ADMINISTRATION OF THYROTROPIN RELEASING HORMONE (TRH) TO RATS RELEASES DOPAMINE IN N. ACCUMBENS BUT NOT N. CAUDATUS D. J. MRC Clinical

Pharmacology

HEAL

Unit.

and A. R. GREEN

Radcliffe

(Accepretl

Infirmary.

13 July

Oxford

OX2 6HE. England

1978)

Summary-Bilateral injection of thyrotropin releasing hormone (TRH; IOpg) into the n. accumbens of rats produced a short-lasting increase in co-ordinated locomotor activity and behavioural changes similar to those produced by injection of dopamine at this site. These effects were potentiated and prolonged by pretreatment with tranylcypromine (5 mg/kg i.p.). Injection of haloperidol (2.5 pg bilaterally) into the n. accumbens blocked the behavioural changes produced by intra-accumbens injection of TRH (1Opg bilaterally. 30min after tranylcypromine 5 mgjkg i.p.). Destruction of the presynaptic dopamine terminals with 6-hydroxydopamine (8 pg bilaterally) abolished the effects of intra-accumbens injection of TRH (IO pg bilaterally) in either untreated or tranylcypromine pretreated rats. This inhibition was not due to non-specific damage to post-synaptic dopamine receptors since these rats showed a normal locomotor response to intra-accumbens Injection of dopamine (5 pg bilaterally. 30 min after tranylcypromine 5 mg/kg). These results suggest that TRH is acting by release of dopamine. Peripheral injection of TRH (20mg/kg) produced behavioural changes similar to those observsed after central administration of this drug. Although these effects were not enhanced by pretreatment with tranylcypromine (5mg/kg. 30min before TRH), they were blocked by peripheral mjectton of haloperidol (1 mg/kg). Injection of thyroid stimulating hormone (TSH: 20mg!kg i.p.) produced no behavioural changes suggesting that peripherally injected TRH is not acting by release of TSH. Injection of TRH (20mg/kg i.p.) to unilateral nigro-striatal lesioned rats failed to induce ctrcling. Furthermore pretreatment with TRH (2 mg.;kg i.p.) did not enhance the turning produced by mcthamphetamine (0.5 mg,‘kg) 3 hr later in tranylcypromine-treated animals. This evidence suggests that in contrast to its effects in the n. accumbens TRH is unable to release dopamine in the n. caudatus.

Immunohistochemical and immunofluorescence studies have shown that thyrotropin releasing hormone (TRH) is widely distributed in extrahypothalamic brain tissues including the n. accumbens (Hokfelt, Fuxe. Johansson. Jeffcoate and White, 1975a) and in certain brain stem nuclei and spinal cord (Hiikfelt. Fuxe, Johansson, Jeffcoate and White, 197513). Recently TRH has been shown to bind to a rat brain membrane preparation (Burt and Snyder. 1976) and to be inactivated by peptidase enzymes from various areas of rabbit brain (Griffiths, Hooper, Jeffcoate and White, 1976). Renaud. Martin and Brazeau (1975) demonstrated that microiontophoretic application of this peptide depressed neuronal firing in both hypothalamic and extra-hypothalamic brain tissue. These widely differing pieces of evidence suggest that TRH may serve a role in the brain distinct from its actions on the hypothalmus-pituitary-thyroid axis. Consistent with this suggestion is the observation that TRH pretreatment potentiates the behavioural changes produced by injection of a monoamine oxidase inhibitor and L-dopa (Plotnikoff, Prange, Breese, Anderson and Wilson, 1972: Huidobro-Toro, Scotti de Carolis and Key words: n. caudatus.

