Behavioral effects of thyrotropin releasing hormone in frontal decorticated rats

Peptides. Vol. 5. pp. 899-903. 1984. ' Ankho InternationalInc. Printed in the U.S.A.

0196-9781/84 $3.110 ÷ .(K)

Behavioral Effects of Thyrotropin Releasing Hormone in Frontal Decorticated Rats GORO KATSUURA, KENJI YOSHIKAWA, AND SIGMUND HSIAO*

SHINJI ITOH I

Shionogi Research Laboratories, Fukushima-ku, Osaka 553, Japan *Department o f Psychology, University o f Arizona, Tucson, A Z 85721 R e c e i v e d 13 O c t o b e r 1983 KATSUURA, G.. K. YOSHIKAWA, S. ITOH AND S. HSIAO. Behavioral effects ofthyrotropin releasing hormone in frontal decorticated rats. PEPTIDES 5(5) 899-903, 1984.--A low dose intracerebroventricular injection of thyrotropin releasing hormone (TRH, 100 rig) changed many behavioral responses in the rat. TRH increased locomotion, scratching, body shaking, piloerection, and rearing, but decreased sniffing, and resting. Ablation of frontal neocortex further enhanced the TRH effects on locomotion and resting. A dose effect of TRH (0, 5, 10, 50, 100 ng) to increase general activity was established and the effect was further enhanced by decortication. In our test situations decortication had no effect by itself. Since the TRH effects became much more pronounced without the frontal neocortex it appears that the cortex exerts a powerful inhibitory effect to moderate the TRH effects. The TRH effect does not depend upon the frontal cortex, actually a cortical function is to dampen the TRH effects on various behavioral responses. Thyrotropin releasing hormone Decortication of frontal neocortex Dose-effect response curve TRH

Behavioral effects

General activity

lated to control of general activity [21], it is likely that the TRH effect on general activity is also mediated by this area. The nucleus accumbens area is associated to mesocortical DA pathways which terminate in the frontal cortex [1]. We recently showed that frontal decortication enhanced the effect of amphetamine to increase general activity but not that of apomorphine [ 16], suggesting a role of frontal cortex in the release of DA in the nucleus accumbens. In this study we observed a variety of responses to study the effects o f TRH and their modification by decortication of frontal neocortex.

T H Y R O T R O P I N releasing hormone (TRH) and its receptors are found to be widely distributed throughout the central nervous system [28]. A TRH receptor binding study shows that no region in the central nervous system appears totally devoid of binding sites [36]. The regional distributions of binding sites, however, vary vastly in densities and appear to be different among species [29, 35, 36]. Administrations of TRH produce various physiological and behavioral effects independent of its action on the pituitary gland and the effects are dependent upon the sites of administrations [2, 26]. TRH has been shown to excite or inhibit the activity of some cortical neurons and to potentiate the activity of some other neurons which are sensitive to acetylcholine, indicating that TRH has both the neurotransmitter and neuromodulator characteristics [2]. The behavioral effects of T R H are to increase a variety of responses including body shakes, limb tremors, repetitive head and limb movements, biting, scratching, an alert appearance as well as locomotion [5]. It can potentiate the excitatory effect of L-DOPA [13,30]. Its effects are similar to those o f amphetamine but apparently are not mediated by the same mechanisms [23,24]. T R H is also known to antagonize the sedative and hypothermic effects of barbiturates, ethanol and several other depressants [ 3 . 4 . 6 . 18.31]. It has been shown that TRH selectively promotes the release of dopamine (DA) in the nucleus accumbens area [8.22]. Because DA activity in the nucleus accumbens is re-

METHOD The subjects were 181 male Wistar rats, weighing about 250 g. They were housed in groups of 3 or 4 in plastic cages in a room with a controlled temperature of 25°C and illumination of 12 hr light and dark cycles. The experiments were conducted during the light phase. The animals were given free access to food (standard rat biscuit) and water. Frontal decortication was performed under pentobarbital anesthesia (50 mg/kg, IP). The extent of lesion was to replicate that reported by Scatton, Worms, Lloyd and Bartholini [34]. The frontal bones were removed to expose almost the entire frontal neocortex except for a narrow strip of midline bone over the sagital venous sinus which was left to provide support for the scalp to protect the brain. After retraction of the dura, the medial, dorsal and lateral areas of

'Requests for reprints should be addressed to S. Itoh. Shionogi Research Laboratories, Fukushima-ku. Osaka 553. Japan.

