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A study of the changes in motor behaviour caused by TRH on intracerebral injection
European Journal of Pharmacology, 53 (1979) 143--150
143
© Elsevier/North-Holland Biomedical Press
A STUDY OF THE CHANGES IN MOTOR BEHAVIOUR CAUSED BY T R H ON I N T R A C E R E B R A L INJECTION BRENDA COSTALL, SIU-CHUN G. HUI, GEOFFREY METCALF * and ROBERT J. NAYLOR Postgraduate School of Studies in Pharmacology, University of Bradford, Bradford, U.K. and • Pharmacology Department, Reckitt and Colman, Pharmaceutical Division, Dansom Lane, Hull, U.K.
Received 17 May 1978, revised MS received 1 August 1978, accepted 12 September 1978
B. COSTALL, S.-C.G. HUI, G. METCALF and R.J. NAYLOR, A study of the changes in motor behaviour caused by TRH on intracerebral injection, European J. Pharmacol. 53 (1979) 143--150. 20/~g TRH injected bilaterally into the caudate-putamen, tuberculum olfactorium, nucleus accumbens, amygdala, lateral ventricles, midbrain or cerebral cortex failed to induce any increase in locomotor activity (measured using photocells), although other behavioural changes were observed after each injection, and included body shakes, limb tremor, repetitive head and limb movements, biting, scratching and an alert appearance. These behavioural changes could result in positive readings from equipment used to measure locomotor activity, but careful investigations focussing on the nucleus accumbens used photocell boxes, activity wheels and Animex recorders to emphasise the' inability of intracerebral TRH (10--40/~g) to enhance locomotor activity. Intraaccumbens TRH also failed to enhance amphetamine hyperactivity or reduce the motor depression caused by haloperidol and analeptic drugs. The data do not support a central locomotor stimulant action for TRH. Intracerebral injections Nucleus accumbens
Locomotor activity
Dopamine
d-Amphetamine
1. I n t r o d u c t i o n
2. Materials and methods
It has been shown in a n u m b e r o f species t h a t T R H can m o d i f y normal m o t o r behaviour and the m o t o r effects induced by drugs which influence cerebral dopamine systems (Mora et al., 1976; P l o t n i k o f f e t al., 1975; Carino et al., 1976). However, it is difficult t o assess the relative involvement o f the different dopamine systems since m os t investigations have been limited to drug administration by the peripheral or ventricular routes. Therefore, in the present experiments T R H is injected directly into a n u m b e r o f dopamine-contalning areas, and special emphasis is placed on the possible role o f t he nucleus accumbens, an area widely implicated in m o t o r control.
2.1. A n i m a l s
TRH
T he studies utilised male, Sprague-Dawley (CFE) rats weighing 250--300 g at t he time of operation. Animals used in peripheral studies only weighed 200 -+ 25 g. 2.2. Intracerebral injection t e c h n i q u e
Bilateral guide cannulae for intracerebral injections (or unilateral guide cannulae for intraventricular injections) were c o n s t r u c t e d from 0.65 mm diameter stainless steel tubing and were implanted stereotaxicaUy in t he brains of rats anaesthetised with chloral
144
A.
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Ant.K) Ve r t.* 0-5 ( 3 . 0 m m ) Lat._+l.5
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Fig. 1. Diagrammatic representation of the location of the injection (e) or guide (A) cannulae tips for intracerebral injections. Stereotaxic coordinates were selected with the aid of the atlas of De Groot (1959). Coordinates given are for the implantation of the guide cannulae tips; the vertical extension of the injection units beyond the guide tips are shown in parentheses. Each diagram was constructed from the histological data obtained from 8--12 rats. A. Nucleus accumbens, B. tuberculum olfactorium, C. caudate-putamen, D. cerebral cortex, E. ventricles, F. nucleus amygdaloideus centralis, G. thalamus and H. midbrain.
hydrate, 300 mg/kg i.p. The guide cannulae were fixed to the skull using retaining screws and acrylic cement, and kept patent b y stainless steel stylets, 0.3 mm diameter, made to extend 0.5 mm below the tips of the guides. Guide cannulae were located at the coordinates shown in fig. 1, and stainless steel (0.3 mm diameter) injection units were made to extend below the guides as indicated. Animals were used 7--14 days following the operation and were used on a maximum of 2 occasions with intervening 7 day recovery periods. Rats were manually restrained during the injection procedure. Drug or solvent was administered bilaterally (simultaneously into both hemispheres) (unilaterally for intraventricular injection) with Agla micrometer syringes in volumes of 1 pl over a 5 sec period, a further
55 sec being allowed for deposition of drug. The injection units were immediately replaced b y the stylets. (In all studies reported in this manuscript and using bilateral drug administration, the dose given refers to the unilateral dose.) On completion of the studies the locations of the guide cannulae were determined histologically (fig. 1).
2.3. Behavioural studies All behavioural studies were carried out between 8 am and 7 pm in sound-proofed, diffusely illuminated rooms maintained at a temperature of 21 + 1 ° C.
2.3.1. Assessment of general motor changes Preliminary studies indicated that T R H
TRH AND MOTOR BEHAVIOUR caused characteristic m o t o r changes on injection into a number of brain regions: shaking, limb tremor, head and limb movements, biting, scratching, alertness. The occurrence of these phenomena was carefully assessed following intracerebral administration of T R H into all regions of study (see fig. 1). Immediately on completion of the intracerebral injections rats were placed in individual perspex observation cages (30 cm X 20 cm and 15 cm high) and the above behavioural changes recorded at 5--10 min intervals, for the duration of effect, as marked, present or absent.
