The effects of thyrotropin-releasing hormone, metabolites and analogues on locomotor activity in rats

Regulatory Peptides, 7 (1983) 97-109

97

Elsevier RPT00229

The effects of thyrotropin-releasing hormone, metabolites and analogues on locomotor activity in rats J.S. Andrews and A. Sahgal MRC Neuroendocrinology Unit, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, U.K.

(Received2 June 1983; revisedmanuscriptreceived 11 July 1983; acceptedfor publication12July 1983)

Summary Thyrotropin-releasing hormone (TRH) has generally been reported to increase locomotor activity in rats; however there are also some negative reports. In order to identify the possible causes for this discrepancy, the effects of intra-cerebroventricular injection of TRH, its metabolites 'acid TRH' (TRH-OH) and His-Pro-diketopiperazine (DKP), and two analogues 3-methyl-His-TRH and RX 77368 (3,3-dimethyl-Pro-TRH), were assessed using photocell activity cages. All compounds were tested in groups of eight rats in the afternoon (1300-1700 h), but in addition TRH and DKP were tested in two further groups of rats during the morning (0900-1230 h). TRH and DKP failed to induce a significant rise in activity during the morning test period, but TRH did have a significant effect when tested in the afternoon. Both TRH and TRH-OH caused dose dependent increases in locomotor activity, whereas DKP and the two analogues had no effect. This stimulation of activity was shown to be at least partly mediated by dopamine since locomotor enhancement was blocked in a second experiment using the dopamine antagonist a-Flupenthixol. The results are discussed in terms of actions on the mesolimbic dopamine system, and the importance of circadian variations within this system to the expression of peptide effects in general. TRH; TRH analogues; Flupenthix01; locomotor activity; circadian rhythms; rats

Introduction

Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) is widely distributed throughout the nervous system and has been accorded the status of a putative 0167-0115/83/$03.00 © 1983 ElsevierSciencePublishersB.V.

98 neurotransmitter or modulator of synaptic function (for a review see Ref. 1). The discovery of specific binding sites concentrated in regions such as the nucleus accumbens [2] and the finding that TRH stimulates dopamine release from this structure [3] suggests that it may play a regulatory role at some dopaminergic synapses. In keeping with this view, there are several reports that TRH stimulates locomotor activity [4-6] which is known to involve the mesolimbic dopamine system. However, some groups have failed to obtain increased locomotor activity with TRH [7,8] and it is uncertain whether all the effects attributed to TRH are due to the peptide itself. This peptide is rapidly degraded by peptidases [9] and produces metabolites that may themselves be active. For example, one such metabolite 'acid-TRH' (TRH-OH, pGlu-His-Pro-OH) is at least as active as TRH in producing shaking behaviour ('wet dog shakes', WDS) [10, 11] when injected into the ventricles. Diketopiperazine (DKP, cyclo-His-Pro), another metabolite, has a different regional distribution in brain to TRH [12], and is reported to affect locomotor activity following local infusion [l 3,14]. TRH is often contaminated with DKP and occasionally TRH-OH, the presence of which may help to explain reported inconsistencies. By using TRH, metabolites and analogues which degrade in a known fashion it should be possible to identify which compounds are behaviourally active. Even more interesting is the possibility that the effects of TRH depend on the time of injection, since animals demonstrate rhythmic diurnal cycles. It could be that T R H effects are particularly sensitive to such rhythms, and this coupled with its short half-life may explain some of the discrepant findings. The present study was undertaken to investigate the influence of testing time on the effects of TRH, its metabolites and two analogues on locomotor activity.