TRH. dopamine. behaviour. n. accumbens. 23

Longo, 1974: Green and Grahame-Smith, 1974), since this effect was demonstrated both in normal and hypophysectomized animals (Plotnikoff er al., 1972). Green, Heal, Grahame-Smith and Kelly (1976) found that TRH had no effect on caudate nucleus adenylate cyclase activity, or its stimulation by dopamine in citro, and selectively potentiated the behavioural effects of metamphetamine, but not apomorphine. These authors concluded. therefore. that TRH was probably acting by a pre-synaptic mechanism. possibly by enhancing dopamine release. The effects of injection of TRH into the n. accumbens of rats have now been studied since this brain region is known to be rich in dopamine nerve terminals (Fuxe, 1965) and injection of dopamine at this site produces behavioural excitation’ and increased locomotor activity (Pijnenburg and Van Rossum, 1973: Jackson, Anden and Dahlstrom, 1975: Costall and Naylor, 1975: Heal, Phillips and Green, 1978). Recently Miyamoto and Nagawa (1977) have suggested that TRH produces its behavioural effects by stimulating the mesolimbic but not the striatal dopamine systems, and this has been investigated by examination of the effects of peripheral injection of TRH to rats with unilateral 6-hydroxydopamine lesions of the substantia nigra.

D. J.

24

HEAL and

METHODS

~n~cieus

of 6-h.ko.x_vdopamine

Iesioned

rats

und

anal~~sis of circliny behaviour

Adult male Sprague-Dawley rats (An&a Laboratory Animals, Alconbury, Huntingdon) weighing 300-3.508 at the time of operation were used in those experiments involving either bilateral implantation of cannulae into the n. accumbens or unilateral 6-hydroxydopamine lesioning of the substantia nigra. All other experiments were performed on rats weighing 130-220 g, obtained from the same supplier. Drugs were obtained from the following sources: dopamine hydrochloride, 6-hydroxydopamine hydrobromide (Sigma, London) haloperidol (Searle, High Wycombe), tranylcypromine (Smith, Kline & French, Welwyn Garden City), TRH (Roche, Welwyn Garden City), methamphetamine (Wellcome. Beckenham) and TSH (Armour, Pheonix, USA). Drugs for intraperitoneal and central injection were dissolved in 0.99; NaCl (saline) except for 6-hydroxydopamine hydrobromide which was freshly dissolved in ice-cold 0.9:; saline containing I mg/ml ascorbic acid and injected intracerebrally as described below. of

GREEK

Preparution

Animals and drugs

Prepuration

A. R.

accumbens

jmplanted

rats and

injection qf drugs

Rats were anaesthetized with Equithesin (JensenSalsbery Laboratories) and positioned in the stereotaxic apparatus. After exposure of the skull two 23-gauge stainless-steel cannulae were simultaneously implanted with their tips in the n. accumbens using the co-ordinates of i-3.2, + 1.7, -6.6 according to the atlas of Pellegrino and Cushman (1967). This was achieved by supporting the cannulae on an inverted U-shaped wire carrier. The distance between the 2 upright parallels of the wire carrier was 3.4mm, equivalent to 1.7 mm either side of the midline. The cannulae were kept patent with 35-gauge stainlesssteel wire stylets. The rats were allowed one week to recover before testing. All drugs were injected bilaterally into the n. accumbens through a 30-gauge needle passed down the cannulae. This needle had a collar which allowed a projection of 0.3 mm beyond the end of the cannulae. The rats were lightly anaesthetized with halothane and TRH and other drugs (all concentrations were dissolved in 1~1 of saline) were injected at a rate of 1~1 in 0.5 min. For the bilateral lesioning of the n. accumbens, 6-hydroxydopamine (see Animals and drugs) was injected using the injection needle described above. Two ~1 of the 6-hydroxydopamine solution (4pg base/PI) were injected at the rate of 1 @/min while the animal was anaesthetized with Equithesin. The rats were left for 10 days before testing. A group of animals which had shown the locomotor response and behavioural changes following injection of TRH were killed and the positions of the cannulae tips were examined. These were all found to be located in the ventral aspect of the n. accumbens (Fig. 1).

