMENICANTONIO ROTIROTI, ANGELA DE SARRO and JOHN D. STEPHENSON ~"

Institute of Pharmacology, Faculty of Medicine, University of Messina, Italy, and t Department of Pharmacology, Institute of Psychiatry, University of London, London, U.K. Received 24 January 1978, revised MS received 14 April 1978, accepted 24 April 1978

G. NISTIC(), D. ROTIROTI, A. DE SARRO and J.D. STEPHENSON, Behavioural, electrocortical and body temperature effects after intracerebral infusion of TRH in fowls, European J. Pharmacol. 50 (1978) 253--260. In fowls TRH given into the III cerebral ventricle (0.25--5 #g) produced intense behavioural stimulation, electrocortical desynchronization and a slight increase in body temperature. In particular, an intense pattern of stereotyped head-neck movements, increase in locomotor activity, repeated pecking and preening, vocalization, erection of the tail feathers and occasionally 'escape responses' were observed. This picture lasted for about 30 rain and was followed by slight behavioural sedation during which stereotypies continued to occur but to a lesser extent. Similar increases in locomotor activity and stereotypies were evoked by infusing TRH into the hypothalamus whereas the unilateral microinfusion into the n. basalis or the n. mesencephalicus profundus, homologous to the mammalian striatum and s. nigra respectively, produced very intense stereotyped head-neck movements, wetdog syndrome and vocalization. TRH given into the other areas of the brain (e.g. hyper~triatum, neostriatum, olfactory ventricle, eminentia basalis and lateral part of the mesencephalon) lacked effects on behaviour and body temperature. The effects o f intraventricular infusion of TRH were antagonized by prior administration of haloperidol and spiperone whereas antagonists at a and ~-adrenoceptors and at 5-HT receptors were ineffective. In addition, TRH reversed sedation induced by intraventricular a-methyl-p-tyrosine. Behavioural and body temperature effects of TRH were independent of its endocrine properties since these were not observed after systemic or intracerebroventricular injection of thyrotropin, triiodothyronine and thyroxine. The increase in body temperature evoked by intracerebroventricular injection of TRH was due to activation of heat production and decrease in thermodispersive mechanisms. TRH

Behaviour

Electrocortical

Body temperature

1. Introduction

Thyrotropin releasing hormone (TRH) was first isolated from porcine hypothalamus (Schally et al., 1966) and subsequently found to possess pharmacological properties in addition to its effect on thyrotrophic hormone. Thus TRH increased spontaneous motor activity, respiratory rate and muscle tone, altered body temperature and reduced both * Correspondence to: Prof. Giuseppe NisticS, Institute of Pharmacology, Faculty of Medicine, Piazza XX Settembre 4, 98100 Medina, Italy.

Dopamine

Stereotypies

the sedative and hypothermic effects of several hypnotics (for references see De Wied, 1977; Miiller et al., 1977). TRH also potentiated the behavioural effects of monoamines and their precursors (Plotnikoff et al., 1972; Green and Grahame~mith, 1974; HuidobroToro et ah, 1975) and increased brain turnover of noradrenaline and dopamine (Keller et al., 1974; Fuxe et al., 1976; Marek and Haubrich, 1977) suggesting a possible involvement of brain catecholamines in some of the above actions of TRH. The present experiments were designed to clarify this relation by first comparing, in an

254

avian species, the behavioural, electrocortical and thermoregulatory effects of intracerebral injection of TRH with those reported after similar injections of noradrenaline, dopamine, clonidine, apomorphine, etc. (Marley and Nisticb, 1972, 1975a,b; Nisticb, 1976). Effects of substances which interfere with monoamines at both pre- and postsynaptic sites on responses to TRH were also studied. A short communication dealing with some aspects of this work has been published (Nisticb et al., 1977).