2.3.2. Measurement of locomotor activity using photocell equipment Immediately after intracerebral injection rats were placed in perspex cages of the above dimensions, each fitted with one photocell unit placed off-centre. Hyperactivity was assessed b y counting the number of interruptions of the light beam occurring each 5 min period. Readings were taken every 10 min throughout the duration of drug effect. The behaviour of the animals was visually observed /vhilst mechanically recorded in order to differentiate measurements of l o c o m o t o r activity from counts caused b y animals tremoring, shaking etc. (as described in 2.3.1.) in the region of the beam. This method of assessment of l o c o m o t o r activity was adopted for the majority of experiments (see Results section).
2.3.3. Measurement of hyperactivity using the activity wheel In these experiments activity was recorded electromechanically from activity wheels 300 mm in diameter and 140 mm wide. Animals were placed in the activity wheels on at least 3 occasions prior to their actual use to obtain a more constant level of activity. Immediately after injection rats were placed in the activity wheels and their activity was recorded in wheel revolutions per 10 min period for the duration of drug effect. Rats were used once only in this manner.
145
2.3.4. Measurement of hyperactivity using the Animex recorder Animals were placed individually in the Animex recorder and allowed 30 min to acclimatise to the new environment. T R H was then administered into the nucleus accumbens and animals returned to the Animex recorder: counts were taken for 9 to 10 min periods and behaviour was also visually assessed. The sensitivity of the apparatus was adjusted to 40 pA.
2.3.5. Measurement of catalepsy Catalepsy was assessed b y placing animals in perspex cages (20 X 15 X 15 cm) fitted with a 10 cm high bar and, after a period of acclimatisation, placing the animal's front limbs carefully over the bar. A normal animal will recover from this position within 6 sec b u t a cataleptic animal maintains the abnormal imposed position for a period of time dependent on the degree of catalepsy. Hence, time may be transduced into intensity and, in order to account for animals which maintain the imposed position for an "infinite period of time", the following scoring system was adopted to express the intensity of catalepsy: 0.1--2.5 min = score 1, 2.6--5.0 min = score 2, 5.1--10.0 min = score 3, 10.1.-20.0 min = score 4, 20.1 min --oo = score 5. After onset, the intensity of catalepsy was assessed at 30 min intervals for 6 h.
2.3.6. Measurement of analeptic activity Analeptic activity was indicated b y a more rapid recovery of the righting reflex following anaesthetisation with chloral hydrate, 200 mg/kg i.p., or sodium pentobarbitone, 30 mg/ kg i.p.
2.4. Drugs For intracerebral injections dopamine hydrochloride (Koch-Light) was dissolved in nitrogen bubbled distilled water neutralised with sodium bicarbonate, and T R H (L-pyroglutamyl-L-histodyl-L-prolineamide L-tartrate monohydrate, thyrotropin releasing hormone)
146
(Reckitt and Colman) in distilled water. All drugs were freshly prepared. For i.p. injections, d-amphetamine sulphate (Sigma), chloral hydrate (BDH) and sodium pentobarbitone (May and Baker) were dissolved in distilled water. Solutions of haloperidol (Janssen) were prepared in distilled water from a stock solution of 10 mg/ml haloperidol in 1% lactic acid. The significance of the results was assessed using the Student's t-test for comparisons with control values. 3. Results
3.1. Changes in locomotor activity (photocell counts) caused by dopamine, TRH and a combination o f dopamine and TRH injected bilaterally into the nucleus accumbens Dopamine injected bilaterally into the nucleus accumbens, in the absence of any pretreatment, caused a dose-dependent hyperactivity which was threshold at 50 pg and maximum at 200 pg when the effect was apparent for up to 4 h (fig. 2). However, 10-4 0 p g T R H injected bilaterally into the nucleus accumbens under similar conditions failed to cause any increase in activity (fig. 2). 80 pg T R H produced inconsistent changes in l o c o m o t o r activity: only 10% of animals treated with this dose (of a total of 60 animals tested) appeared hyperactive, b u t the concurrent development of repetitive head and limb movements, tremor etc. as described in 3.5. prevented a differentiation of hyperactivity counts. Further, the combination of 20 pg T R H with the threshold dose of dopamine for hyperactivity induction ( 5 0 p g ) failed to initiate a hyperactivity response (fig. 2). Similarly, 40 pg T R H failed to enhance the l o c o m o t o r hyperactivity established to a dose of 100 pg dopamine.
3.2. The effect of intra-accumbens TRH on the hyperactivity (photocell counts) induced by peripherally administered d.amphetamine d-Amphetamine caused a dose-dependent hyperactivity response (0.5--1.5 mg/kg i.p.,
B. C O S T A L L E T AL,
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Hours after injection Fig. 2. Changes in l o c o m o t o r activity c a u s e d b y dopamine, TRH and a combination of dopamine and TRH i n j e c t e d bilaterally i n t o t h e n u c l e u s a c c u m b e n s . In t h e l a t t e r e x p e r i m e n t s d o p a m i n e was given 30 rain b e f o r e t h e i n j e c t i o n o f TRH. H y p e r a c t i v i t y is e x p r e s s e d as t h e n u m b e r of i n t e r r u p t i o n s o f a p h o t o cell b e a m o c c u r r i n g w i t h i n a 5 rain period. E a c h value is t h e m e a n o f d e t e r m i n a t i o n s f r o m 8 rats. S t a n d a r d errors are less t h a n 20% o f t h e means.
maximum counts being 50--117 respectively, S.E.M.s <20%). Hyperactivity established to a submaximal dose of 1 . 0 m g / k g d-amphetamine was n o t modified b y a subsequent bilateral intra-accumbens injection of 20 pg T R H (P > 0.05).
3.3. The effect of d-amphetamine administered peripherally and TRH administered into the nucleus accumbens on wheel activity of rats Activity wheels were shown to detect the ability of d-amphetamine to cause a dose-
TRH AND MOTOR BEHAVIOUR
dependent hyperactivity (0.5--1.5 mg/kg i.p., maximum wheel revolutions/10 min being 10--25 respectively, S.E.M.s <20%). However, this technique failed to show any ability of intra-accumbens TRH to increase activity (2.5--40 #g, P > 0.05 compared with the activity of normal rats, or rats receiving intraaccumbens solvent).