Rats were anaesthetized with Equithesin. positioned in the stereotaxic apparatus and after exposure of the skull, injected unilaterally with 6-hydroxydopamine through a 30 gauge stainless-steel cannula aimed at the substantia nigra. The co-ordinates of the cannulae tips were - 2.8; + 2.0: - 8.0 (Pellegrino and Cushman, 1967). Four /iI of the 6-hydroxydopamine solution (2 mg base/ml) were injected at the rate of f pl/min. The rats were allowed 10 days to recover. Circling activity was measured in glass rotameter bowls measuring 28 cm in diameter and 26cm in height. Circling during 1 min was recorded at IOmin intervals. Only rats circling more than 5 turns;min for the 90min period following methamphetamine (2mgjkg i.p.1 were used. Measurement

of locomotor

acriritj

Following injection of a drug directly into the n. accumbens each rat was placed in a cage on an LKB Animex activity meter (sensitivity and tuning: 30pAf. Counts for the first 5min after injection were discarded as the animal was still recovering from the halothane anaesthesia. Movements/min were then recorded and plotted as total movements/lOmin, thus, the 15min value shows the movements in the period 5-ISmin after injection. Results are plotted as mean fS.E.M. with the number of observations (each a different animal) noted. While there was a large variation between individual rats as is shown in Figure 3, the variation of an individual animal was not greater than 45”,;, of the mean from experiment to experiment. For statistical analysis a Student’s paired t-test was used. For behavioural studies on the effect of intraperitoneal injection of TRH, rats were tested in groups of 3. Activity was measured on LKB Animex meter (sensitivity and tuning: 30 @A) and recorded as movementsi5min with the counts for the first 5min discarded. Results are plotted as mean average movements/min + S.E.M. with the number of experiments noted. Where possible the groups of rats were used as their own experimental controls and results were analysed using the Student’s paired t-test. Where this was not possible the unpaired t-test was used.

RESULTS

Effect of injection of TRH

into the nucleus

accumbens

Injection of TRH (IOpg bilaterally) produced an increase in co-ordinated locomotor activity (Fig. 2). The locomotion was generally performed with the back arched, held held down and abdomen ileld clear of the floor of the cage. Other behavioural changes were also observed. These were licking and chewing movements, sniffing, piloerection, Straub tail and head weaving. These behavioural changes bear a strong similarity to those seen following injection of

TRH, dopamine

and behaviour

Fig. 1. A vertical section through the rat forebrain showing cannulae placement. The figure shows the section 3.4mm anterior to the bregma according to the atlas of Pellegrino and Cushman (1967) from which this figure is reproduced. The placement of the cannulae tips in 8 animals is shown by the paired symbols. The three other animals examined had the cannulae tips 3.2mm anterior to the bregma but lying on the same horizontal plane. The caudate is shown by the symbol CPU.

15

25

35 Time

after

45 injection

55

65

75

85

95

(min)

Fig. 2. Effect of bilateral injection of TRH into the n. accumbens on locomotor activity. Rats were briefly anaesthetized with halothane and then either saline 1~1 bilaterally (O---O) or TRH lOfig bilaterally (M) was injected into the n. accumbens. The activity (total movements/lOmin period) is shown plotted against time after injection (min). The counts for the first 5 min period were discounted because the rats were still recovering from the effects of the anaesthesia. The results are plotted as the mean +S.E.M. for the locomotor responses from 5 different animals.

D. J. HEAL and A. R. GREED

26 801

0

15

25 Time

35

45

after

injection

55

65

15

85

95

(min)

Fig. 3. Effect of injection of tranylcypromme on the locomotor activity produced by injection of TRH into the n. accumbens. Rats were injected with tranylcypromine (5 mg/kg) and 30min later either saline 1~1 bilaterally (O---O) or TRH IOpg bilaterally (04) was injected into the n. accumbens. The results are plotted as the mean +S.E.M. of the locomotor responses of 5 different animals. Figure I showing the effect of injection of either saline 1PI bilaterally (n--A) or TRH lOp(g bilaterally (A-A) into the n. accumbens of untreated rats is plotted here for comparison. into the n. accumbens of tranylcyprominepretreated animals. A marked increase in locomotor activity occurred only for the first 2Omin after the TRH injection. After this time the activity was only slightly greater than that of the control group. However some of the other behavioural changes such as licking. chewing and sniffing were observed up to 90min. When rats were pretreated with tranylcypromine (5 mg/kg i.p.) 30 min before injection of TRH (1Opg bilaterally) into the n. accumbens the behavioural activity. including locomotion, was greatly potentiated both in magnitude and duration (Fig. 3). dopamine

