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Thyrotropin-releasing hormone and amphetamine produce different patterns of behavioral excitation in rats
European Journal of Pharmacology, 72 (1981) 35--43 Elsevier/North-Holland Biomedical Press
35
THYROTROPIN-RELEASING HORMONE AND AMPHETAMINE PRODUCE DIFFERENT PATTERNS OF BEHAVIORAL EXCITATION IN RATS * GREGORY N. ERVIN **, STEPANIE A. SCHMITZ, CHARLES B. N E M E R O F F and ARTHUR J. PRANGE, Jr.
Biological Sciences Research Center, Departments of Psychiatry and ~ychology, and the Neurobiology Program, University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. Received 25 November 1981, revised MS received 4 Feburary 1981, accepted 9 March 1981
G.N. ERVIN, S.A. SCHMITZ, C.B. N E M E R O F F and A.J. PRANGE, Jr., Thyrotropin-releasing hormone and amphetamine produce different patterns of behavioral excitation in rats, European J. Pharmacol. 72 (1981) 35--43. We compared the arousal and hyperactivity produced by intraperitoneal (i.p) injections of thyrotropin-releasing hormone (TRH, pGIu-His-Pro-NH2; 10, 20, 30 and 60 mg/kg) and 0.3 and 2 mg/kg d-amphetamine (low and moderate amph., respectively)by measuring the occurrence of discrete behavioral items with a behavioral sampling and scoring method. To minimize extraneous variables affecting activity, rats were caged singly inside isolated observation chambers and tested in the daytime after a 2.5 h period of habituation. Under these conditions, vehicle (0.9% NaCl)-treated rats were inactive and either rested or slept through 80% o f all time samples taken in the hour after injection. Both TRH and amph. produced significant arousal from sleeping, but TRH, at all doses tested, produced less arousal than moderate amph. and a pattern of behavioral responses which differed from both low and moderate amph. Moderate amph. produced marked increases in forward locomotion and rearing, but low amph. and TRH did not. Both TRH and low amph. increased grooming (perhaps simply b y increasing wakefulness), b u t TRH failed to increase sniffing, a cardinal feature of amph.-induced excitement. Unlike amph., TRH produced wet-dog shakes, piloerection, tail elevation and teeth chattering. Both mod. amph. and TRH significantly produced increased activity when compared to controls as assessed with photocell counts, though the amph. effect was more robust. The lack of arousal after i.p. injections of thyroid-stimulating hormone (10 I.U./kg) or melanocyte-stimulating hormone release-inhibiting factor (Pro-Leu-Gly-NH2; 60 mg/kg) is evidence that TRH-induced arousal is neither mediated b y activation of the pituitary-thyroid axis nor b y a non-specific effect of tripeptides generally. Thyrotropin-releasing hormone
Neuropeptides
1. Introduction Pyroglutamyl-l-histidyl-l-prolineamide is that tripeptide which was isolated from extracts of ovine and bovine hypothalami by the groups of Guilleman and Schally (Burgus et al., 1969; Boler et al., 1969) in their * This research was supported b y NIMH Grants MH32316, MH-22536, MH-33127 and NICHHD HD03110. ** Correspondence should be sent to Gregory N. Ervin, University of North Carolina School of Medicine, Biological Sciences Research Center 220-H, Chapel Hill, NC 27514, U.S.A.
d-Amphetamine
searches for a substance which controlled pituitary secretion of thyroid-stimulating hormone (TSH). Thus named thyrotropin-releasing hormone (TRH), it was the first hypothalamic releasing or release-inhibiting hormone to be chemically characterized (for reviews, see Guillemin, 1978; Schally et al., 1973). In retrospect, the naming of TRH was somewhat misleading since other pituitary effects of TRH have been discovered. Furthermore, wide phylogenetic and regional brain distributions of TRH support the hypothesis that, in addition to its role as a hypothalamic hormone, TRH also may function as a neuro-
0 014-2999/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
36 transmitter (Brownstein et al., 1974; Jackson and Reichlin, 1974; Johansson and HSkfelt, 1980). Administration of TRH causes behavioral effects and changes in responses to psychoactive drugs which are not observed after administration of TSH or thyroid hormones, and which persist even after hypophysectomy (Prange et al., 1978, 1979; Yarbrough, 1979). Finally, microiontophoresis of TRH has been shown to alter firing rates of neurons in several different brain regions (Renaud, 1977). Many of the effects of centrally or peripherally administered TRH appear as increases in arousal. In a variety of mammals that have been tested, including mice, rats, hamsters, gerbils, dogs and monkeys, TRH antagonizes barbiturate- and ethanol-induced sedation (Prange et al., 1974; Breese et al., 1974; 1975), potentiates L-DOPA and L-5-HTPinduced excitation, and increases wakefulness and alertness (see Nemeroff et al., 1978; Prange et al., 1979; Morley, 1979, for reviews). In rats, the behavioral excitation observed after peripheral TRH administration has been reported as increased grooming, shaking and tail elevation (Schenkel-Hulliger et al., 1974; Wei et al., 1975). Some authors have reported increased locomotion, rearing and sniffing (Miyamoto and Magawa, 1977). These last three behaviors are also major behavioral components of d-amphetamine sulfate (amph.)-induced arousal (Ervin e t al., 1977; Ayhan and Randrup, 1973). Nucleus accumbens septi contains dopamine (DA) terminals where amph. acts to stimulate locomotion by releasing DA (Kelly et al., 1975), and nucleus accumbens also contains TRH-like immunoreactivity (Johansson and HSkfelt, 1980). Both TRH and DA injected into nucleus accumbens reportedly cause locomotion, rearing and sniffing (Miyamoto and Nagawa, 1977). On these behavioral grounds, TRH has been postulated to release DA, like amph. (Heal and Green, 1979). However, behavioral similarities between TRH and amph. have usually been inferred from photocell or electronic activity meters, or have been