Experiment 1 Materials and methods Animals and surgery 56 male Wistar rats (Bantin and Kingrnan, Hull, U.K.), weighing 275-300 g at the beginning of the experiment were individually housed after surgery with free access to food and water under diurnal lighting conditions (lights on 0700-1900 h). Stainless steel, 23 gauge cannulae (Plastic Products Inc., VA, U.S.A.) were implanted under general anaesthesia (Nembutal). The co-ordinates measured from Bregma were LV + 1.3, AP - 1 , HV 4.5 mm. At the conclusion of the experiment, all placements were verified by injection of Evan's blue dye into the ventricles, followed by inspection of gross sections. Drugs In addition to TRH, the following homologues were used: TRH-OH, DKP, 3Me-TRH, pGlu-[3-methyl-His]-Pro-NH2; RX 77368, pGlu-His-(3,3-dimethyl)-ProN H 2. TRH, TRH-OH, and DKP were obtained from Cambridge Research Biochemicals, Cambridge, U.K.; 3Me-TRH from Merseyside Laboratories, Warrington,

99 U.K. and RX 77368 from Reckitt and Coleman, Hull, U.K. The purity of these peptides was tested by high performance liquid chromatography (HPLC). They were dissolved in 0.9% saline and 2 /11 injected into the lateral ventricle through an injection cannula, immediately prior to testing.

Apparatus Locomotor activity was assessed in 8 black Perspex cages measuring 40 x 20 × 20 cm fitted with a metal mesh floor. Each cage was equipped with 2 red light photocells (20 cm apart and 4 cm above the floor), connected to a micro-computer (Acorn Computers Ltd., Cambridge, U.K.) running on ONLIBASIC software [15]. Procedure Seven days after surgery, the rats were placed in the activity boxes for 90 rain per day for three consecutive days. On the fourth day o f habituation each animal received an intracerebroventricular (icv) injection of 2/~1 of saline. This was repeated the following day, which became the first data day. Rats were tested with the relevant drug every other day in order to minimise possible carry-over effects. The protocol was a reverse block design, the peptide being administered and data collected in this order for TRH, TRH-OH and DKP: saline, 10/~g, 100/~g, 1 ~g, 1 /~g, 100/~g, 10 #g, saline. For the two analogues: saline, 1/~g, 10/~g, 0.1/~g, 0.1 #g, 10/~g, 1 /~g, saline. These doses were selected on the basis of previous reports [e.g. 16], and on unpublished observations in our laboratory. Thus, each subject yielded 8 data days, 2 for each dose, and the data from each identical pair were subsequently averaged to minimise order effects. Each o f five groups of eight rats received one of the drugs and were tested during the afternoon (1300-1700 h); in addition, two further groups received either TRH or DKP and were tested in the morning (0900-1230 h) using an identical procedure. The computer recorded (a) counts per photocell over 18 successive 5-min time bins, and (b) all counts with a duration of less than 0,2 s, i.e., very brief interruptions (VBIs). It has often been noticed that high bursts of counts are restricted to one photocell and these could be caused by grooming or shaking rather than ambulation. TRH has been reported to enhance these activities: this could lead to spuriously high counts, suggesting increased ambulation. For example, Costall et al. [7] found no effect of TRH on overall activity but did note that rats were more alert and repetitive movements such as grooming often occurred. Collecting data as total counts and VBIs may yield information as to the possible cause of any changes in overall activity. If differences disappear when VBIs are subtracted from the total counts, then any increase was probably not due to locomotor effects. The data were analysed by a two factor (dose × time) repeated measures analysis of variance (ANOVA), and a posteriori comparisons by Tukey's HSD (A) test at the 0.05 significance level [ 17]. Results The results of the two factor repeated measures ANOVA's for total counts are shown in Table I; the VBI data gave an identical distribution of results (which, for

100

TABLE I

Results of two factor repeated measures analysis of variance on the total counts recorded in photocell activity cages Dose

Time

D o s e × time

F(3, 21)

F(17, 119)

F(51,357)

TRH m

2.242

8.049 **

DKP m

0.262

16.302 **

1.79 *

24.087 **

2.275 **

33.337 **

1.974 **

TRH a

15.325 **

DKP a

1.578

TRH-OH 3Me-TRH

8.482 ** 0.619

R X 77368

0.924

8.18 ** 5.215 ** 10.772 **

1.096

1.457 * 2.02 ** 0.804

Significant results are represented by asterisks: * = P < 0.05; ** = P < 0.01; m = morning testing, a = afternoon testing. See text for further details.