Following

tranylcypromine Table

pretreatment

the locomo-

I. Etfect of injection of haloperidol activity produced by injection

tor activity showed no decline during the 90min test period and was still visible in the rats several hours after testing, as were the other behavioural changes reported above. eflkct

of‘ neuroleptic

pretreutment

on

TRH-induced

locomotion

The locomotor activity produced by injection of TRH (10 pg bilaterally) into the n. accumbens 30 min after tranylcypromine (5 mg/kg i.p.) was markedly inhibited by injection of haloperidol (2.5pg bilaterally, 15 min before TRH) at the same site (Table 1). This dose of haloperidol has previously been shown by

into the n. accumbens of rats on the locomotor of TRH at the same site 15min later First injection

Second

Saline (I ~1 bilaterally)

injection

TRH (IOpg bilaterally. after tranylcypromine 5 mg,‘kg i.p.)

3Omin

4529 + 863 (4)

Haloperidol (2.5 pg bilaterally) P < 0.02

1956 f 5X3 (4)

Results are the mean f S.E.M. of the total movements in the 90min period following intra-accumbens injection of TRH after discounting the first 5 min period. The number of observations are shown in brackets. Results were analysed using the Student’s paired r-test. The total movements for the 90min period following intra-accumbens injection of saline (1 {cl bilaterally) 30 min after tranylcypromine (5 mg/kg i.p.) was 1114 + 146 for these 4 rats. When this result is compared to that of the haloperidol-treated group the total movements for the 2 groups are not significantly different.

TRH. Table

2. Effect of bilateral

Injection

dopamine

6-hydroxydopamine dopamine-induced

lesioning of the nucleus locomotor activity

into n. accumbens

Saline

(1PI bilaterally)*

TRH

(IO pg bilaterally)*

Control

30 min 5 mg/kg 30min 5 mg/kg

Saline (1 ~1 bilaterally. after tranylcypromine

30 min 5 mg!kg

i.p.)*

accumbens

on TRH

and

6-Hydroxydopamine lesioned rats

rats

33oi71 (41 P < 0.01 956 f I31 (4)

Saline (I ~1 bilaterally. after tranylcypromine TRH (IOpg bilaterally. after tranylcypromine

21

and behaviour

N.S. P < 0.01

1150 + 155 (4) P < 0.01

N.S.

4422 f 841 (4)

P < 0.05

745 + 85 (5) P < 0.01 2269 It 304 (5)

N.S.

384 f 61 (4) N.S. 421 f 57 (41 1305 f 228 (4) N.S. 1696 f 208 (4)

i.p.)*

i.p.)t Dopamine (5 pg bilaterally. 30 min after tranylcypromine 5 mg/kg i.p.)t

930 _+ 149 (4) P < 0.02 2591 + 438 (4)

N.S.

The results are the mean f S.E.M. of the total number of movements in: * 90 min period following injection of TRH or t 60min period following injection of dopamine after discounting the first 5 min period. The number of observations are shown in brackets. N.S. Not significant. Results were analysed usinp the Student’s paired r-test except for lines marked t which were analysed with Stud&t’s unpaired t-test. Heal et al. (1978) to have no effect on control locomotor activity following injection of saline (1 ~1 bilaterally) into the n. accumbens.

J$‘iict of bilateral 6-hydroxydopamine accumbens

on the TRH-induced

lesions of the n.

locomotor

actkit)