G.N. ERVIN ET AL. based upon unsystematic observations. Photocell and electronic measurements of activity have often been shown to be ambiguous since a variety of behavioral items can produce the same measurable effect (e.g., Krsiak et al., 1970). Behavioral observations of TRH have been inconsistent, and Costall et al. (1979) did not report increased locomotion, rearing and sniffing after intra-accumbens injections of TRH as others did (Miyamoto and Nagawa, 1977; Heal and Green, 1979). In the present study we used a time-sampling and behavioral scoring method to further compare TRH and amph. Both TRH and amph. produced arousal, but the behavioral excitation caused by TRH was markedly different from the excitation caused by amph. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (Zivic-Miller Laboratories, Allison Park, PA) weighing 250500 g were housed in groups of four in a controlled environment animal facility (ambient temperature 23-25°C; lights on, 06:0018:00 h) and received laboratory chow and water ad libitum. 2.2. Apparatus Rats were observed in plexiglass test cages (21 X 44 X 20 cm high) that had wire lids and wire mesh floors. Each test cage was mounted inside a ventilated wooden box (52 X 66 X 46 cm high) with a 14 cm X 66 cm smoked plexiglass window on one wall which allowed viewing of rats from 20-40 cm. Each box was illuminated by a 7.5 W bulb; the test room was dark. Each box was equipped with a near infrared light source and photocell (Lafayette Instrument Co., Lafayette, IN) positioned 2 cm above the floor and halfway along the length of the plexiglass test cage. The sensitivity of photocell apparatus was adjusted so the beam was interrupted by two fingers (3.8 cm in width) but not by a pencil (0.7 cm in width).
BEHAVIORAL
PROFILES OF TRH AND AMPHETAMINE
2.3. Drugs
Rats were injected intraperitoneally (i.p.) with either TRH, amph., bovine thyroidstimulating hormone (TSH), melanocytestimulating hormone release inhibiting factor (MIF-I) or sterile0.970 NaCl alone. All drugs were dissolved in sterile0.970 NaCI, and injection volume was always 2 ml/kg body weight. T R H (lot No. 21-220-AL) was a giftof Abbott Laboratories, North Chicago, IL; MIF-I Clot No. 15487) was purchased from U.S. Biochemical Corporation, Cleveland, OH: and amph. was purchased from Sigma Chemical Co., St. Louis, M O .
37
the hindlimbs to groom the head was included in this category. ( 7 ) G r o o m i n g of the hindquarters: Licking of the hindlimbs and body, or the tail. Use of the forelimbs to groom the hindquarters was included in this category. (8) Chewing or teeth chattering: Rapid, repetitive movement of the jaws not associated with self-biting or biting of other objects (such as the wire mesh floor). (9) Resting: Any resting position with the eyes simultaneously open throughout a 10 sec interval. (10)Sleeping: Any of the resting positions with the eyes closed throughout a 10 sec interval. Forward locomotion and rearing scores are the number of times these behaviors were observed. All other behavioral items received a
2.4. Time samples of behavior taken a't 10 rain in tervals TABLE 1
Between 08:00 and 1 0 : 0 0 h , rats were transferred from the colony room to observation boxes in group cages. The observation cages were devoid of food, water, or other objects. Rats were habituated to observation cages for at least 2 h prior to the first behavioral observation. Three consecutive 10sec behavioral samples were observed every ten rain starting 30 min before and lasting 2 h beyond i.p. injection of T R H (10, 20, 30, 60 mg/kg), MIF-I (60 mg/kg)., TSH (10 IU/ kg), amph. (0.3 or 2 mg/kg) or 0.9% NaC1 (2 ml/kg), and occurrence of the following behavioral items were noted as previously described (Ervin et al., 1977). (1) Forward locomotion: The movement of the animal from one end of the cage to the other. (2) Rearing: The vertical extension of the head, b o d y , and forelimbs, excluding such postures associated with grooming. (3) Circling: Complete 360 ° turn of the animal's b o d y axis. Direction of circling was also noted. (4) Sniffing: Repetitive m o v e m e n t of the snout and nostrils directed toward some cage surface. ( 5 ) H e a d stereotypy: Rapid movement or bobbing of the head n o t directed toward any cage surface. (6) Grooming of the head or forelimbs: Any licking, washing, or scratching of the forelimbs or head. Use of
Effects o f t h y r o t r o p i n - r e l e a s i n g h o r m o n e a n d a m p h e t a m i n e o n arousal f r o m resting a n d sleeping. R a t s were scored for resting or sleeping if t h e y p e r f o r m e d such b e h a v i o r t h r o u g h o u t a I 0 sec behavioral sample. S h o w n is t h e m e d i a n p e r c e n t a g e a n d range of observ a t i o n t i m e in t h e h o u r a f t e r i n j e c t i o n t h r o u g h w h i c h rats o f each g r o u p (n = 8) e i t h e r rested or slept. T r e a t m e n t (i.p.)