899

K A T S U U R A ET AL.

900 TABLE 1

BEHAVIORAL EFFECTS OF TRH IN FRONTAL-DECORTICATED AND SHAM-OPERATED RATS

Neofrontal-decorticated

Sham-operated Behavior Locomotion Scratching Body shaking Piloerection Rearing Head stereotypy Grooming Yawning Biting Sniffing Teeth chattering Resting

Saline

TR! I

Saline

TRH

15 ± 2.7 4 _ 1.5 2 ± 0.4 2 ± 0.6 30 --- 7.7 26 --- 7.0 1 9 - 5.1 2 - 0.6 2 - 0.8 30 --- 6.3 I ± 0.4 233 ± 17.3

36 ~- 5.1" 27 --- 11.6,~ 26 ... 4.0, 11 - 1.1~ 44 _ 7.5 27 ± 7.4 32--- 5.8 1 ± 0.4 9 ± 2.4* 6 - 1.4t 3 ± 1.2 155 - 18.2t

20 ± 8.1 7 ___ 3.0 3 ± I.I 3 --- 0.5 27 ± 6.9 22 ± 2.4 25 ± 6.4 0 - 0.4 2 ± 1.I 22 ± 6.0 2 - 0.4 248 ___ 14.8

91 ~ 16.5-54 ___ 18.7+ 39 ± 5.0+ II ± 1.175 ± 13.5+ 41 ± 4.9* 31 ± 5.8 0 ~ 0.1 3 ± 1.5 10 ± 2.3 3 _+ 1.0 67 ± 11.St+~

Sixty rain scores (mean - SE) following ICV injection at a dose of 100 ng are summarized. Ten rats were used in each group. *p<0.05, tp<0.01 TRH vs. respective saline control, '~p<0.01 decorticated TRH-treated vs. sham-operated TRH-treated by Duncan's multiple range test.

frontal neocortex were removed bilaterally by aspiration using a stainless steel pipette. Particular care was taken not to damage structures deeper than the neocortex. After the aspiration, the frontal bones were replaced and the scalp was closed with wound clips. Sham operated animals were anesthetized, the scalp incised, the frontal bones removed and replaced, and the scalp sutured with wound clips. A stainless steel guide cannula was affixed on the skull with dental cement in an appropriate position for insertion of an injection cannula into the left lateral ventricle for an intracerebroventricular (ICV) injection of TRH. Details of the cannula fixture is described in an earlier report [12]. A period of 9 days was provided for recovery from the surgery. The animals were then given insertion of the injection cannula every day for 5 days to make them accustomed to this procedure to minimize the emotional reaction in preparation for the behavioral test. The amount of TRH given varied from zero (saline) to 100 ng per rat dissolved in a volume of 5/xi physiological saline and was injected at a rate of 0.5/zl/sec with a microsyringe. Following the test 5 p.l of 1% Evans blue solution was injected into the ventricle to serve as a marker for examination of proper placement of the cannula postmortem. The brain was examined to ascertain that no subcortical structures were damaged by aspiration. No animal was eliminated due to improper surgical procedures. Experiment I involved 20 sham-operated and 20 decorticated animals. They were subdivided into 4 groups of l0 each and given saline or TRH (100 ng) followed by the test. The experimental design was a 2 by 2 factorial with sham versus decortication in one factor and saline versus TRH in the other factor. The test situation was a plastic cage (21 x 4 4 x 2 0 cm high) with 2 cm layer of clean sawdust covering the floor. A subject was placed in the cage for 75 rain to become habituated to the novel environment before an injection of saline or TRH was given. The following responses were then scored for a period of 60 min; (a) Locomotion: number of times that a rat moved from one end of the cage to the other, (b} Scratching: number of scratching bouts involv-