The observation that the behavioural effects of TRH could be potentiated by a monoamine oxidase inhibitor and abolished by the dopamine receptor antagonist haloperidol suggested that TRH might be producing the behavioural changes by releasing dopamine. This possibility was investigated by examining the effect of destruction of the presynaptic dopamine terminals in the n. accumbens using 6-hydroxydopamine on the TRH-induced behavioural changes. A group of rats with cannulae implanted into the n. accumbens were tested for increased locomotor response induced by injection of TRH (10 pg bilaterally) either alone or 30min after administration of tranylcypromine (5 mg/kg i.p.) (Table 2). The n. accumbens of these rats were then bilaterally lesioned with 6-hydroxydopamine (see Methods) and l&15 days later the animals were retested as described above. The 6-hydroxydopamine lesioning did not affect the control response following the bilateral injection of ! ~1 saline. The TRH mediated locomotor response, however, was abolished by the lesioning (Table 2). Similar changes were seen in the tranylcypromine pretreated rats, the lesioning not affecting the control response but abolishing the effect of the TRH (Table 2). In order to establish whether the 6-hydroxydopamine lesions had caused non-specific damage to the post-synaptic dopamine receptors, 4 of the rats used were injected with dopamine (5 pg bilaterally) after tranylcypromine pretreatment. The activity observed showed no significant differences in the locomotor response between this lesioned group and another group of non-lesioned controls (Table 2), suggesting that the post-synaptic receptor site was undamaged.

&fecr.s of peripheral

injection 01 TRH

Intraperitoneal injection of TRH (20 mg/kg) also induced a series of behavioural changes, including locomotion (Fig. 4). These changes occurred almost immediately after injection and consisted of licking and chewing facial movements, wet-dog shakes, grooming of the face with the forepaws, grooming of the other rats present, sniffing, licking and nibbling of the forelimbs, ptosis, Straub tail and the characteristic locomotion described earlier. One striking feature of the behaviour was the rats standing on 3 legs with one forelimb held out, the limb held out being sometimes changed. These observations confirm the previous results of Goujet, Simon, Chermat and Boissier (1975) and Miyamoto and Nagawa (1977). In contrast to the potentiating effects of tranylcypromine on the behavioural effects of TRH injected into the n. accumbens, pretreatment with this drug (5 mg/kg i.p.) 30min before peripheral injection of TRH (20mg/kg i.p.) did not enhance the locomotor activity (Table 3). Indeed pretreatment with tranylcypromine actually reduced the increased activity produced by TRH when compared to the effects of TRH administration alone (Table 3). Certain features of the behaviour were also modified, with head jerking and padding now being seen and wet-dog shakes almost totally absent. The locomotor activity produced by intraperitoneal injection of TRH (20 mg/kg) was almost totally abolished by injection of haloperidol (1 mg/kg i.p.) 15 min earlier, although this dose of haloperidol also markedly reduced the spontaneous locomotor activity following injection of saline (Table 4). EfSects

of TRH

on unilateral

nigro-striatal

lesioned

rats

TRH (20mg/kg i.p.) did not cause any rotational behaviour in unilateral nigro-striatal lesioned rats

28

D.

J. HEAL

and

A. R.

GREEN

80

60 .-C E ‘;; z i

40

z E ,” ..1 ‘5 4

20

0 Time

after

injection

(min)

Fig. 4. Effect of intraperitoneal injectlon of TRH on locomotor activity. Groups of 3 rats were injected with saline (O-O) and the activity was measured for 60min. Following this the rats were injected with TRH 20mg/kg i.p. (04) and tested for a further 60min. Activity (average movements/min) is plotted against time after injection (min). The counts for the first 5 min were discounted because the rats were still recovering from the effects of being handled. The results are plotted as the mean +S.E.M. for the locomotor responses of 3 groups of 3 rats.

Table

3. Effect of tranylcypromine by intraperitoneal

on the increased behavioural activity injection of TRH 30min later

produced

First injectlon Second Saline TRH

(20 mg/kg

i.p.)

Increase in total of movements

‘Tranylcypromine (5 mgikg i.p.)

Saline

injection

number

892 + 165 (3) P < 0.01 1768 f 187 (3)

N.S. N.S.

697 + 212 (3) P < 0.01 1114 f 132 (3)

876 f 26 (3)

P i 0.01

447 f 89 (3)

Results are the mean + S.E.M. of the total number of movements in the 60min period following intraperitoneal injection of TRH after discounting the first 5min. The number of observations on groups of 3 rats are shown in brackets. Results were analysed using the Student’s paired r-test for vertical columns and the Student’s unpaired I-test for horizontal lines. Table 4. EITect of peripheral injection duced by intraperitoneal

of haloperidol on the locomotor injection of TRH 30min later First

Second

injection

Saline TRH (20mg/kg

Lp.)

pro-

injection Haloperidol (1 mg/kg i.p.)