Sampling time spent resting or sleeping %
Range
{A) Experiment 1. Three behavioral samples every 10 rain NaCI, 0.9% (vehicle) T R H , 10 m g / k g T R H , 20 m g / k g T R H , 30 m g / k g TRH, 60 mg/kg TSH, I 0 l.U./kg MIF, 60 m g / k g AMPIi, 0.3 m g / k g AMPH, 2 m g / k g
83 61 ,17 28 42 67 78 22 0
a • b b
b b
50--83 33--83 17--72 17--44 6-56 28 - 8 3 67--94 0 56
(B) Experiment 2. Three behavioral samples every 5rain
NaCI, 0.9% (vehicle) TRH, 30 mg/kg AMPH, 2 mg/kg
78 28 b 0 b
53--97 6--44
a P < 0.05 c o m p a r e d to vehicle g r o u p ' s score with M a n n - W h i t n e y U-test. b p < 0.01 c o m p a r e d to vehicle g r o u p ' s score with M a n n - W h i t n e y U-test.
38
G.N. ERVIN ET AL.
score of 1 when they occurred, however often, in a 10-sec interval. Thus the total score of these behavioral items was the number of 10-sec intervals in which that behavior occurred, and the m a x i m u m possible score for a particular behavioral item in 1 h was 18. Resting and sleeping were scored only when t h e y persisted throughout a 10-second interval. The sum of the resting and sleeping scores is an inverse measure of arousal. 2.5. Time samples o f behavior taken at 5 min intervals
Rats were injected i.p. with TRH (30 mg/ kg), amph. (2 mg/kg) or vehicle (0.9% NaC1).
The design of this experiment was identical to the design described in 2.4 with the following exceptions: (1)Behavior was observed for only l h after drugs. (2) Behavior was sampled for 30 sec every five, rather than every 10 min. (3) The occurrence of wet dog shakes, tail elevation and piloerection was also noted. (4) General activity was also measured automatically by counting interruptions of a photocell beam. 2.6. Data analysis
All behavioral data were expressed as medians and ranges; statistical comparisons were performed using the Mann-Whitney Utest (Conover, 1971).
TABLE 2 Effects of thyrotropin-releasing hormone and d-amphetamine on behavioral excitation. Behavioral item
NaC1 0.9%
TRH
d-Amph
10 mg/kg
20 mg/kg
30 mg/kg
60 mg/kg
0.3 mg/kg
2 mg/kg
MIF 60 mg/kg
TSH 10 IU/kg
Forward locomotion
0 (0)
0 (0--1)
0 (0--3)
0.5 (0--2)
0 (0--4)
0 (0--2)
4b (0--9)
0 (0)
0 (0--1)
Rearing
0 (0--1)
0.5 (0--3)
0.5 (0--6)
0 (0--3)
0 (0--2)
1 (0--3)
4.5 b (2--11)
0 (0--3)
0 (0--5)
Sniffing
1.5 (0--5)
1.5 (0--4)
1 (0--4)
3 (0--7)
1.5 (0--4)
7b (0--12)
17 b (13--17)
0 (0--6)
2.5 (0--4)
Circling
0 (0)
0 (0)
0 (0)
0 (0--1)
0 (0--1)
0 (0)
0 (0-6)
0 (0)
0 (0--1)
Head stereotypy
0 (0--1)
0 (0)
0 (0)
0 (0--2)
0 (0--2)
0 (0)
2.5 a (0--6)
0 (0)
0 (0)
Grooming of head and forelimbs
0.5 (0--5)
3a (0--8)
3a (1--7)
3a (0--7)
4b (3--15)
4a (1--9)
1.5 (0-6)
1 (0--4)
0 (0--3)
Grooming of hindquarters Chewing/teeth chattering
0 (0--2) 0.5 (0--5)
1 (0--3) 1.5 (0--2)
0.5 (0--5) 3 (0--6)
0 (0--1) 7.5 b (2--15)
0 (0--2) 9.5 b (8--18)
1.5 (0--5) 0 (0--2)
0 (0--2) 0 (0--3)
0.5 (0--3) 0.5 (0-6)
1 (0--3) 0 (0--1)
Resting
4 (0--12)
3 (0--12)
2.5 (0--7)
3.5 (0--5)
2.5 (0--7)
4 (0--9)
0a (0)
4 (0--14)
7 (3--15)
Sleeping
9 (0--15)
7 (0--12)
6 (0--11)
3b (0--3)
2.5 b (0--10)
0b (0--3)
0b (0)
9 (3--15)
4.5 (0--9)
After being habituated to observation cages for 2.5 h rats were injected intraperitoneally and observed for 30 sec every 10 min for 2 h. Eight rats were in each group. Data shown are the median behavioral scores over the first hour after injection. Numbers in parentheses are the ranges of behavioral scores recorded for that item. Suffixes indicate significant differences from NaCl-injected rats: a p < 0.05; b p < 0.01, Mann-Whitney U-test.