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3.4. The effects of TRH administered into the nucleus accumbens and measured by the Animex recorder 10
10--40 pg TRH injected into the nucleus accumbens caused a general "hyperactivity" associated with repetitive head and limb movements, tremor etc. as indicated in 3.5. Counts were obtained from the Animex recorder which were significantly different from control values, P < 0.05--P < 0.001, during the 30--40 min period following injection (fig. 3). However, these "counts", which were not dose-dependent, could not be differentiated to give any indication of an increased locomotor activity. 3.5. The effects on general behaviour and on locomotor activity (photocell counts) o f TRH injected into several brain regions 20 #g TRH injected bilaterally into the caudate-putamen, tuberculum olfactorium, nucleus accumbens, amygdala, lateral ventricles, midbrain, cerebral cortex or thalamus failed to induce any increase in locomotor activity ( P < 0.05 compared with animals receiving intracerebral solvent). However, this dose of TRH was shown to be adequate to cause a number of other behavioural changes from each brain area analysed, the response observed varied from area to area both in nature and severity as indicated in table 1, and included body shakes, limb tremor, repetitive head and limb movements, biting, scratching (at body and environment), and a degree of alertness which was quite distinct from the behaviour of control animals. Generally, these behavioural changes were most marked
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Fig. 3. The effect of 10 =-=, 20 -* • and 40 • " pg TRH, or solvent o o, injected bilaterally into t h e n u c l e u s a c c u m b e n s as r e c o r d e d by an " A n i m e x " in c o u n t s / 1 0 min. 6--8 rats were used at each dose level. Mean values are given ±S.E.M.
during the 20--30 min period following onset. It is stressed that animals exhibiting these responses to TRH, even those appearing unusually alert, at no time exhibited movements about the observation cages which could be differentiated in frequency from those of control animals. The behaviottral changes described here for a dose of 20 #g TRH were also apparent at 10 #g TRH: the intensity, onset and duration of effects could not be distinguished from those described. As the dose of TRH was further reduced the change was observed in the number of animals responding, not in the nature of the response: 60--6.5% of animals responded to a dose of 5/~g TRH but no response was observed at 2.5 #g. 3.6. The effect o f intra-accumbens TRH on haloperidol catalepsy Haloperidol, 0.5--4 mg/kg i.p., induced a dose-dependent catalepsy (scored 1--5 respectively, S.E.M.s <20%). The weak intensity catalepsy induced by 0.5 mg/kg i.p. haloperidol was not enhanced (or in any way signifi-
148
B. C O S T A L L E T A L
TABLE 1 B e h a v i o u r a l e f f e c t s o f T R H i n j e c t e d b i l a t e r a l l y i n t o d i f f e r e n t b r a i n areas. T h e d i f f e r e n t b e h a v i o u r a l s t a t e s are d e s i g n a t e d as a b s e n t (0), p r e s e n t ( + ) o r r e l a t i v e l y m a r k e d (++). n = 8 f o r e a c h g r o u p . Brain area
Nucleus accumbens Tuberculum olfactorium Thalamus Midbrain Cerebral cortex Caudate-putamen Lateral ventricles Amygdala
B e h a v i o u r o b s e r v e d a f t e r i n t r a c e r e b r a l i n j e c t i o n o f T R H (20 pg) Shakes
Limb tremor
Head and limb movement
Biting
Scratching
Alertness
+ + ++ + + + + ++
+ 0 ++ + 0 + ++ +
+ + ++ + + + ++ +
0 0 ++ 0 0 ++ + 0
+ + + + 0 ++ + +
+ + ++ ++ 0 ++ + ++
cantly modified, P > 0.05) by a subsequent bilateral intra-accumbens injection of 20 pg TRH. 3.7. Effect o f intra-accumbens T R H on the sleeping time to chloral hydrate and sodium pentobarbitone
TRH (20 pg injected bilaterally into the nucleus accumbens) showed no analeptic activity. The times of onset of "sleep" (loss of righting reflex) (2.84 + 0.37 min for chloral hydrate, 4.8 + 0.07 min for sodium pentobarbitone in control animals) to recovery of the righting reflex (duration of loss 35.2 + 8.1 min for chloral hydrate, 107.7 +- 19.4 min for sodium pentobarbitone in control animals) did n o t differ significantly between control animals, animals receiving intraaccumbens solvent and those receiving intraaccumbens TRH (P > 0.05 in all experimental situations).