2. Materials and methods Rhode Island Red fowls (1.5--2 kg) housed at 20--25°C and young chicks (16--18 days old) maintained at an ambient temperature of 29--31°C were used. All operative procedures were under halothane anaesthesia. The outer or guide cannula was implanted stereotactically into the IIIrd ventricle, olfactory ventricle, hypothalamus, n. mesencephalicus profundus (homologous to the s. nigra), nucleus basalis (homologous to the mammalian striatum) and other brain areas of adult fowls, according to the atlas of Van Tienhoven and J u h ~ z (1962) or into the hypothalamus of young chicks, as described by Marley and Stephenson (1970). Methods for implanting electrocortical and nuchal electromyographic recording electrodes, for implanting a thermistor subcutaneously between the scapulae and for recording skin temperature from the unfeathered tarsometatarsal region of a hind-limb have been described (Dewhurst and Marley, 1965; Allen and Marley, 1967; Marley and Stephenson, 1970; Marley and Nisticb, 1972, 1975b). In some animals 2 cannulae were implanted, one into the IIIrd ventricle, the other into the hypothalamus. Young chicks were tested when recovery was complete and at least 24 h after the operative procedures. Adult fowls were not tested until at least one week after the operative procedures and thereafter at intervals of at least one week. Cannulae posi-

G. NISTIC0 ET AL.

tions were verified histologically at post mortem. Fowls were tested in a chamber (Angelantoni, Milan) the ambient temperature of which was maintained within (21--24°C) or below (0 ° ) their thermoneutral range. Electrocortical activity was automatically integrated at one minute intervals, large amplitude potentials producing high integral counts and alert low voltage electrocortical patterns giving low integrals (Marley and Stephenson, 1970). For recording carbon dioxide elimination, chicks were placed in a 1 litre chamber, maintained at thermoneutrality (31 ° C), through which CO:-free air, also at 31 ° C, was passed at a-rate of 1 litre/minute. The percentage of CO2 in the expired air, measured with an infrared analyzer (Hartmann and Bran) was recorded continuously. Drug infusions were made at a rate of 1 pl/ min. Those into the IIIrd ventricle were of 5 #1 volume; all other infusions were of 1 pl volume or less. Control infusion of the same volume of dist. H20 lacked effects on behaviour, electrocortical activity and body temperature. Drugs used were TRH (Pyr-His-Pro-NH2; Biodata S.p.A. Roma), thyroxine, triiodothyronine, thyrotropin, phentolamine HC1, phenoxybenzamine H C 1 , propranolol HC1, a-methyl-p-tyrosine methylester, methysergide bimaleate, haloperidol and spiperone.

3. Results 3.1. Infusion o f TRH into the IIIrd ventricle 3.1.1. Behaviour and electrocortical activity TRH infused into the IIIrd cerebral ventricle of adult fowls (number of experiments given in parentheses) in doses of 0.25 (3), 0.5 (3), 1 (5), 2 (6), 3 (3), 4 (3) and 5 #g (3) produced intense behavioural stimulation, electrocortical desynchronization and an associated decrease in electrocortical integrals (fig. 1); smaller d o s e s w e r e ineffective. Within

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3.1.2. Body and leg temperature, postural changes and respiratory rate

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Fig. 1. Effects of TRH (5 pg) infused into the IIIrd cerebral ventricle on electrocortical and electromyographic activity in an unanaesthetized adult fowl. (A) Control electrocortical activity. (B) Lower amplitude higher frequency potentials 7 min after TRH aasodated with behavioural excitation. (C) Electrocortical potentials returning to baseline activity 40 min after TRH. (D) Control EMG activity. (E) Presence of periodic muscle artifacts interrupting electromyographic activity and corresponding to head-neck movements 12 min after TRH. At the bottom: histogram of integrated minute by minute electrocortical activity showing in comparison to pretreatment values (180--280/mini a sustained decrease after TRH lasting about 30 min.

Concomitant with the above behavioural changes, intracerebroventricular infusion of TRH (total number of experiments = 12) in fowls kept at ambient temperatures within the thermoneutral range (21--24 ° C) produced a slight increase in body temperature; the mean increase was 0 . 7 6 -+ 0.19°C (mean -+ S.E.M.) with return to baseline values 45.5 +- 11.3 rain after infusion. At the same time leg temperature fell (maximum mean fall 1.54 ° C) and returned to control values after 92 -+ 20 min; respiratory rate was not modified (fig. 2). The temperature response was unrelated to the dose. Thus, only small increases were usually observed after each dose but in a few cases marked hyperthermic responses (maximum increase over 1.5°C) A ~.