BEHAVIORAL PROFILES OF TRH AND AMPHETAMINE
formed significantly more chewing and teeth chattering, and slept significantly less. TRH did not increase sniffing--a cardinal feature of amph.-induced excitation. TRH resembled amph. only because low amph. (0.3 mg]kg) - - like TRH-- significantly increased grooming (of the head and forelimbs) and decreased sleeping, but not resting. Animals injected with the moderate dose of amph. (2 mg/kg) exhibited significant increases in locomotion, rearing, sniffing, and head stereotypy, and significant decreases in resting as well as in sleeping. In the second hour after injections, rearing, sniffing, head stereotypy and insomnia were still significantly elevated by amph. (2 mg/kg; P < 0.05, data not shown), but only chewing/teeth chattering was still elevated by TRH (60 mg/kg, P < 0.05).
3. Results 3.1. Time samples o f behavior taken at 10minute intervals
Prior to i.p. injections, all rats behaved similarly - - t h e y rested or slept, but displayed few other behaviors (data not shown). Injections of TRH (10-60 mg/kg i.p.) and amph. (0.3 and 2.0 mg/kg i.p.) produced arousal, but injections of vehicle, MIF and TSH did not (table 1A). After vehicle, MIF and TSH, rats quickly resumed resting or sleeping. TRH and amph. produced different pat~ terns of behavioral arousal (table 2). After TRH, rats groomed the head and forelimbs significantly more. In addition, after higher doses of TRH (30 and 60 mg/kg), rats per-
35
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Fig. 1. Behavioral profiles o f TRI-I and a m p h e t a m i n e . As in e x p e r i m e n t 1, animals were a l l o w e d t o h a b i t u a t e t o cages f o r 2.5 h p r i o r t o (i.p.) injections. AnimaLs were observed f o r 30 see every 5 rain f o r 1 h. Data s h o w n are m e d i a n scores f o r 8 rats over the entire o b s e r v a t i o n period. P h o t o c e l l score given is actual c o u n t divided b y 10. With the e x c e p t i o n o f scores f o r l o c o m o t i o n and rearing ( w h i c h are actual f r e q u e n c y o f behavior), m a x i m a ] score possible f o r a p a r t i c u l a r behaviors] i t e m is 36. * P < 0.05; * * P < 0.01 c o m p a r e d t o vehic|e (0.9% NaC|)
&coup's score with Mann-Whitney U-test.
40 3.2. Time samples o f behavior taken at 5 min intervals
Both TRH (30 mg/kg) and amph. (2 mg/ kg) elicited significant increases in photocell counts when compared with vehicle-treated controls (fig. 1), though the amph. effect was significantly greater than the TRH effect (P < 0.01). As in experiment 1, TRH and amph. produced arousal (table 1B) but different patterns of behavioral activation (fig. 1). Amph. elicited large increases in forward locomotion, rearing, sniffing and head stereotypy, but TRH did not. However, increasing the frequency of behavioral sampling did increase the sensitivity of our technique, because it was demonstrated that TRH does, in fact, increase forward locomotion to a slight degree (though significantly less than amph., P < 0.01). TRH elicited large increases in grooming (of head and forelimbs), teeth chattering, wet dog shakes, tail elevation, and piloerection, but amph. did not. Both amph. and TRH produced insomnia, but only amph. significantly decreased resting behavior.