4. Discussion Whilst m a n y authors have c o m m e n t e d on the ability of TRH to induce a "general arousal" reaction (Carino et al., 1976; Havli-
Onset (rain)
5--10 ~10 5--10 -5 -10 10--15 -10 -10
Duration
430 -20 -40 -35 -10 -40 -45 -40
cek et al., 1976; Horita et al., 1977), three groups have reported their results in terms of "locomotor activity" (intra-accumbens TRH) (Miyamoto and Nagawa, 1977; Green and Heal, 1978) or "spontaneous locomotor activity" (i.p. TRH administration) (Agarwal et al., 1976). The nucleus accumbens has been shown to be intimately involved with locomotor responding (Pijnenburg and Van Rossum, 1973) and contains high concentrations of endogenous TRH (HSkfelt et al., 1975; Schenkel et al., 1974). Therefore, this area was subject to more intensive investigation as a possible site of TRH action. However, TRH failed to induce a locomotor hyperactivity following injection into the nucleus accumbens, whether measurements were from photocells or activity wheels. Both techniques were shown to be sensitive in detailing a locomotor hyperactivity, for example, marked hyperactivity was recorded using the photocell technique both after intra-accumbens dopamine and peripheral amphetamine, and the locomotor stimulant effect of amphetamine was also detected using the activity wheels. In the studies quoted above where a " l o c o m o t o r activity" was recorded after intra-accumbens TRH, the techniques used to measure "activity" record body movements or dis-
TRH AND MOTOR BEHAVIOUR
placement. This is considered an important factor since, as clearly shown in this paper and elsewhere, intracerebral (including intraaccumben~s) T R H causes b o d y shaking/head and limb movements which must inevitably be recorded to a greater or lesser degree. This was shown to be so in the present studies using an Animex recorder, when an increased " c o u n t " coincided with the development and disappearance of a "hyper-reactivity" response associated with b o d y shakes and other repetitive movements. Our data would support the conclusion of Agarwal et al. (1976) that the "counts... reflect the general state of excitability of the rat" rather than a true expression of l o c o m o t o r activity. Further studies reported in the present paper emphasise an apparent inability of intra-accumbens TRH to modify l o c o m o t o r activity; thus, intraaccumbens T R H failed to increase the locom o t o r count induced by peripheral amphetamine or intra-accumbens dopamine administration, failed to reverse the catalepsy induced by peripheral haloperidol treatment or to shorten the duration of anaesthesia induced 'by peripheral injections of chloral hydrate or sodium pentobarbitone. Although T R H injected into the nucleus accumbens caused a state of general excitation characterised b y b o d y shakes, limb tremor, repetitive head and limb movements, scratching and an increased alertness, no claims for a locus specificity of action may be made since similar behavioural changes were recorded when the bilateral T R H injections were made into the tuberculum olfactorium, thalamus, midbrain, cerebral cortex, caudateputamen, amygdala and lateral ventricles. The onset and duration of effects were generally similar irrespective of the site of injection, and one may argue that if there a single site for initiation of the behavioural effects described, then variations in onsets should occur and relate to the distance from the "active site". Further, the data obtained would not appear to reflect a rapid diffusion of T R H since a study of the distribution of 3H-TRH after intracerebral administration
149
into various brain areas indicated little diffusion at relevant time periods (Carino et al., 1976). Therefore, at the present time it would appear impossible to define a suitable diffuse neurophysiological substrate which could be activated to trigger the various m o t o r effects. However, it is interesting to note here that the effects of T R H were particularly marked from the thalamus since this area has previously been implicated in the development of "shaking behaviour" (Wei et al., 1975). In conclusion, T R H can induce a variety of motor effects (shakes, limb tremor, repetitive head and limb movements, biting, scratching and an alertness) on intraventricular and intracerebral injection in the rat. Of the dopaminecontaining areas examined, the nucleus accumbens was no more or less sensitive than the other areas, and a l o c o m o t o r hyperactivity could n o t be evoked from this region. It would appear unlikely that a single brain area or mechanism is "responsible" for the changes in motor behaviour which can be induced b y TRH.
Acknowledgements Haloperidol was donated by Janssen Pharmaceutica. T R H was supplied by Dr B.A. Morgan of Reckitt and Colman.
References Agarwal, R.A., R.B. Rastogi and R.L. Singhal, 1976, Changes in brain catecholamines and spontaneous locomotor activity in response to thyrotropin releasing hormone, Res. C o m m u n . Chem. Pathol. Pharmacol. 15, 743. Carino, M.A., J.R. Smith, B.G. Weick and A. Horita, 1976, Effects of thyrotropin-releasing hormone (TRH) microinjected into various brain areas of conscious and pentobarbitol-pretreated rabbits, Life Sci. 19, 1687. De Groot, J., 1959, The rat forebrain in stereotaxic coordinates, Verh. K. Ned. Akad. Wet. 52, 11. Green, A.R. and D.J. Heal, 1978, Release of dopamine in the nucleus accumbens of rats by thyrotrophin releasing-hormone (TRH), Brit. J. Pharmacol. (in press).
150 Havlicek, V., M. Rezek and H. Friesen, 1976, Somatostatin and thyrotropin releasing hormone: central effect on sleep and m o t o r system, Pharmacol. Biochem. Behav. 4 , 4 5 5 . H/Jkfelt, T., K. Fuxe, O. Johansson, S. Jeffcoate and N. White, 1975, Distribution of thyrotropinreleasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry, European J. Pharmacol. 34, 389. Horita, A., M.A. Carino and H. Lal, 1977, Influence of catecholamine antagonists and depletors on the CNS effects of TRH in rabbits, Progr. Neuropsychopharmacol. 1 , 1 0 7 . Miyamoto, M. and Y. Nagawa, 1977, Mesolimbic involvement in the l o c o m o t o r stimulant action of thyrotropin-releasing hormone (TRH) in rats, European J. Pharmacol. 44, 143. Mora, S., A. Loizzo and V.G. Longo, 1976, Central
B. COSTALL ET AL. effects of thyrotropin-releasing factor (TRF): interaction with some antipsychotic drugs, Pharmacol. Biochem. Behav. 4 , 2 7 9 . Pijnenburg, A.J.J. and J.M. Van Rossum, 1973, Stimulation of l o c o m o t o r activity following injection of dopamine into the nucleus accumbeus, J. Pharm. Pharmacol. 25, 1003. Plotnikoff, N.P., G.R. Breese and A.J. Prange, 1975, Thyrotropin releasing hormone (TRH): DOPA potentiation and biogenic amine studies, Pharmacol. Biochem. Behav. 3, 665. Schenkel-Hulliger, L., W.P. Koella, A. Hartmann and L. Maftre, 1974, Tremorogenic effect of thyrotropin releasing hormone in rats, Experientia 30, 1168. Wei, E., S. Sigel, H. Loh and E.L. Way, 1975, Thyrotrophin-releasing hormone and shaking behaviour in rat, Nature 253, 739.
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© Elsevier/North-Holland Biomedical Press
A STUDY OF THE CHANGES IN MOTOR BEHAVIOUR CAUSED BY T R H ON I N T R A C E R E B R A L INJECTION BRENDA COSTALL, SIU-CHUN G. HUI, GEOFFREY METCALF * and ROBERT J. NAYLOR Postgraduate School of Studies in Pharmacology, University of Bradford, Bradford, U.K. and • Pharmacology Department, Reckitt and Colman, Pharmaceutical Division, Dansom Lane, Hull, U.K.