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Fig. 1. The effects of icy injection of T R H on locomotor activity after morning (top), or afternoon (bottom) administration to groups of eight rats. Key: 0 ~ saline; * ~ 1 t~g of TRH; • = 10 ~g TRH; • = 100/~g TRH. Inset diagram shows mean total counts and S.E.M: for each drug condition, significant differences from saline indicated by an asterisk. See text for further details.

101 the sake of brevity, are not shown). All groups showed a significant time effect; activity generally decreased. TRH N o dose gave a significant increase over saline control values, when administered to animals tested in the morning. However, a clear dose dependent effect on activity was seen when T R H was given to animals tested in the afternoon. A posteriori testing of factor A (dose) revealed both 10 and 100/~g to be significantly higher than saline. The effect of T R H on activity counts for both morning and afternoon administration is illustrated in Fig. 1. As the VBI data also showed a similar effect, the increase seen in the total counts could have been due to abnormally high VBIs, reflecting grooming and shaking, and not to locomotor activity per se. Thus two further analyses were undertaken: VBIs as a percentage of total counts increased in a dose dependent manner (dose F(3, 21)= 7.684, P < 0.01). However, the effect of T R H on locomotor activity was preserved even when VBIs were subtracted from the total counts data (dose F(3, 21)= 18.975, P < 0.01, time /7(17, 119) = 24.535, P < 0.01), dose x time F(51,357) = 2.43, P < 0.01). Thus, we can conclude that the observed increase in counts after T R H administration in the afternoon were due to a rise in locomotor activity concurrent with, and not because of, an increase in VBIs. DKP D K P had no significant effect on overall activity (Table I) or VBIs whether administered in the morning or the afternoon. TRH-OH T R H - O H had a significant effect on locomotor activity (Table I) during the afternoon test period; a posteriori testing revealed that all doses (1, 10 and 100/~g) differed significantly from saline in a dose dependent manner. Analysis of VBI data revealed a similar pattern and again a posteriori testing showed all doses to be significantly different from saline. The data were further analysed to discover to what extent the increase in total counts was due to an increase in locomotor activity and not to an increase in VBIs. Although the percentage of VBIs in the total counts increased, this was not significant, and, as the increase in locomotor activity was preserved when they were subtracted from the total counts (dose F(3, 21)= 9.647 P < 0.01), we can conclude that the increase is due to a rise in locomotor activity. The effects of T R H - O H and D K P when tested in the afternoon are illustrated in Fig. 2. R X 77368 This analogue had no significant effect on locomotor activity (Table I) or VBIs during the afternoon test period. All three doses produced marked changes in the animals' appearance and behaviour. Extreme piloerection was seen, and the two higher doses (1 and 10/~g) produced a hunched posture and stereotypic behaviour, including biting; these effects continued for several hours after testing.

102

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~ ~ ~ ~ 6 ~ 8 6 61'11~~1~-Y8 Time 5 rain bins Fig. 2. The effects of icy injection of DKP (top diagram) or T R H - O H (bottom) on locomotor activity. Key: 0 = saline, * = 1 #g; • = 10 #g; • = 100 #g. Inset diagram shows mean total counts and S.E.M. for each drug condition, significant differences from saline indicated by an asterisk: See text for further details.

3Me- T R H 3Me-TRH had no significant effect on locomotor activity (Table I) or VBIs during the afternoon test period. Unlike RX 77368 there were no striking behavioural effects and thus no obvious interference with activity from stereotypic responses. Discussion

Originally, it was hoped to compare directly the effects of T R H and its homologues on locomotor activity. However, as this study was carried out over several months, the animals used originated from different breeding stock and did not show the same baseline rates; essentially rats split into two comparable groups, the four T R H / D K P groups and the T R H - O H / a n a l o g u e groups of rats. As the baselines were not strictly comparable, no formal statistical test between the groups was undertaken. However, since each rat served as its own control, the different groups may be regarded as self-contained experiments; the results are discussed in this context.