4. Discussion Both TRH and amph. produced arousal, but vehicle, TSH and MIF did not (table 1). The low dose of amph. produced arousal much like TRH; rats were more wakeful, but still restful. After the higher dose of amph., rats neither slept nor rested (table 2 and fig. 1). Electroencephalographically, TRH arousal and amph. arousal are similar (Itil et al,, 1975). However, EEG arousal and behavioral arousal are separable (Jones et al., 1978; Bradley and Elks, 1957), and the behavioral arousal produced by TRH and amph. are strikingly different. The most pronounced behavioral effect of TRH was grooming of the head and forepaws. Grooming is the most frequently observed behavior of laboratory rats during wakefulness (Bolles, 1960). Thus, frequent grooming after
G.N. ERVIN ET AL. TRH may have simply been due to increased wakefulness. Grooming was also frequent after low amph., but not after the higher dose of amph. which elicited behaviors which would clearly compete with grooming (table 2 and fig. 1; Ayhan and Randrup, 1973). The higher dose of amph. markedly increased the frequency of forward locomotion and rearing, but low amph. and TRH did not. Although we did not detect any increase in locomotion after TRH when behavior was observed at 10-minute intervals, we did detect a very slight increase in locomotion when behavior was observed at five-minute intervals. Thus the sensitivity of time-sampling methods is clearly dependent on observation frequency. Many groups have emphasized locomotor hyperactivity after peripheral or intracerebral administration of TRH, but the data were general 'activity' measurements from photocells or electronic devices (Miyamoto and Nagawa, 1977; Vogel et al., 1979; Heal and Green, 1979) and these devices measure certain behaviors in addition to locomotion (Krsiak et al., 1970; Ljungberg, 1978). Sniffing was a cardinal characteristic of amph.-induced excitation. Although low amph. was like TRH in many respects (e.g., both increased grooming and neither markedly increased locomotion or rearing), low amph. elicited sniffing but TRH did not (table 2). After TRH, but not after amph., we observed chewing and teeth chattering, tail elevation, wet-dog shakes .and piloerection. The higher d o s e of amph., 2 mg/kg, was threshold for stereotypies because head bobbing was slightly increased. After higher doses of amph., stereotypies (e.g., biting, licking and stereotyped sniffing) become prominent and decrease forward locomotion and rearing (Fog, 1972; Kelly et al., 1975; Ervin et al., 1981). TRH does not elicit such stereotyped behavior (Vogel et al., 1979; Miyamoto and Nagawa, 1977). Although TRH, like amph., produces arousal, this cannot explain its potency in many pharmacological tests. For example, TRH (1 mg/kg) is as effective as amph. (3
BEHAVIORAL PROFILES OF TRH AND AMPHETAMINE
mg/kg) in reversing pentobarbital sedation in mice (Prange et al., 1975), but amph. (3 mg/ kg) produces intense arousal in otherwise untreated rats while TRH (1 mg/kg) does not (Miyamoto and Nagawa, 1977). Also, TRH and amph. both potentiate L-DOPA-induced excitation (Plotnikoff et al., 1974; N. Plotnikoff, personal communication), but TRH is as effective as more arousing doses of amph. Moreover, TRH, unlike amph., is effective after immediate arousal effects have concluded. The potentiation of L-DOPA-induced excitation is also affected by substances, such as MIF and TSH, which alone do n o t produce arousal (tables 1 and 2). Rats apparently perceive T R H and amph. arousal differently. Jones et al. (1978) demon° strated that both T R H and amph. act as cues in a two-lever operant discrimination task, but generalization between T R H (10, 20 or 30 mg/kg, i.p.)and amph. (0.8 or 1.6 mg/kg, i.p.) does not occur. Other reported differences between T R H and amph. are: (1) T R H reverses ethanol-induced sedation; amph. either exerts no effect or enhances it (Breese et al., 1974); (2) T R H possesses considerable activity in an anti-conflict activity whereas arnph, exerts no such effect (Vogel et al., 1980), and (3) T R H reverses oxotremorineinduced hypothermia whereas amph. does not. Several other differences between T R H and amph. have been reported, and these have been reviewed (Manberg et al., 1979), Amph. exerts its major behavioral effects through the presynaptic release of catecholamines (especially DA), and TRH has been shown to cause release of DA in nucleus accumbens as well as in nucleus caudatus in vitro (Miyamoto et al., 1979). Kerwin and Pycock (1979) have reported that TRH releases [3H IDA from slices of the nucleus accumbens but not from nucleus caudatus. The present results and failure of others to observe amph.like stereotypies after TRH administration (Vogel et al., 1979; Miyamoto and Nagawa, 1977) suggest that TRH does n o t exert arnph.like neurochemical effects in vivo. Despite the numerous pharmacological
41
effects of TRH which have been reported, the physiological roles of central TRH have yet to be established. References Ayhan, I.H. and A. Randrup, 1973, Behavioral and pharmacological studies on morphine-induced excitation of rats. Possible relation to brain catecholamines, Psyehopharmaeologia 29, 317. Boler, J., F. Enzmann, K. Folkers, C.Y. Bowers and A.V. Schally, 1969, The identity of chemical and hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl-proline-amide, Biochem. Biophys. Res. Commun. 37,705. Bolles, R.C., 1960, Grooming behavior in the rat, J. Comp. Physiol. Psych. 53, 306. Bradley, P.B. and J. Elks, 1957, The effect of some drugs on the electrical activity of the brain, Brain 80, 77. Breese, G.R., J.M. Cott, B.R. Cooper, A.J. Prange, Jr. and M.A. Lipton, 1974, Antagonism of ethanol narcosis by thyrotropin-releasing hormone, Life Sci. 14, 1053. Breese, G.R., J.M. Cott, B.R. Cooper, A.J. Prange, Jr., M.A. Lipton and N.P. Plotnikoff, 1975, Effects of thyrotropin-releasing hormone (TRH) on t h e actions of pentobarbital and other centrally acting drugs, J. Pharmacol. Exp. Ther. 193, 11. Brownstein, M.T., M. Palkovits, J~I. Saavedra, R.M. Bassiri and R.D. Utiger, 1974, Thyrotropin-releasing hormone in specific nuclei of rat brain, Science 195, 267. Burgus, R., T.F. Dunn, D. Desiderio and R. Guillemin, 1969, Molecular structure of the hypothalamic thyrotropin-releasing factor (TRF) of ovine origin, demonstration o f the pyroglytamyl-histidyl-prolinamide sequence by mass spectrometry, C.R. Acad. Sci. (Paris) 263, 1870. Conover, W.J., 1971, Practical Nonparametric Statistics (Wiley, New York). Costall, B., G.H. Siv-Chun, G. Metcalf and R.J. Naylot, 1979, A study of the changes in motor behavior caused by TRH on intracerebral injection, European J. Pharmacol. 53, 143. Ervin, G.N., L.S. Birkemo, C.B. Nemeroff and A.J. Prange, Jr., 1981, Neurotensin blocks certain amphetamine-induced behaviors, Nature (in press). Ervin, G.N., J.S. Fink, R.C. Young and G.P. Smith, 1977, Different behavioral responses t o L-DOPA after anterolateral o r posterolateral hypothalamic injections of 6-OHDA, Brain Res. 132,507. Fog, R., 1972, On stereotypy and catalepsy: Studies on the effects of amphetamines and neurolepties in rats, Acta Neurol. Scand. 48, 7.