Received 17 May 1978, revised MS received 1 August 1978, accepted 12 September 1978
B. COSTALL, S.-C.G. HUI, G. METCALF and R.J. NAYLOR, A study of the changes in motor behaviour caused by TRH on intracerebral injection, European J. Pharmacol. 53 (1979) 143--150. 20/~g TRH injected bilaterally into the caudate-putamen, tuberculum olfactorium, nucleus accumbens, amygdala, lateral ventricles, midbrain or cerebral cortex failed to induce any increase in locomotor activity (measured using photocells), although other behavioural changes were observed after each injection, and included body shakes, limb tremor, repetitive head and limb movements, biting, scratching and an alert appearance. These behavioural changes could result in positive readings from equipment used to measure locomotor activity, but careful investigations focussing on the nucleus accumbens used photocell boxes, activity wheels and Animex recorders to emphasise the' inability of intracerebral TRH (10--40/~g) to enhance locomotor activity. Intraaccumbens TRH also failed to enhance amphetamine hyperactivity or reduce the motor depression caused by haloperidol and analeptic drugs. The data do not support a central locomotor stimulant action for TRH. Intracerebral injections Nucleus accumbens
Locomotor activity
Dopamine
d-Amphetamine
1. I n t r o d u c t i o n
2. Materials and methods
It has been shown in a n u m b e r o f species t h a t T R H can m o d i f y normal m o t o r behaviour and the m o t o r effects induced by drugs which influence cerebral dopamine systems (Mora et al., 1976; P l o t n i k o f f e t al., 1975; Carino et al., 1976). However, it is difficult t o assess the relative involvement o f the different dopamine systems since m os t investigations have been limited to drug administration by the peripheral or ventricular routes. Therefore, in the present experiments T R H is injected directly into a n u m b e r o f dopamine-contalning areas, and special emphasis is placed on the possible role o f t he nucleus accumbens, an area widely implicated in m o t o r control.
2.1. A n i m a l s
TRH
T he studies utilised male, Sprague-Dawley (CFE) rats weighing 250--300 g at t he time of operation. Animals used in peripheral studies only weighed 200 -+ 25 g. 2.2. Intracerebral injection t e c h n i q u e
Bilateral guide cannulae for intracerebral injections (or unilateral guide cannulae for intraventricular injections) were c o n s t r u c t e d from 0.65 mm diameter stainless steel tubing and were implanted stereotaxicaUy in t he brains of rats anaesthetised with chloral
144
A.
E.
B. COSTALL ET AL Ant.9.4 Vert.+ 2.5(2.5mm) Lat.-+ 1.6
B.
Ant. 8.0
E
Ve r t . + 4 . 0 ( 2 . 0 m m ) L a t . - 1.5
Ant.9-0 Vert.*4.0(6'7mm) Lat.±2.5
C,
Ant.8-o V e t t.*3v0 (1.5ram) Lat.±3t0
D,
A n t . 8O V e r t.+ 5.5(1-0mm) Lat.+- 3.5
Ant.5.6
G.
A nt.4.6 V e r t.* 1-5(1.5ram) Lat.+-2.5
H.
Ant.K) Ve r t.* 0-5 ( 3 . 0 m m ) Lat._+l.5
Ve r t.+ 2 . 0 ( 3 . ' / m m ) L a t . i 4,5
Fig. 1. Diagrammatic representation of the location of the injection (e) or guide (A) cannulae tips for intracerebral injections. Stereotaxic coordinates were selected with the aid of the atlas of De Groot (1959). Coordinates given are for the implantation of the guide cannulae tips; the vertical extension of the injection units beyond the guide tips are shown in parentheses. Each diagram was constructed from the histological data obtained from 8--12 rats. A. Nucleus accumbens, B. tuberculum olfactorium, C. caudate-putamen, D. cerebral cortex, E. ventricles, F. nucleus amygdaloideus centralis, G. thalamus and H. midbrain.
hydrate, 300 mg/kg i.p. The guide cannulae were fixed to the skull using retaining screws and acrylic cement, and kept patent b y stainless steel stylets, 0.3 mm diameter, made to extend 0.5 mm below the tips of the guides. Guide cannulae were located at the coordinates shown in fig. 1, and stainless steel (0.3 mm diameter) injection units were made to extend below the guides as indicated. Animals were used 7--14 days following the operation and were used on a maximum of 2 occasions with intervening 7 day recovery periods. Rats were manually restrained during the injection procedure. Drug or solvent was administered bilaterally (simultaneously into both hemispheres) (unilaterally for intraventricular injection) with Agla micrometer syringes in volumes of 1 pl over a 5 sec period, a further
55 sec being allowed for deposition of drug. The injection units were immediately replaced b y the stylets. (In all studies reported in this manuscript and using bilateral drug administration, the dose given refers to the unilateral dose.) On completion of the studies the locations of the guide cannulae were determined histologically (fig. 1).
2.3. Behavioural studies All behavioural studies were carried out between 8 am and 7 pm in sound-proofed, diffusely illuminated rooms maintained at a temperature of 21 + 1 ° C.
2.3.1. Assessment of general motor changes Preliminary studies indicated that T R H
TRH AND MOTOR BEHAVIOUR caused characteristic m o t o r changes on injection into a number of brain regions: shaking, limb tremor, head and limb movements, biting, scratching, alertness. The occurrence of these phenomena was carefully assessed following intracerebral administration of T R H into all regions of study (see fig. 1). Immediately on completion of the intracerebral injections rats were placed in individual perspex observation cages (30 cm X 20 cm and 15 cm high) and the above behavioural changes recorded at 5--10 min intervals, for the duration of effect, as marked, present or absent.