103

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Key:

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for each drug condition.

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(bottom) mean

on locomotor

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See text for further details.

TRH had no significant effect on locomotor activity when administered in the morning, but both TRH and TRH-OH significantly increased locomotor activity when tested in the afternoon. DKP had no effect whether administered in the morning or the afternoon. Although one of the two analogues (RX 77368) was observed to have gross behavioural effects, neither analogue had any overall effect on locomotor activity. The fact that TRH significantly increased locomotor activity only when administered in the afternoon may help to explain the discrepancies in the reported effects of TRH administration. It can be concluded that the effects of TRH are dependent upon circadian rhythms. Although a diurnal rhythm has been reported for TRH [18], there is evidence to suggest that the differential effect described here was due to changes in the dopamine (DA) system. Locomotor activity is known to be dependent on the activation of the mesolimbic DA system. Since TRH promotes the release of DA from mesolimbic terminal areas such as the nucleus accumbens [3] and induces locomotor activity when injected directly into this area [5,14], TRH induced activity is probably due to its action on this system.

104 Amphetamine, which increases locomotor activity (regardless of time of injection), is also especially effective in the afternoon [19]. These workers have reported that TRH enhances amphetamine-induced activity in the morning, but depresses it in the afternoon, a result which appears to contradict the present finding. A consideration of rate-dependent effects (see Robbins [20] for a review) provides a solution: it is known that stimulants often enhance low, but suppress high rates of responding, and the effects of a combined amphetamine and TRH injection may therefore be facilitatory only when TRH has little or no effect (morning). Such administration may result in a reduction of activity in the afternoon, since both agents are now exerting a powerful stimulatory effect', perhaps producing (stereotypic) behaviour incompatible with locomotion. DKP has been reported to depress spontaneous locomotor activity [13] and antagonize TRH induced activity [14]. As DKP was tested in both the morning and afternoon the lack of a behavioural response may not be due to circadian changes. Although no long term behavioural effects were observed (either enhancing or suppressing), immediately following injection animals appeared much more active in comparison to administration of the other compounds. However, this effect was very short lived and was probably not seen because of the few minutes taken to inject each animal and begin the recording. Unless DKP is rapidly removed from circulation, either by uptake or metabolism, one conclusion must be that DKP is behaviourally inactive and the actual function of DKP remains to be clarified. Studies in this laboratory (unpublished observations) have occasionally demonstrated striking behavioural effects of DKP; however, stock samples analysed by HPLC have then been found to be contaminated with several other compounds. Previous reports of DKP effects may not have taken into account the level of impurities in many commercially available preparations. The analogues used here had no measurable effect on locomotor activity. However, RX 77368 did cause stereotypic behaviour similar to that induced by dopamine agonists such as apomorphine. Similar results, after systemic administration, have been reported [8]. The wide variation in the activity scores of these animals reflects the fact that much of the activity recorded was stereotyped behaviour, which will only be recorded if the rat is stereotyping in front of a (single) photocell. The exceptional length of time that these behaviours persisted can be partly attributed to RX 77368's resistance to enzymatic degradation. This compound does not appear to form a de-amidated 'free acid' metabolite or a significant quantity of the DKP form when incubated with rat brain tissue [9]. The lack of significant effects for 3Me-TRH was surprising. This analogue has been reported to possess a much greater affinity for the TRH receptor [21] and a correspondingly greater potency for producing WDS [16], although in this study, very little WDS was seen. The failure of 3Me-TRH to stimulate locomotor activity has also been noted by others (Griffiths, personal communication), and one explanation for this may be that 3Me-TRH is active at only some TRH receptor sites.

105

Experiment 2 If the effects of T R H and T R H - O H are dependent on the D A system, then they ought to be blocked by a specific D A antagonist such as a-Flupenthixol (Flu). Conversely, if the effects of the analogues were due to stereotypy caused by D A over-stimulation, then D A blockade m a y result in enhanced locomotor activity, since incompatible responses m a y be attenuated.