42 Guillemin, R., 1978, Biochemical and physiological correlates of hypothalamic peptides. The endocrinology of the neuron, in: The Hypothalamus, eds. R.J. Baldessarini and J.B. Martin (Raven Press, New York) p. 155. Heal, D.T. and A.R. Green, 1979, Administration of thyrotropin-releasing hormone (TRH) to rats releases dopamine in nucleus accumbens but not nucleus eaudatus, Neuropharmacoh 18, 23. Itil, T.M., C.D. Patterson, N. Polvar, A. Bigelow and B. Bergey, 1975, Clinical and CNS effects of oral and I.V. thyrotropin-releasinghormone in depressed patients, Dis. Nerv. Syst. 36, 529. Jackson, I.M.D. and S. Reichlin, 1974, Thyrotropinreleasing hormone (TRH): Distribution in hypothalamic and extrahypothalamic brain tissues of mammalian and submammalian chordates, Endocrin. 95,854. Johansson, O. and T. H6kfelt, 1980, Thyrotropin releasing hormone, somatostatin and enkephalin: Distribution studies using immunohistochemical techniques, J. Histochem. Cytochem. 28,364. Jones, C.N., L.D. Grant and A.J. Prange, Jr., 1978, Stimulus properties of thyrotropin-releasing hormone, Psychopharmacol. 59,217. Kelly, P.H., P.W. Seviour and S.D. Iversen, 1975, Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum, Brain Res. 94,507. Ljungberg, T., 1978, Reliability of two 'activity boxes' commonly used to assess drug-induced behavioral changes, Pharmacol. Biochem. Behav. 8,191. Kerwin, R.W. and C.J. Pycock, 1979, Thyrotropinreleasing hormone stimulates release of [3H]dopamine from slices of rat nucleus accumbens in vitro, Br. J. Pharmacol. 67,323. Krsiak, M., H. Steinberg and I.P. Stolerman, 1970, Uses and limitations of photocell activity cages for assessing effects of drugs, Psychopharmacologia 17, 258. Manberg, P.J., C.B. Nemeroff and A.J. Prange, Jr., 1979, Thyrotropin-releasing hormone and amphetamine: A comparison of pharmacological profiles in animals, Prog. Neuropsychopharmacol. 3,303. Miyamoto, M. and Y. Nagawa, 1977, Meso!imbic involvement in the locomotor stimulant action of thyrotropin-releasing hormone (TRH) in rats, European J. Pharmacol. 44,143. Miyamoto, M., S. Narumi, Y. Nagai, T. Shuma and Y. Nagawa, 1979, Thyrotropin-releasing hormone: Hyperactivity and mesolimbic dopamine system in rats, Jap. J. Pharmacol. 29, 335. Morley, J.E., 1979, Extrahypothalamic thyrotropinreleasing hormone (TRH) -- Its distribution and its functions, Life Sci. 25, 1539. Nemeroff, C.B., P.T. Loosen, G. Bissette, P.J. Man-
G.N. ERVIN ET AL. berg, I.C. Wilson, M.A. Lipton and A.J. Prange, Jr., 1978, Pharmaco-behavioral effects of hypothalamic peptides in animals and man: Focus on thyrotropin-releasing hormone (TRH) and neurotensin, Psychoneuroendocrinol. 3, 279. Plotnikoff, N.P., A.J. Prange, Jr., G.R. Breese, M.S. Anderson and I.C. Wilson, 1974, Effects of thyrotropin-releasing hormone on DOPA response in normal, hypophysectomized, and thyroidectomized animals, in: The Thyroid Axis, Drugs and Behavior. ed. A.J. Prange, Jr. (Raven Press, New York) p. 103. Prange, A.J., Jr., G.R. Breese, J.M. Cott, B.R. Martin, B.R. Cooper, I.C. Wilson and N.P. Plotnikoff, 1974, Thyrotropin releasing hormone: Antagonism of pentobarbital in rodents, Life Sci. 14. 447. Prange, A.J., Jr., G.R. Breese, G.D. Jahnke, B.R. Martin, B.R. Cooper, J.M. Cott, I.C. Wilson, L.B. Alltop, M.A. Lipton, G. Bh~sette, C.B. Nemeroff and P.T. Loosen, 1975, Modification of pentobarbital effects by natural and synthetic polypeptides: Dissociation of brain and pituitary effects, Life Sci. 16, 1907. Prange, A.J., Jr., C.B. Nemeroff, M.A. Lipton, G.R. Breese and I.C. Wilson, 1978, Peptides and the central nervous system, in: Handbook of Psychopharmacology, Vol. 13, eds. L.L. Iversen, S.D. Iversen and S.H. Snyder (Plenum Press, New York) p. 1. Prange, A.J., Jr., C.B. Nemeroff, P.T. Loosen, G. Bissette, A.J. Osbahr III, I.C. Wilson and M.A. Lipton, 1979, Behavioral effects of thyrotropin-releasing hormone in animals and man: A review, in: Central Nervous System Effects of Hypothalamic Hormones and Other Peptides, ed. R. Collu (Raven Press, New York), p. 75. Renaud, L.P., 1977, TRH, LHRH and somatostatin: Distribution and physiological action in neural tissue, in: Society for Neuroscience Symposia II: Approaches to the Cell Biology of Neurons, eds. W.M. Cowan