Saline 1041 * 168 (3) P < 0.05 1768 + 187 (3)

activity

P < 0.01 P < 0.01

300 f 127 (3) N.S. 339 + 94 (3)

Results are the mean + S.E.M. of the total movements in the 60 min ing intraperitoneal injection of TRH after discounting the first 5 min. of observations on groups of 3 rats are shown in brackets. Results using the Student’s paired t-test for horizontal lines and the Student’s for vertical columns.

period followThe numbers were analysed unpaired t-test

TRH. dopamine and behaviour when administered either alone or 30min after injection of tranylcypromine (5 mg/kg i.p.). Green et al. (1976) demonstrated that 3 hr after peripheral administration of TRH (I-2mg/kg) the amphetamine-induced locomotor activity was potentiated in rats only if they had been pretreated with tranylcypromine. However the rotational behaviour of unilateral nigro-striatal lesioned rats produced by injecting methamphetamine (OS mg/kg i.p.) 30 min after tranylcypromine (5 mg,kg i.p.) was unaffected by pretreatment with TRH (2mg/kg i.p.) 3 hr before methamphetamine. Eflecr of peripheral injection of thyroid stimulating hormone (TSH) To eliminate the possibility that TRH was producing its behavioural effects by release of TSH rats were injected with TSH (20mgbg i.p.). TSH produced neither an increase in locomotor activity nor any of the behavioural changes seen after TRH. DISCUSSION

The behavioural changes and the increase in locomotor activity observed following injection of TRH into the n. accumbens of rats closely resemble those produced by injection of dopamine at this site, confirming previous findings (Miyamoto and Nagawa, 1977). Pretreatment with tranylcypromine potentiates both the magnitude and the duration of the behavioural response to the injection of TRH. Monoamine oxidase inhibition produces a similar enhancement of the behavioural effects of intra-accumbens injection of dopamine (Costall and Naylor, 1976). Furthermore injection of the dopamine antagonist haloperidol, blocks the behavioural effects of TRH. Finally it was found that destruction of the pre-synaptic dopaminergic terminals in the n. accumbens with 6-hydroxydopamine abolishes the locomotor response and behavioural changes produced by injection of TRH at this site. All these data indicate that the behavioural changes procued by injection of TRH into the n. accumbens are due to the release of dopamine by this peptide. In agreement with previous studies (Goujet er al., 1975; Kruse, 1975; Miyamoto and Nagawa, 1977) it was found that injection of high doses of TRH intraperitoneally cause both behavioural excitation, and increased locomotor activity. These behavioural changes were very similar to those produced by injection of TRH or dopamine directly into the n. accumbens. The present experiments on the effect of TRH on unilateral nigro-striatal lesioned animals suggest, however, that the behavioural changes following peripherally administered TRH do not result from changes in dopaminergic activity in the striatum. Similarly Miyamoto and Nagawa (1977) observed no rotational responses to intraperitoneal or intravenous TRH in mice with unilateral caudate lesions. The fail-