Materials and Methods Animals The subjects were the five groups of 8 Wistar rats tested in the afternoon (Experiment 1).

Apparatus The equipment was the same as in Experiment 1.

Drugs and procedure 48 h after the final saline day, each rat was injected with 0.5 m g / k g of a-Flupenthixol (Lundbeck, Copenhagen, D K ) 30 rain prior to testing. After an intervening rest day, they were injected with Flu as before, and then immediately before testing injected with the high dose (10 or 100/xg in 2/xl of saline, icv) of the T R H homologue they had previously received in Experiment 1. Statistical analysis was by a two factor A N O V A (drug × time) to c o m p a r e the second saline day with Flu and F l u / T R H - a n a l o g u e data; a posteriori testing was by T u k e y ' s H S D test [17].

Results The results of the A N O V A ' s are summarised in Table II. As in Experiment 1, time was always significant reflecting the fact that activity generally decreased

TABLE II Results of two factor repeated measures analysis of variance on total activity counts after a-Flupenthixol treatment

TRH DKP TRH-OH 3Me-TRH RX 77368

Dose F(2, 14)

Time F(17, 119)

Dose × time F(34, 238)

13.008 ** 9.328 ** 0.162 3.757 * 4.74 *

19.092 ** 26.754 ** 6.731 ** 6.531 ** 2.184 **

1.649 * 3.654 ** 1.868 ** 1.603 * 1.405

Significant results are represented by asterisks: * = P < 0.05; ** = P < 0.01. See text for further details.

106

500 ¸

I

±

35 300

TRH

DKP

TRH-OH

RX 77368

3 Ho-TRH

Fig. 4. The effects of 0.5 mg/kg of Flu on the ability of icv administration of TRH and homologues to increase locomotor activity. Each histogram shows the mean and S.E.M. for eight rats. Key: horizontal stripes = saline; unshaded = 0.5 mg/kg of Flu; vertical stripes = 0.5 mg/kg Flu/100 #g TRH, DKP, TRH-OH or 0.5 mg/kg Flu/10 gg RX "17368, 3Me-TRH; * = significant difference from saline. See text for further details. The difference Imtween the TRH and DKP saline scores in comparison to the other groups is striking. This effect is partly due to these two groups being abnormally active at this stage of the design. All other groups of rats tested show a decrease in activity between the first and second saline days averaging around 255; the other two groups show a 10~ increase suggesting some disturbance. As each animal is its own control, this does not affect the result of the experiment. However, a strictly formal comparison between drug groups would be difficult as the baseline controls of the groups are different.

during the test session. Fig. 4 shows the average total counts for each of the groups under the different drug conditions. With the exception of TRH-OH, all peptides showed a significant drug effect. In the TRH group of animals, Flu depressed overall activity, and also abolished TRH induced increase. For the DKP treated group, Flu again depressed activity; DKP given together with Flu did not reverse this blockade. In the 3Me-TRH treated group of rats Flu/3Me-TRH treatment did not differ significantly from saline, but reversed the slight depression of activity induced by Flu alone. The RX 77368 treated rats were significantly more active after joint F l u / R x 77368 administration than on saline or Flu treatment days: as in Experiment 1, the counts recorded were not locomotor activity per se, but stereotypic movements generally confined to one area. Although Flu did not block the intense stereotypy, in comparison with the previous study very little biting was observed. Discussion

a-Flupenthixol blocked the effects of both TRH and TRH-OH, and this provides evidence that their effects are mediated via a similar mechanism. The apparent contradiction between the results of the two experiments using RX

107

77368 (no effect in comparison to saline in Experiment 1, significant difference in Experiment 2) could be due to its ability to produce marked and persistent stereotypy, which is not blocked by the dose of Flu used. Moreover, nearly all animals show a reduction in activity between the first and second saline days, due to continuing habituation to the test environment. Taken together, these effects may contribute to the discrepancy.