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~ 40.5~ a few minutes of infusion, fowls showed intense stereotyped movements of the head and neck, repeated preening and pecking, erection of the tail feathers and an increase in locomotor activity which occasionally cul. minated in escape responses. Vocalization, uncommon in adult fowls isolated in the observation box, was frequent. Artifacts caused by the stereotyped head-neck movements were evident in the nuchal EMG (cf. fig. 1D and 1E). The above behaviour persisted for approximately 30 min and was followed by behavioural sedation lasting some 2 h during which stereotypies continued but to a lesser extent. The above effects were dose-related inasmuch as the number of head-neck movements within the first 30 rain after the infusion increased with increasing doses of TRH (from 0.5 to 3 #g). After 4 and 5/~g the number of

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Fig. 2. Effects of intraventricular TRH (0.5 #g) on body temperature, leg temperature and respiratory rate in an adult unanaesthetized fowl kept at an ambient temperature of 21°C. Immediately after TRH infusion, body temperature increased 0.55°C with return to baseline values after 30 min (A) while leg temperature decreased 3°C with return to baseline values after 160 min (B); respiratory rate did not change (C). In B: 'After 60 min'ffi 60 min after the interrupted line I.

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infusing TRH (0.1 and 0.5/~g) into the hypothalamus (3 experiments for each dose). The increase in body temperature (maximum (0.8 °) lasted 30-=40 min. Stereotyped behaviour (head-neck contralateral movements, etc.) wet
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Fig. 3. Effects o f intraventricular TRH ( 2 # g ) on body temperature, leg temperature and respiratory rate in an adult unanaesthetized fowl kept at an ambient temperature o f 21°C. As a consequence o f a large increase in body temperature (max. increase 2.45°C) (A), after an initial fall (--1.5°C) there was a rise in leg temperature (max increase 3°C) (B) and a sustained increase in respiratory rate (C). In B: 'After 120 min' = 120 min after the interrupted line//.

occurred after TRH 0.25 pg and 2 pg. In these instances the respiratory rate increased (120-150/min) during t h e fever, the wings were abducted about 45 ° from the trunk and there was a peripheral vasodilatation, as evidenced by an increase in leg temperature (fig. 3). 3.1.3. Body temperature effects below thermoneu trality TRH (1, 2 and 4 ~g) given into the IIIrd cerebral ventricle of fowls (3 experiments for each dose) at an environmental temperature of 0 °C produced slight hyperthermic responses, similar to those seen at thermoneutrality (see 3.1.2.). 3.2. Infusion of TRH into the hypothalamus, n. basalis, n. mesencephalicus profundus and lateral ventricle Similar motor, electrocortical and body temperature changes were also evoked after

3.3. Effects on carbon dioxide elimination At a thermoneutral environmental temperature of 31°C, TRH (1 and 2 ;zg) infused into the hypothalamus of chicks (3 experiments for each dose) increased locomotor activity, produced stereotypies, vocalization, hyperthermia (maximum increase 0.8°C + 0.1), etc. and increased carbon dioxide elimination by a mean of 49% from 43.4 + 0.09 ml/kg/min in the control period to a peak of 64.7 ml/kg/min 8 min after infusion with recovery some 10-15 min later (fig. 4). 3.4. Infusion o f TRH into other brain areas TRH (0.5, 1 and 2 ~g) infused into the hyperstriatum, neostriatum, olfactory ventricle, eminentia basalis and mesencephalon (at the level of n. rotundus) had no effect on

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TRH CENTRAL EFFECTS

257

behaviour and body temperature (at least 3 experiments for each site).

3.5. Antagonism of the effects of TRH Haloperidol (0.5 mg/kg i.v.) and spiperone (0.05 mg/kg i.v.) given 15 min before TRH (0.5, 1, 2 and 3 #g) either markedly attenuated or completely abolished the increases in locomotor activity, vocalization and stereotypies and the increase in body temperature caused by the peptide; alone haloperidol and spiperone had no effect on behaviour (see also Ranje and Ungerstedt, 1977). Haloperidol and spiperone, given in the same doses 15 min after intraventricular TRH (2/~g) antagonized behavioural and hyperthermic effects of the peptide (2 experiments for each antagonist). Antagonism by haloperidol (0.5 mg] kg i.v. given 15 min before) of the stereotyped head-neck movements caused by TRH (0.5--3/~g) given into the IIIrd cerebral ventricle is illustrated in fig. 5. No stereotypies occurred after 0.5 #g of TRH in fowls pretreated with haloperidol. 18(I

The effects of TRH on behaviour and body temperature were not antagonized by intracerebroventricular injection of p h e n o x y b e n z a m i n e (0.5/~mol) or by systemic administration of either propranolol (5/~mol/kg i.v.) or methysergide (1/~mol/kg i.m.).