2.3.2. Measurement of locomotor activity using photocell equipment Immediately after intracerebral injection rats were placed in perspex cages of the above dimensions, each fitted with one photocell unit placed off-centre. Hyperactivity was assessed b y counting the number of interruptions of the light beam occurring each 5 min period. Readings were taken every 10 min throughout the duration of drug effect. The behaviour of the animals was visually observed /vhilst mechanically recorded in order to differentiate measurements of l o c o m o t o r activity from counts caused b y animals tremoring, shaking etc. (as described in 2.3.1.) in the region of the beam. This method of assessment of l o c o m o t o r activity was adopted for the majority of experiments (see Results section).
2.3.3. Measurement of hyperactivity using the activity wheel In these experiments activity was recorded electromechanically from activity wheels 300 mm in diameter and 140 mm wide. Animals were placed in the activity wheels on at least 3 occasions prior to their actual use to obtain a more constant level of activity. Immediately after injection rats were placed in the activity wheels and their activity was recorded in wheel revolutions per 10 min period for the duration of drug effect. Rats were used once only in this manner.
145
2.3.4. Measurement of hyperactivity using the Animex recorder Animals were placed individually in the Animex recorder and allowed 30 min to acclimatise to the new environment. T R H was then administered into the nucleus accumbens and animals returned to the Animex recorder: counts were taken for 9 to 10 min periods and behaviour was also visually assessed. The sensitivity of the apparatus was adjusted to 40 pA.
2.3.5. Measurement of catalepsy Catalepsy was assessed b y placing animals in perspex cages (20 X 15 X 15 cm) fitted with a 10 cm high bar and, after a period of acclimatisation, placing the animal's front limbs carefully over the bar. A normal animal will recover from this position within 6 sec b u t a cataleptic animal maintains the abnormal imposed position for a period of time dependent on the degree of catalepsy. Hence, time may be transduced into intensity and, in order to account for animals which maintain the imposed position for an "infinite period of time", the following scoring system was adopted to express the intensity of catalepsy: 0.1--2.5 min = score 1, 2.6--5.0 min = score 2, 5.1--10.0 min = score 3, 10.1.-20.0 min = score 4, 20.1 min --oo = score 5. After onset, the intensity of catalepsy was assessed at 30 min intervals for 6 h.
2.3.6. Measurement of analeptic activity Analeptic activity was indicated b y a more rapid recovery of the righting reflex following anaesthetisation with chloral hydrate, 200 mg/kg i.p., or sodium pentobarbitone, 30 mg/ kg i.p.
2.4. Drugs For intracerebral injections dopamine hydrochloride (Koch-Light) was dissolved in nitrogen bubbled distilled water neutralised with sodium bicarbonate, and T R H (L-pyroglutamyl-L-histodyl-L-prolineamide L-tartrate monohydrate, thyrotropin releasing hormone)
146
(Reckitt and Colman) in distilled water. All drugs were freshly prepared. For i.p. injections, d-amphetamine sulphate (Sigma), chloral hydrate (BDH) and sodium pentobarbitone (May and Baker) were dissolved in distilled water. Solutions of haloperidol (Janssen) were prepared in distilled water from a stock solution of 10 mg/ml haloperidol in 1% lactic acid. The significance of the results was assessed using the Student's t-test for comparisons with control values. 3. Results
3.1. Changes in locomotor activity (photocell counts) caused by dopamine, TRH and a combination o f dopamine and TRH injected bilaterally into the nucleus accumbens Dopamine injected bilaterally into the nucleus accumbens, in the absence of any pretreatment, caused a dose-dependent hyperactivity which was threshold at 50 pg and maximum at 200 pg when the effect was apparent for up to 4 h (fig. 2). However, 10-4 0 p g T R H injected bilaterally into the nucleus accumbens under similar conditions failed to cause any increase in activity (fig. 2). 80 pg T R H produced inconsistent changes in l o c o m o t o r activity: only 10% of animals treated with this dose (of a total of 60 animals tested) appeared hyperactive, b u t the concurrent development of repetitive head and limb movements, tremor etc. as described in 3.5. prevented a differentiation of hyperactivity counts. Further, the combination of 20 pg T R H with the threshold dose of dopamine for hyperactivity induction ( 5 0 p g ) failed to initiate a hyperactivity response (fig. 2). Similarly, 40 pg T R H failed to enhance the l o c o m o t o r hyperactivity established to a dose of 100 pg dopamine.
3.2. The effect of intra-accumbens TRH on the hyperactivity (photocell counts) induced by peripherally administered d.amphetamine d-Amphetamine caused a dose-dependent hyperactivity response (0.5--1.5 mg/kg i.p.,
B. C O S T A L L E T AL,
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Hours after injection Fig. 2. Changes in l o c o m o t o r activity c a u s e d b y dopamine, TRH and a combination of dopamine and TRH i n j e c t e d bilaterally i n t o t h e n u c l e u s a c c u m b e n s . In t h e l a t t e r e x p e r i m e n t s d o p a m i n e was given 30 rain b e f o r e t h e i n j e c t i o n o f TRH. H y p e r a c t i v i t y is e x p r e s s e d as t h e n u m b e r of i n t e r r u p t i o n s o f a p h o t o cell b e a m o c c u r r i n g w i t h i n a 5 rain period. E a c h value is t h e m e a n o f d e t e r m i n a t i o n s f r o m 8 rats. S t a n d a r d errors are less t h a n 20% o f t h e means.
maximum counts being 50--117 respectively, S.E.M.s <20%). Hyperactivity established to a submaximal dose of 1 . 0 m g / k g d-amphetamine was n o t modified b y a subsequent bilateral intra-accumbens injection of 20 pg T R H (P > 0.05).