and J.A. FerendeUi (Soc. Neurosci. Pub., Bethesda) p. 265. Schally, A.V., A. Arimura, L. Debeljuk, T.W. Redding and J.J. Reeves, 1973, Hypothalamic hormones regulating pituitary function; their physiology and biochemistry, in: Hypothalamic Hypophysiotropic Hormones, eds. C. Gual and E. Rosemberg (Excerpta Medica, Amsterdam) p. 1. Schenkel-Hulliger, L., E.P. Koella, A. Hartmann and L. Maitre, 1974, Tremorogenic effects of thyrotropin releasing hormone in rats, Experientia 30, 1168. Vogel, R.A., B.R. Cooper, T.S. Barlow, A.J. Prange, Jr., R.A. Mueller and G.R. Breese, 1979, Effects of thyrotropin-releasing hormone on locomotor activity operant performance, and ingestive behavior, J. Pharmacol. Exp. Ther. 208,161. Vogel, R.A., G.D. Frye, J.H. Wilson, C.M. Kuhn, R.B.
BEHAVIORAL PROFILES OF TRH AND AMPHETAMINE Mailman, R.A. MueUer and G.R. Breese, 1980, Attenuation of the effect of punishment by thyrotropin-releasing hormone: Comparisons with chlordiazepoxide, J. Pharmacol. Exp. Ther. 212, 153. Wei, E., S. Siegel, H. Loh and E.L. Way, 1975, Thyro-
43
tropin-releasing hormone and shaking behavior in rat, Nature 253, 739. Yarbrough, G.C., 1976, Studies on the neuropharmacology of thyrotropin-releasing hormone (TRH), Prog. Neurobiol. 12,291.
35
THYROTROPIN-RELEASING HORMONE AND AMPHETAMINE PRODUCE DIFFERENT PATTERNS OF BEHAVIORAL EXCITATION IN RATS * GREGORY N. ERVIN **, STEPANIE A. SCHMITZ, CHARLES B. N E M E R O F F and ARTHUR J. PRANGE, Jr.
Biological Sciences Research Center, Departments of Psychiatry and ~ychology, and the Neurobiology Program, University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. Received 25 November 1981, revised MS received 4 Feburary 1981, accepted 9 March 1981
G.N. ERVIN, S.A. SCHMITZ, C.B. N E M E R O F F and A.J. PRANGE, Jr., Thyrotropin-releasing hormone and amphetamine produce different patterns of behavioral excitation in rats, European J. Pharmacol. 72 (1981) 35--43. We compared the arousal and hyperactivity produced by intraperitoneal (i.p) injections of thyrotropin-releasing hormone (TRH, pGIu-His-Pro-NH2; 10, 20, 30 and 60 mg/kg) and 0.3 and 2 mg/kg d-amphetamine (low and moderate amph., respectively)by measuring the occurrence of discrete behavioral items with a behavioral sampling and scoring method. To minimize extraneous variables affecting activity, rats were caged singly inside isolated observation chambers and tested in the daytime after a 2.5 h period of habituation. Under these conditions, vehicle (0.9% NaCl)-treated rats were inactive and either rested or slept through 80% o f all time samples taken in the hour after injection. Both TRH and amph. produced significant arousal from sleeping, but TRH, at all doses tested, produced less arousal than moderate amph. and a pattern of behavioral responses which differed from both low and moderate amph. Moderate amph. produced marked increases in forward locomotion and rearing, but low amph. and TRH did not. Both TRH and low amph. increased grooming (perhaps simply b y increasing wakefulness), b u t TRH failed to increase sniffing, a cardinal feature of amph.-induced excitement. Unlike amph., TRH produced wet-dog shakes, piloerection, tail elevation and teeth chattering. Both mod. amph. and TRH significantly produced increased activity when compared to controls as assessed with photocell counts, though the amph. effect was more robust. The lack of arousal after i.p. injections of thyroid-stimulating hormone (10 I.U./kg) or melanocyte-stimulating hormone release-inhibiting factor (Pro-Leu-Gly-NH2; 60 mg/kg) is evidence that TRH-induced arousal is neither mediated b y activation of the pituitary-thyroid axis nor b y a non-specific effect of tripeptides generally. Thyrotropin-releasing hormone
Neuropeptides
1. Introduction Pyroglutamyl-l-histidyl-l-prolineamide is that tripeptide which was isolated from extracts of ovine and bovine hypothalami by the groups of Guilleman and Schally (Burgus et al., 1969; Boler et al., 1969) in their * This research was supported b y NIMH Grants MH32316, MH-22536, MH-33127 and NICHHD HD03110. ** Correspondence should be sent to Gregory N. Ervin, University of North Carolina School of Medicine, Biological Sciences Research Center 220-H, Chapel Hill, NC 27514, U.S.A.