General Discussion

The results of these experiments demonstrate that both TRH and its metabolite TRH-OH increase locomotor activity in a dose dependent manner, and that this response is probably mediated via the DA system since the stimulatory effects are blocked by a specific DA antagonist. The study also demonstrates the importance of diurnal rhythms in regulating peptide effects. A similar type of result has recently been described for the effects of substance P on general activity [22]. Thus, circadian variation may play a critical role, and is therefore of importance when assessing the effects of peptides, not only on behaviour, but also other regulatory effects. TRH and TRH-OH have similar effects on shaking behaviour when centrally injected [10,11]. Since TRH is rapidly converted to TRH-OH in the central nervous system, it can be argued that the effects of TRH are in fact due to TRH-OH. An alternative hypothesis is that TRH-OH exerts its behavioural effects by being re-amidated to TRH [23]. There is some support for this: TRH-OH injected into the periaqueductal grey area in rats takes longer than TRH to induce shaking [11]. If TRH-OH was the active metabolite, then it might be expected to act more, and not less quickly. In this study, the magnitude of the response to TRH-OH was less than that to TRH (Figs. 1 and 2), and RX 77368, which is not metabolised, was visibly the most behaviourally active compound. Although this can be seen as support for the re-amidation hypothesis, an alternative interpretation is that TRH-OH is itself active, perhaps at the same site, and that initial activity is due to TRH, but the effects continue at a reduced level due to the less active metabolite. The differential effect of morning and afternoon administration of TRH may then be due to the DA system being less sensitive to TRH and virtually unresponsive to its metabolite. It appears that TRH and TRH-OH, while stimulating behaviour, are not as powerful as traditional DA agonists. An obvious difference is the fact that TRH is readily metabolised, whereas amphetamine for example is not. Although circadian rhythms influence the magnitude of the response to amphetamine [cf. 19], it is such a potent stimulant that time of administration may be essentially unimportant for its effects. Our familiarity with relatively stable DA agonists could have hindered understanding of the more subtle, time dependent effects of neuropeptides. In this context, the stable TRH analogue RX 77368 may be especially useful. As a stimulant, this analogue is more potent than the classic DA agonists amphetamine and apomorphine, at least when centrally administered. If it acts at the TRH receptor (no binding studies have yet been reported), then like amphetamine time of administration may well prove to be unimportant.

108

The TRH analogue 3Me-TRH had little effect on locomotor activity with respect to saline in both experiments. However, as it reversed the slight depression of activity caused by Flu it cannot be considered as completely inactive. The reason for 3Me-TRH's overall lack of effect on locomotor activity is not easy to explain. Although 3Me-TRH may bind as well as the naturally occurring peptide to TRH receptors in all brain areas, it does not necessarily follow that it is active at all receptor sites. Therefore, 3Me-TRH may emulate only some of the effects of the naturally occurring peptide, the control of which is localised to one area and one particular type of TRH receptor. This may explain why in the present study the previously reported ability of a compound to induce WDS does not predict its ability to increase locomotor activity. Finally, the data illustrate a problem often associated with the comparison between separate groups in multiple drug studies spread over a period of time. Different baseline locomotor activity rates prevent a direct comparison between groups; however, we may regard each group as an independent experiment, and then draw relative conclusions. We conclude that future studies involving TRH, and perhaps other behaviourally active peptides should consider interactions not only with classical transmitter systems but also with circadian rhythms. Moreover, the results demonstrate that TRH metabolites may themselves be active, and this has important implications for behavioural studies.

Acknowledgements We thank Dr. G. Metcalf of Reckitt and Colman Ltd., for supply of the RX 77368 analogue, Professor J.A. Edwardson, Dr. J. McDermott, Messrs. P. Huddie and A. Smith and Miss C. Wright for their help. J.S.A. holds a Medical Research Council scholarship.