3.3. The effect of d-amphetamine administered peripherally and TRH administered into the nucleus accumbens on wheel activity of rats Activity wheels were shown to detect the ability of d-amphetamine to cause a dose-
TRH AND MOTOR BEHAVIOUR
dependent hyperactivity (0.5--1.5 mg/kg i.p., maximum wheel revolutions/10 min being 10--25 respectively, S.E.M.s <20%). However, this technique failed to show any ability of intra-accumbens TRH to increase activity (2.5--40 #g, P > 0.05 compared with the activity of normal rats, or rats receiving intraaccumbens solvent).
147
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3.4. The effects of TRH administered into the nucleus accumbens and measured by the Animex recorder 10
10--40 pg TRH injected into the nucleus accumbens caused a general "hyperactivity" associated with repetitive head and limb movements, tremor etc. as indicated in 3.5. Counts were obtained from the Animex recorder which were significantly different from control values, P < 0.05--P < 0.001, during the 30--40 min period following injection (fig. 3). However, these "counts", which were not dose-dependent, could not be differentiated to give any indication of an increased locomotor activity. 3.5. The effects on general behaviour and on locomotor activity (photocell counts) o f TRH injected into several brain regions 20 #g TRH injected bilaterally into the caudate-putamen, tuberculum olfactorium, nucleus accumbens, amygdala, lateral ventricles, midbrain, cerebral cortex or thalamus failed to induce any increase in locomotor activity ( P < 0.05 compared with animals receiving intracerebral solvent). However, this dose of TRH was shown to be adequate to cause a number of other behavioural changes from each brain area analysed, the response observed varied from area to area both in nature and severity as indicated in table 1, and included body shakes, limb tremor, repetitive head and limb movements, biting, scratching (at body and environment), and a degree of alertness which was quite distinct from the behaviour of control animals. Generally, these behavioural changes were most marked
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Fig. 3. The effect of 10 =-=, 20 -* • and 40 • " pg TRH, or solvent o o, injected bilaterally into t h e n u c l e u s a c c u m b e n s as r e c o r d e d by an " A n i m e x " in c o u n t s / 1 0 min. 6--8 rats were used at each dose level. Mean values are given ±S.E.M.
during the 20--30 min period following onset. It is stressed that animals exhibiting these responses to TRH, even those appearing unusually alert, at no time exhibited movements about the observation cages which could be differentiated in frequency from those of control animals. The behaviottral changes described here for a dose of 20 #g TRH were also apparent at 10 #g TRH: the intensity, onset and duration of effects could not be distinguished from those described. As the dose of TRH was further reduced the change was observed in the number of animals responding, not in the nature of the response: 60--6.5% of animals responded to a dose of 5/~g TRH but no response was observed at 2.5 #g. 3.6. The effect o f intra-accumbens TRH on haloperidol catalepsy Haloperidol, 0.5--4 mg/kg i.p., induced a dose-dependent catalepsy (scored 1--5 respectively, S.E.M.s <20%). The weak intensity catalepsy induced by 0.5 mg/kg i.p. haloperidol was not enhanced (or in any way signifi-
148
B. C O S T A L L E T A L
TABLE 1 B e h a v i o u r a l e f f e c t s o f T R H i n j e c t e d b i l a t e r a l l y i n t o d i f f e r e n t b r a i n areas. T h e d i f f e r e n t b e h a v i o u r a l s t a t e s are d e s i g n a t e d as a b s e n t (0), p r e s e n t ( + ) o r r e l a t i v e l y m a r k e d (++). n = 8 f o r e a c h g r o u p . Brain area
Nucleus accumbens Tuberculum olfactorium Thalamus Midbrain Cerebral cortex Caudate-putamen Lateral ventricles Amygdala
B e h a v i o u r o b s e r v e d a f t e r i n t r a c e r e b r a l i n j e c t i o n o f T R H (20 pg) Shakes
Limb tremor
Head and limb movement
Biting
Scratching
Alertness
+ + ++ + + + + ++
+ 0 ++ + 0 + ++ +
+ + ++ + + + ++ +
0 0 ++ 0 0 ++ + 0
+ + + + 0 ++ + +
+ + ++ ++ 0 ++ + ++
cantly modified, P > 0.05) by a subsequent bilateral intra-accumbens injection of 20 pg TRH. 3.7. Effect o f intra-accumbens T R H on the sleeping time to chloral hydrate and sodium pentobarbitone
TRH (20 pg injected bilaterally into the nucleus accumbens) showed no analeptic activity. The times of onset of "sleep" (loss of righting reflex) (2.84 + 0.37 min for chloral hydrate, 4.8 + 0.07 min for sodium pentobarbitone in control animals) to recovery of the righting reflex (duration of loss 35.2 + 8.1 min for chloral hydrate, 107.7 +- 19.4 min for sodium pentobarbitone in control animals) did n o t differ significantly between control animals, animals receiving intraaccumbens solvent and those receiving intraaccumbens TRH (P > 0.05 in all experimental situations).