29

ure of Green er a/. (1976) to observe enhancement by TRH of methamphetamine-induced circling in unilateral nigro-striatal lesioned rats was therefore probably due to the fact that TRH appears to have no effect on striatal dopaminergic systems rather than that the animals had not been pretreated with a monoamine oxidase inhibitor, as was suggested by these authors. While this study did not observe any enhancement of tranylcypromine/methamphetamine induced circling by TRH, Green et al. (1976) found that TRH tranylcypromine/methamphetamine did enhance locomotor activity. Since Kelly, Seviour and Iversen (1975) have suggested that locomotion is associated with the mesolimbic dopamine system, while circling is associated with the striatal dopamine system these effects also point to a selective action of TRH on the mesolimbic system. Miyamoto and Nagawa (1977) also believe that the behavioural responses following peripheral TRH administration are mediated by dopamine receptors in the n. accumbens, since the hyperactivity is blocked by injection of haloperidol at this site but not by the a-adrenergic antagonist phenoxybenzamine. In this study we have shown that the effects of peripheral injection of TRH are blocked by peripheral injection of haloperidol. Although the dose of haloperidol used markedly reduced spontaneous locomotor activity following saline injection, the degree of inhibition of the TRH response was such that it could not be explained by a haloperidol blockade of control activity. The observation that pretreatment with tranylcypromine did not enhance the locomotor response produced by intraperitoneal injection of TRH was surprising, and at present no satisfactory explanation for this apparently contradictory result can be offered. This failure may be associated with the finding that this drug also altered the behavioural changes observed after peripheral injection of TRH. Horst and Spirt (1974) showed that whilst TRH was capable of releasing [3H]-dopamine from striatal synaptosomes in t’itro the concentration of TRH necessary to produce this effect was very high and Miyamoto and Nagawa (1977) found that in vice injection of TRH into the striatum failed to mimic the effects of dopamine. It is possible that the observation that TRH can apparently release dopamine in the n. accumbens but not n. caudatus has physiological significance since Hijkfelt et a/. (1975a) using immunohistofluorescence techniques showed that there were dense TRH containing fibres in the n. accumbens but not in the striatum. Clearly TRH does not appear to be acting through release of TSH since this peptide had no effect on behaviour. Noradrenaline occurs in the n. accumbens (Koob, Balcom and Meyerhoff, 1974) and Keller, Bartholini and Pletscher (1974) have shown that high doses of TRH (10 mg/kg) decrease noradrenaline turnover, probably as a result of increased noradrenaline release. While Pijnenburg, Honig and Van Rossum

30

D. _I. HEAL and A. R.

(1975) demonstrated that intra-accumbens injection of noradrenaline produced weak locomotor stimulation, their data indicated that this was due to stimulation of dopamine receptors. Furthermore, locomotor activity produced by peripheral injection of TRH was unaffected by intra-accumbens injection of phenoxybenzamine, but blocked by injection of haloperidol at this site (Miyamoto and Nagawa, 1977). Although the 6-hydroxydopamine denervated rats were not pretreated with desmethylimipramine to prevent damage to noradrenergic fibres (Breese and Traylor, 1971) the data above and the dopamine-like appearance of the behavioural changes makes it unlikely that TRH exerts its major effects on the noradrenergic system in the n. accumbens. In agreement with Miyamoto and Nagawa (1977) it was found that following peripheral injection of TRH, rats showed mild self-biting of the forelimbs. These

authors

have suggested

that

this behaviour

may

be mediated by a noradrenergic system which is present in neither the n. accumbens nor the caudate nucleus. The behavioural effects of TRH have also been linked with the putitative neurotransmitter GABA. Cott and Engel (1977) have shown that the GABAmimetics and amino-oxyacetic acid. in general antagonize the locomotor and other central effects of TRH and suggested that GABA pathways may mediate some actions of TRH. However, since GABA-transaminase inhibitors block the behavioural effects of intra-accumbens injection of both dopamine (Pycock and Horton, 1976) and dibutyryl cyclic AMP (Heal er al., 1978) then the actions of these GABAergic drugs may adequately be explained by an inhibition of the effects of TRH-released dopamine. The amphetamine-like behavioural effects produced by injection of a high dose (20mg/kg) of this peptide intraperitoneally occur almost immediately and only last for about 20min. These effects may therefore be separate from the enhancement of some dopaminemediated behaviours which occur some hours after pretreatment with a peripheral injection of a low dose of TRH (Plotnikoff et al., 1972: Huidobro-Toro et al., 1974: Green and Grahame-Smith, 1974). In conclusion both biochemical and behavioural evidence indirectly suggest that TRH exerts some of its behavioural effects by presynaptic release of dopamine and that this selectively occurs in the n. accumbens

and

not

the n. caudatus.

Ackna~ledgrmmts~We thank Dr E. T. Rolls (Department of Experimental Psychology. University of Oxford) for performing the histological examination of the brains. We would also like to thank Smith, Kline & French and Roche for the generous donation of drugs for this study and DI L. J. Pellegrino and Appleton-Century-Crofts for permission to reproduce Figure 1. REFERENCES

GREEN

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