3.6. Reversal of ~-methyl-p-tyrosine-induced sedation Intracerebroventricular injection of a-methyl-p-tyrosine (2 injections of 100 pg each separated by an interval of 24 h) to 6 fowls produced behavioural depression and/or sleep squatting, and decreased locomotor activity, a syndrome persisting for more than 2 h after the second injection (see also Papeschi and Randrup, 1973). This symptomatology was reversed by infusion of TRH (1--2/~g) 15--20 min after a-methyl-p-tyrosine. Thus fowls were aroused and exhibited increased locomotor activity, valocalization and stereotypies.

3.7. Effects of triiodothyronine, thyroxine and thyrotropin

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Fig. 5. Antagonism by haloperidol (0.5 mg/kg i.v. 15 min before) o f stereotyped head-neck movements evoked by various doses of TRH given into the IIIrd cerebral ventricle. At each dose the top circles show the number of head-neck movements in animals receiving only TRH and the bottom circles those after pretreatment with haloperidol. Each circle indicated the mean values ± S.E. (the number of experiments is given in parentheses). In fowls pretreated with haloperidol no stereotypies occurred after 0.5 #g of TRH. The difference between the number of stereotypies in TRH-treated fowls after or without a pretreatment with haloperidol was significant at all the doses tested (P < 0.01).

Intracerebroventricular injection (0.5 and 1 /~g) of triiodothyronine (2 experiments for each dose), or systemic administration (0.1 mg/kg i.m. in 3 fowls), produced immediate sedation lasting 3--4 h followed by a rise in body temperature of approximately I°C. Thyroxine (0.5, 1 and 2 #g) given into the IIIrd cerebral ventricle (2 experiments for each dose)produced slight behavioural sedation without changes in body temperature whereas given intramuscularly, thyroxine (1 mg/kg in 3 fowls) produced marked sedation and lowered body temperature by I°C for approximately 1 h. Thyrotropin (2 and 4 I.U./kg i.m.) did not affect behaviour or body temperature (2 experiments for each dose). 4. Discussion

TRH given into the IIIrd cerebral ventricle or into selected brain areas of young and

258

adult fowls produced behavioural effects similar to those seen in mammals after systemic administration of large doses (Plotnikoff et al., 1972; Segal and Mandell, 1974; Goujet et al., 1975; Kruse, 1975, 1977; Cott and Engel, 1977), although in some studies there was either no change or a decrease in locomotor activity (Breese et al., 1974; Kulig, 1975). Electrocortical arousal was also seen after TRH thus supporting the suggestion (Dyer and Dyball, 1974; Renaud et al., 1975) that TRH and related peptides are involved in the modulation of central neuronal activity. Increased motor activity, together with activation of heat production mechanisms (tremor, shivering) and decrease in thermodispersive mechanisms produced an increase in body temperature. Similar temperature responses have been obtained in rabbits (Metcalf, 1974; Horita and Carino, 1975) and are consistent with the finding that peripheral injections of TRH antagonize the hypothermic effects of various sedatives (Prange et al., 1974; Breese et al., 1975; Horita and Carino, 1975; Kruse, 1977). The reason for the discrepancy between these findings and those in cats in which intraventricular injection of TRH produces hypothermia (Metcalf and Myers, 1976) is not known. However Metcalf and Myers (1976) injected a volume of 200 pl. This injection volume is known to flood the entire ventricular system and subarachnoid space (McCarthy and Borison, 1966) and the hypothermia might therefore have resulted from action at distant sites. The above effects of TRH were a property of the peptide since they were not mimicked by TSH, thyroxine or triiodothyronine. The behavioural syndrome evoked by TRH (increased locomotor activity, stereotyped head-neck movements, preening and vocalization) resembled that following intracerebroventricular or intrahypothalamic injection of apomorphine suggesting dopamine receptor activation (Nistic6, 1976; Koc and Marley, 1977). However, and in contrast to apomorphine, intracerebral injection of dopamine produced behavioural and electrocortical