4. Discussion Whilst m a n y authors have c o m m e n t e d on the ability of TRH to induce a "general arousal" reaction (Carino et al., 1976; Havli-
Onset (rain)
5--10 ~10 5--10 -5 -10 10--15 -10 -10
Duration
430 -20 -40 -35 -10 -40 -45 -40
cek et al., 1976; Horita et al., 1977), three groups have reported their results in terms of "locomotor activity" (intra-accumbens TRH) (Miyamoto and Nagawa, 1977; Green and Heal, 1978) or "spontaneous locomotor activity" (i.p. TRH administration) (Agarwal et al., 1976). The nucleus accumbens has been shown to be intimately involved with locomotor responding (Pijnenburg and Van Rossum, 1973) and contains high concentrations of endogenous TRH (HSkfelt et al., 1975; Schenkel et al., 1974). Therefore, this area was subject to more intensive investigation as a possible site of TRH action. However, TRH failed to induce a locomotor hyperactivity following injection into the nucleus accumbens, whether measurements were from photocells or activity wheels. Both techniques were shown to be sensitive in detailing a locomotor hyperactivity, for example, marked hyperactivity was recorded using the photocell technique both after intra-accumbens dopamine and peripheral amphetamine, and the locomotor stimulant effect of amphetamine was also detected using the activity wheels. In the studies quoted above where a " l o c o m o t o r activity" was recorded after intra-accumbens TRH, the techniques used to measure "activity" record body movements or dis-
TRH AND MOTOR BEHAVIOUR
placement. This is considered an important factor since, as clearly shown in this paper and elsewhere, intracerebral (including intraaccumben~s) T R H causes b o d y shaking/head and limb movements which must inevitably be recorded to a greater or lesser degree. This was shown to be so in the present studies using an Animex recorder, when an increased " c o u n t " coincided with the development and disappearance of a "hyper-reactivity" response associated with b o d y shakes and other repetitive movements. Our data would support the conclusion of Agarwal et al. (1976) that the "counts... reflect the general state of excitability of the rat" rather than a true expression of l o c o m o t o r activity. Further studies reported in the present paper emphasise an apparent inability of intra-accumbens TRH to modify l o c o m o t o r activity; thus, intraaccumbens T R H failed to increase the locom o t o r count induced by peripheral amphetamine or intra-accumbens dopamine administration, failed to reverse the catalepsy induced by peripheral haloperidol treatment or to shorten the duration of anaesthesia induced 'by peripheral injections of chloral hydrate or sodium pentobarbitone. Although T R H injected into the nucleus accumbens caused a state of general excitation characterised b y b o d y shakes, limb tremor, repetitive head and limb movements, scratching and an increased alertness, no claims for a locus specificity of action may be made since similar behavioural changes were recorded when the bilateral T R H injections were made into the tuberculum olfactorium, thalamus, midbrain, cerebral cortex, caudateputamen, amygdala and lateral ventricles. The onset and duration of effects were generally similar irrespective of the site of injection, and one may argue that if there a single site for initiation of the behavioural effects described, then variations in onsets should occur and relate to the distance from the "active site". Further, the data obtained would not appear to reflect a rapid diffusion of T R H since a study of the distribution of 3H-TRH after intracerebral administration
149
into various brain areas indicated little diffusion at relevant time periods (Carino et al., 1976). Therefore, at the present time it would appear impossible to define a suitable diffuse neurophysiological substrate which could be activated to trigger the various m o t o r effects. However, it is interesting to note here that the effects of T R H were particularly marked from the thalamus since this area has previously been implicated in the development of "shaking behaviour" (Wei et al., 1975). In conclusion, T R H can induce a variety of motor effects (shakes, limb tremor, repetitive head and limb movements, biting, scratching and an alertness) on intraventricular and intracerebral injection in the rat. Of the dopaminecontaining areas examined, the nucleus accumbens was no more or less sensitive than the other areas, and a l o c o m o t o r hyperactivity could n o t be evoked from this region. It would appear unlikely that a single brain area or mechanism is "responsible" for the changes in motor behaviour which can be induced b y TRH.
Acknowledgements Haloperidol was donated by Janssen Pharmaceutica. T R H was supplied by Dr B.A. Morgan of Reckitt and Colman.
References Agarwal, R.A., R.B. Rastogi and R.L. Singhal, 1976, Changes in brain catecholamines and spontaneous locomotor activity in response to thyrotropin releasing hormone, Res. C o m m u n . Chem. Pathol. Pharmacol. 15, 743. Carino, M.A., J.R. Smith, B.G. Weick and A. Horita, 1976, Effects of thyrotropin-releasing hormone (TRH) microinjected into various brain areas of conscious and pentobarbitol-pretreated rabbits, Life Sci. 19, 1687. De Groot, J., 1959, The rat forebrain in stereotaxic coordinates, Verh. K. Ned. Akad. Wet. 52, 11. Green, A.R. and D.J. Heal, 1978, Release of dopamine in the nucleus accumbens of rats by thyrotrophin releasing-hormone (TRH), Brit. J. Pharmacol. (in press).
150 Havlicek, V., M. Rezek and H. Friesen, 1976, Somatostatin and thyrotropin releasing hormone: central effect on sleep and m o t o r system, Pharmacol. Biochem. Behav. 4 , 4 5 5 . H/Jkfelt, T., K. Fuxe, O. Johansson, S. Jeffcoate and N. White, 1975, Distribution of thyrotropinreleasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry, European J. Pharmacol. 34, 389. Horita, A., M.A. Carino and H. Lal, 1977, Influence of catecholamine antagonists and depletors on the CNS effects of TRH in rabbits, Progr. Neuropsychopharmacol. 1 , 1 0 7 . Miyamoto, M. and Y. Nagawa, 1977, Mesolimbic involvement in the l o c o m o t o r stimulant action of thyrotropin-releasing hormone (TRH) in rats, European J. Pharmacol. 44, 143. Mora, S., A. Loizzo and V.G. Longo, 1976, Central
B. COSTALL ET AL. effects of thyrotropin-releasing factor (TRF): interaction with some antipsychotic drugs, Pharmacol. Biochem. Behav. 4 , 2 7 9 . Pijnenburg, A.J.J. and J.M. Van Rossum, 1973, Stimulation of l o c o m o t o r activity following injection of dopamine into the nucleus accumbeus, J. Pharm. Pharmacol. 25, 1003. Plotnikoff, N.P., G.R. Breese and A.J. Prange, 1975, Thyrotropin releasing hormone (TRH): DOPA potentiation and biogenic amine studies, Pharmacol. Biochem. Behav. 3, 665. Schenkel-Hulliger, L., W.P. Koella, A. Hartmann and L. Maftre, 1974, Tremorogenic effect of thyrotropin releasing hormone in rats, Experientia 30, 1168. Wei, E., S. Sigel, H. Loh and E.L. Way, 1975, Thyrotrophin-releasing hormone and shaking behaviour in rat, Nature 253, 739.