ing clawing the body with the hindlimbs, (c) Body shaking: number of times of vigorous shaking of the whole body or "wet-dog" shakes, (d) Pih~erection: slight degree of hair raising was given a score of 1, moderate degree a score of 2, and marked degree a score of 3, (e) Rearing: vertical extention of the head, body and forelimbs, (f) Head stereotypy: rapid sideway movements or bobbings of the head. (g) Grooming: repetitive licking and nibbling of the torso and limbs and washing movements on the head, (h) Yawning: wide opening of the mouth, (i) Biting: biting or nibbling some objects other than own body, (j) Sn(ff[ng: repetitive movements of the snout toward the cage floor and walls, (k) Teeth chattering: rapid and rhythmic movements of the jaws without biting any objects, (!) Resting: assuming resting positions without movements but with eyes open. A score of I was assigned when resting, head stereotypy, grooming, sniffing or biting was observed for a 10-sec duration. The other responses were scored by counting the number of times that each occurred. Experiment 2 involved 76 sham operated and 65 decorticated animals. Each group was subdivided into 6 groups and injected (ICV) TRH in a dose of 0 (saline), 5, 10, 20, 50, or 100 ng in a volume of 5 /zl to study the effect on general activity. General activity level was quantified by using an Automex activity monitor (Columbus Instruments Co.) which recorded vertical and horizontal displacements of a single animal placed in a cage. The animal was placed into the test cage for 75 min before a dose of TRH was injected and activity counts were recorded every 10 min for a total of 60 rain. The preinjection activity count was also recorded for a 10 min period prior to an injection. Analysis of variance (ANOVA) was used to evaluate the main and interaction effects followed by Duncan's multiple range test for further comparisons of relevant groups. RESULTS

Table I presents a summary of the data indicating the effects of TRH in frontal decorticated and sham-operated

TRH AND FRONTAL DECORTICATION

901

2000 Decorticctted group

.~**

1500

,ooo

<~ i-

500

,tl I 0 10 2O

I

I

50 IOOng TRH FIG. I. The dose effect of thyrotropin releasing hormone (TRH) on the level of general activity in neofrontal decorticated and shamoperated rats. *(/7<0.05) and **(p<0.01) indicate the comparison ~sainst the saline level of each surgical group and ++(p<0.01) indicates the comparison against the sham-operated level of respective doses of TRH.

animals. ANOVA (2)<2) of each response showed that the main effect of sham versus decortication was significant for locomotion, F(1,36)=11.51, p<0.01, and yawning scores, F(!,36)= 11.5, p<0.01, the main effect of saline versus TRH was significant for locomotion, F(1,36)--27.06, p<0.01, scratching, F(1,36)=9.86, p<0.01, body shaking, F(1,36)--84.91,/7<0.01, piioerection, F(1,36)-- 180.75,/7<0.01, rearing, F(1,36)= 11.50,/7<0.01, sniffing F(1,36)=21.73,/7<0.01, and resting scores, F(1,36)=64.53, p<0.01, and the interaction effect was significant for locomotion, F(1,36)--7.66, /7<0.05, and resting, F(1,36)= 10.21,/7<0.01. The results of Duncan's test, comparing the response scores of TRHtreated animals and those of saline-treated animals in the same surgical condition and the response scores of TRHtreated decorticated animals and those of TRH-treated sham-operated animals are shown in Table 1. Particularly noteworthy are locomotion and resting scores. TRH increased locomotion and frontal decortication interacted with TRH to further enhance the effect. TRH decreased resting and frontal decortication interacted with TRH to further enhance the effect. The differences between the shamoperated and the decorticated groups injected with saline were not significant (ps>0.05) for all responses observed. The dose response curves of general activity in shamoperated and decorticated animals following TRH treatments are given in Fig. 1. ANOVA (2×6) of the 60 min scores indicated that the main effect of sham versus decortication was significant, F(1,120)--39.90,p<0.01, the main effect of the TRH doses was significant, F(5,120)=56.29,p <0.01, and so was the interaction effect, F(5,120)--18.30, p<0.01. One way ANOVA of the preinjection data indicated that there was no significant differences for all of the dose groups (ps>0.05). Duncan's test was used to compare the scores of the sham-operated and decorticated groups at each dose to evaluate the decortication effect and to compare the TRH scores and the saline scores within the same surgical condition to evaluate the TRH effect. The result indicated that the decortication effect was there at the dose of 10 rig or higher

FIG. 2. The representative photographs of frontal decorticated and sham-operated brains. The medial, dorsal, and lateral aspects of the cortex immediately behind the olfactory bulbs were lesioned. The entry spot of the cannula is visible on the le~ hemisphere.