G. NISTIC() ET AL.

sleep, hypothermia and stereotyped headneck movements in fowls pretreated with an amine oxidase inhibitor (Marley and Nistic6, 1972). Thus of the two types of dopamine receptor described in the avian CNS (Koc and Marley, 1977) those sensitive to apomorphine appear to mediate the effect of TRH. This is supported by the finding that the stereotyped head-neck movements, also seen after apomorphine and dopamine, and the behavioural excitation also seen after apomorphine, were abolished by haloperidol, a selective antagonist at apomorphine-sensitive dopamine receptors (Cools and Van Rossum, 1976}. Similar results have been obtained in mammalian species (Kulig, 1975; Cohn et al., 1975; Miyamoto and Nagawa, 1977}. A direct action on these receptors rather than an indirect action via dopamine release (Cohn et al., 1975), seems likely since TRH produced its characteristic effects in fowls in which catecholamine synthesis had been blocked by ~-methyl-p-tyrosine. Antagonists at a and 13 adrenoceptors as well as at 5-HT receptors exclude a participation of other catecholamines and tryptamines in central effects of TRH. The sites from which TRH produced its stereotypies and locomotor effects, the nucleus basalis and nucleus mes. profundus, homologous with the mammalian striatum and substantia nigra respectively, and the hypothalamus all contain large amounts of dopamine (Juorio and Vogt, 1967; Calling,ham and Sharman, 1970; Gargiulo and Nisticb, 1975) and would appear to be functionally related. However, this does not exclude the possibility that other sites (cortical, subcortical and spinal) not investigated could also be TRH sensitive. Biochemical studies, with one exception (Breese et al., 1974) have shown that TRH increased dopamine turnover (Keller et al., 1974; Fuxe et al., 1976; Marek and Haubrich, 1977). Another tripeptide, MIF, also increases dopamine turnover in the caudate nucleus of rats after its intracerebroventricular injection (Versteeg et al., 1978). The reason for this

TRH CENTRAL EFFECTS increase in d o p a m i n e t u r n o v e r is n o t k n o w n . I t is possible t h a t d o p a m i n e r g i c n e u r o n e s are a c t i v a t e d as p a r t o f a s e q u e n c e o f events initiated by TRH; recent data have shown that r e c e p t o r s t o o t h e r p e p t i d e s e.g. e n k e p h a l i n are l o c a t e d o n d o p a m i n e r g i c n e r v e t e r m i n a l s in t h e r a t s t r i a t u m (Pollard e t al., 1 9 7 7 ) . H o w ever, if so, it is d i f f i c u l t t o e x p l a i n t h e ~ - m e t h y l - p - t y r o s i n e ineffectiveness in p r e v e n t i n g T R H e f f e c t s . Since a high a f f i n i t y b i n d i n g f o r T R H has b e e n d e s c r i b e d in t h e p i t u i t a r y , h y p o t h a l a m u s a n d o t h e r b r a i n areas ( B u t t a n d S n y d e r , 1 9 7 5 ) it s e e m s likely t h a t a c t i v a t i o n o f a p o m o r p h i n e sensitive r e c e p t o r s is an e v e n t in a s e q u e n c e i n i t i a t e d b y specific T R H receptors. Acknowledgements Partial support from the Italian Ministry of Education (Rome) is gratefully acknowledged. References Allen, D.J. and E. Marley, 1967, Effect of sympathomimetic and allied amines on temperature and oxygen consumption, Brit. J. Pharmacol. Chemotherap. 31,290. Breese, G.R., J.M. Cott, B.R. Cooper, A.J. Prange, Jr. and M.A. Lipton, 1974, Antagonism of ethanol narcosis by thyrotropin releasing hormone, Life Sci. 14, 1053. Breese, G.R., J.M. Cott, B.R. Cooper, A.J. Prange, Jr., M.A. Lipton and N.P. Plotnikoff, 1975, Effects of thyrotropin-releesing acting drugs, J. Pharmacol. Exptl. Therap. 193, 11. Butt, D.R. and S.H. Snyder, 1975, Thyrotropin releasing hormone (TRH): apparent receptor binding in rat brain membranes, Brain Res. 93,309. Cailingham, B.A. and D.F. Sharman, 1970, The concentration of catecholamines in the brain of the domestic fowl (Gallus domesticus), Brit. J. Pharmacol. 40, 1. Cohn, M.L., M. Cohn and F.H. Taylor, 1975, Thyrotropin releasing factor (TRF) regulation of rotation in the non-lesioned rat, Brain Res. 96,134. Cools, A.R. and J.M. Van Rossum, 1976, Excitationmediating and inhibition-mediating dopamine receptors: a new concept towards a better understanding of electrophysiological, biochemical, pharmacological, functional and clinical date, Psychopharmacologia 45,243.

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