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109 8 Metcalf, G., Dettmar, P.W., Fortune, D., Lynn, A.G. and Tulloch, I.F., Neuropharmacological evaluation of RX 77368 - - a stabilised analogue of thyrotropin-releasing hormone (TRH), Regul. Peptides, 3 (1982) 193-206. 9 Griffiths, E.C., McDermott, J.R. and Smith, A.I., Mechanisms of brain inactivation of centrally acting thyrotrophin-releasing hormone (TRH) analogues: a high performance liquid chromatography study, Regul. Peptides, 5 (1982) 1-11. 10 Boschi, G., Launay, N. and Rips, R., Induction of wet-dog shakes by intracerebral 'acid' TRH in rats, Neurosci. Lett., 16 (1980) 209-212. 11 Webster, V.A.D., Griffiths, E.C. and Slater, P., Induction of wet-dog shaking in rats by analogues and metabolites of thyrotropin-releasing hormone (TRH), Regul. Peptides, 5 (1982) 43-51. 15 Prasad, C., Mori, M., Wilber, J.F., Pierson, W., Pegues, J. and Jayaraman, A., Distribution and metabolism of cycio (His-Pro): a new member of the neuropeptide family, Peptides, 3 (1982) 591-598. 13 Bhargava, H.M. and Matwyshyn, G.A., Influence of thyrotropin releasing hormone and histidyl-proline diketopiperazine on spontaneous locomotor activity and analgesia induced by Ag-tetrahydrocan nabinol in the mouse, Eur. J. Pharmacol., 68 (1980) 147-154. 14 Webster, V.A.D. and Griffiths, E.C., TRH degradation: an example of neuropeptide biotransformation. In E.C. Griffiths and G.W. Bennett (Eds.), Thyrotropin-Releasing Hormone, Raven Press, New York, 1983, pp. 95-102. 15 Fray, P.J., ONLIBASIC, a system for experimental control, Trends Neurosci., 3 (1980) XIII-XIV. 16 Wei, E., Loh, H. and Way, E.L., Potency of the N3im-methyl analog of TRH in the induction of shaking movements in the rat, Eur. J. Pharmacol., 36 (1976) 227-229. 17 Winner, B.J., Statistical principles in experimental design, second edition, McGraw-Hill, New York, 1971, p. 907. 18 Kerdelhue, B., Palkovits, M., Karteszi, M. and Reinberg, A., Circadian variations in substance P, luliberin (LH-RH) and thyroliberin (TRH) contents in hypothalamic and extra-hypothalamic brain nuclei of adult male rats, Brain Res., 206 (1981) 405-413. 19 Breese, G.R., Cooper, B.R., Prange, A.J., Cott, J.M. and Lipton, M.A., Interactions of thyrotropin-releasing hormone with centrally acting drugs. In A.J. Prange, Jr. (Ed.), The Thyroid Axis, Drugs, and Behaviour, Raven Press, New York, 1974, pp. 115-127. 20 Robbins, T.W., Behavioural determinants of drug action: rate-dependency revisited. In Cooper, S.J. (Ed.), Theory in Psychopharmacology, Volume 1, Academic Press, London, 1981, pp. 1-63. 21 Taylor, R.L. and Burt, R., Properties of [3H](3ME-His)TRH binding to apparent TRH receptors in sheep central nervous system, Brain Res., 218 (1981) 207-217. 22 Treptow, K., Oehme, P., Gabler, E. and Bientert, M., Modulation of locomotor activity by substance P in rats, Regul. Peptides, 5 (1983) 343-351. 23 McKelvy, J.F., Lin, C., Chan, L., Joseph-Bravo, P., Charli, J., Pacheco, M., Paulo, M., Neale, J. and Barker, J., Biosynthesis of brain peptides. In A.M. Gotto, Jr., E.J. Peck, Jr. and A.E. Boyd, III (Eds.), Brain Peptides: A New Endocrinology, Proceedings of the 4th Argenteuil Symposium, Waterloo, Belgium, October 29-31, 1978, Elsevier/North-Holland Biomedical Press, Amsterdam, 1979, pp. 183-196.