and that the TRH effect started at the 10 ng dose with decortication and the 20 ng dose without decortication. The difference between the decorticated and the shmn-operated groups injected with saline was not significant. It took TRH to make the difference between the two groups a ~ m e n t . The result of the decortication procedure was depicted in Fig. 2 which gives a representative sample of the decorticated and of the sham-operated brain. The medial, dorsal and lateral aspects of the frontal cortex immediately posterior to the olfactory bulbs were removed. The spot where the guide cannula penetrated the cortex was also visible at the middle part of the left cortex. DISCUSSION At a dose of 100 ng TRH was very potent in changin8 (mostly increasing) many responses. ANOVA indicated tha~ frontal decortication affected locomotion and resting but the effect was due to the TRH administration since without it, or with saline alone, there was no effect of decortication. TRH had a variety of effects and the effects on locomotion and resting were further enhanced by the decortication. The interaction effect of TRH and decortication on locomotion occurred with the lowest dose of 10 ng TRH as shown in Fig. 1. The locomotion score and the resting effects were inversely related but not linearly since TRH affected many other responses which interfered with resting. The TRH effect for locomotion was a 355% increase in the decorticated group versus a 140~ increase in the sham-operated group and for resting it was only a 73% decrease in the decorticated group versus a 33% decrease in the sham-operated group as shown in Table 1. This potent effect of TRH on locomotion was further detailed in Fig. 1. Decortication and saline alone did not induce any difference in the responses we observed. Frontal cortical lesioning has been shown to increase spontaneous response in a novel environment and the response decreases over time more slowly relative to the control lesioned group but eventually reaches the same low level in

902

KATSUURA ET AL.

about one hour [17]. In our study the animals were habituated for more than one hour before the treatments were given, thus the responses which we observed were not those related to a novel environment. The responses which we observed were thus directly related to the stimulation provided by the TRH treatments. We did not expect a difference between the responses of the sham-operated and the decorticated groups without the TRH treatments with our habituation procedure. As shown in Fig. 2 the frontal decortication largely replicated that by Scatton et al. [34] which resulted in altered responses to DA manipulations. TRH and DA have been shown to stimulate locomotion when they are bilaterally injected into the nucleus accumbens, but not into the caudate nucleus and that either peripheral or intra-accumbens injections of haloperidol or pimozide in low doses effectively block the effect [27]. Thus, locomotion is thought to involve the accumbens DA system and the TRH effects may be mediated by the nucleus accumbens. The TRH effect here is similar to the amphetamine effect in that decortication enhanced the effect of amphetamine on locomotion [16]. Although TRH as well as amphetamine can promote release of DA in the nucleus accumbens area [8, 21, 22, 27] to facilitate the general activity level the effects of TRH and amphetamine are not identical [7,24]. For example, in a-methyltyrosine-treated animals TRH is effective in increasing responses and so is TRH in 6-hydroxydopamine-treated animals although those animals are not sensitive to amphetamine [9, 23, 37]. Two explanations are that TRH may be more potent and pure to promote the presynaptic release

of DA than amphetamine and that the TRH effect may not be dependent upon the presynaptic release of DA. TRH has been shown to interact with the activities of acetylcholine [38,391, enkephaline [281,/3-endorphin [t51. cholecystokinin octapeptide [14, 15, 181, serotonin [t0,141. noradrenaline 120. 25, 31], and glutamate [331 and has multiple effects on the cortical neurons [21. Since the TRH effect became much more pronounced without the frontal neocortex we could conclude that the cortex exerts an inhibitory effect to moderate the effect of TRH. Thus, the TRH effect is not dependent upon the cortex, rather the cortical effect is dependent upon TRH since in our test situations the decortication by itself did not have an effect on the responses. A cortical effect appears to be to dampen the excitatory effect of TRH. It appears that the TRH effect is opposite to that of cholecystokinin octapeptide on locomotion [19] and that some TRH effects are antagonized by cholecystokinin octapeptide [14, 15, 18]. However, it was recently shown that cholecystokinin tetrapeptide could increase general locomotor responses [10] and thus the effect is similar to that of TRH. We are currently investigating the interaction effects of the neuropeptides on behaviors. The TRH effect may be mediated or regulated by cholecystokinins and other neurochemicals in selected areas of the brain. ACKNOWLEDGEMENTS We thank Ms. Y. Maeda of Shionogi Research Laboratories for her skillful technical assistance.

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TRH AND FRONTAL

DECORTICATION

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