d-Amphetamine
searches for a substance which controlled pituitary secretion of thyroid-stimulating hormone (TSH). Thus named thyrotropin-releasing hormone (TRH), it was the first hypothalamic releasing or release-inhibiting hormone to be chemically characterized (for reviews, see Guillemin, 1978; Schally et al., 1973). In retrospect, the naming of TRH was somewhat misleading since other pituitary effects of TRH have been discovered. Furthermore, wide phylogenetic and regional brain distributions of TRH support the hypothesis that, in addition to its role as a hypothalamic hormone, TRH also may function as a neuro-
0 014-2999/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
36 transmitter (Brownstein et al., 1974; Jackson and Reichlin, 1974; Johansson and HSkfelt, 1980). Administration of TRH causes behavioral effects and changes in responses to psychoactive drugs which are not observed after administration of TSH or thyroid hormones, and which persist even after hypophysectomy (Prange et al., 1978, 1979; Yarbrough, 1979). Finally, microiontophoresis of TRH has been shown to alter firing rates of neurons in several different brain regions (Renaud, 1977). Many of the effects of centrally or peripherally administered TRH appear as increases in arousal. In a variety of mammals that have been tested, including mice, rats, hamsters, gerbils, dogs and monkeys, TRH antagonizes barbiturate- and ethanol-induced sedation (Prange et al., 1974; Breese et al., 1974; 1975), potentiates L-DOPA and L-5-HTPinduced excitation, and increases wakefulness and alertness (see Nemeroff et al., 1978; Prange et al., 1979; Morley, 1979, for reviews). In rats, the behavioral excitation observed after peripheral TRH administration has been reported as increased grooming, shaking and tail elevation (Schenkel-Hulliger et al., 1974; Wei et al., 1975). Some authors have reported increased locomotion, rearing and sniffing (Miyamoto and Magawa, 1977). These last three behaviors are also major behavioral components of d-amphetamine sulfate (amph.)-induced arousal (Ervin e t al., 1977; Ayhan and Randrup, 1973). Nucleus accumbens septi contains dopamine (DA) terminals where amph. acts to stimulate locomotion by releasing DA (Kelly et al., 1975), and nucleus accumbens also contains TRH-like immunoreactivity (Johansson and HSkfelt, 1980). Both TRH and DA injected into nucleus accumbens reportedly cause locomotion, rearing and sniffing (Miyamoto and Nagawa, 1977). On these behavioral grounds, TRH has been postulated to release DA, like amph. (Heal and Green, 1979). However, behavioral similarities between TRH and amph. have usually been inferred from photocell or electronic activity meters, or have been
G.N. ERVIN ET AL. based upon unsystematic observations. Photocell and electronic measurements of activity have often been shown to be ambiguous since a variety of behavioral items can produce the same measurable effect (e.g., Krsiak et al., 1970). Behavioral observations of TRH have been inconsistent, and Costall et al. (1979) did not report increased locomotion, rearing and sniffing after intra-accumbens injections of TRH as others did (Miyamoto and Nagawa, 1977; Heal and Green, 1979). In the present study we used a time-sampling and behavioral scoring method to further compare TRH and amph. Both TRH and amph. produced arousal, but the behavioral excitation caused by TRH was markedly different from the excitation caused by amph. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (Zivic-Miller Laboratories, Allison Park, PA) weighing 250500 g were housed in groups of four in a controlled environment animal facility (ambient temperature 23-25°C; lights on, 06:0018:00 h) and received laboratory chow and water ad libitum. 2.2. Apparatus Rats were observed in plexiglass test cages (21 X 44 X 20 cm high) that had wire lids and wire mesh floors. Each test cage was mounted inside a ventilated wooden box (52 X 66 X 46 cm high) with a 14 cm X 66 cm smoked plexiglass window on one wall which allowed viewing of rats from 20-40 cm. Each box was illuminated by a 7.5 W bulb; the test room was dark. Each box was equipped with a near infrared light source and photocell (Lafayette Instrument Co., Lafayette, IN) positioned 2 cm above the floor and halfway along the length of the plexiglass test cage. The sensitivity of photocell apparatus was adjusted so the beam was interrupted by two fingers (3.8 cm in width) but not by a pencil (0.7 cm in width).
BEHAVIORAL
PROFILES OF TRH AND AMPHETAMINE
2.3. Drugs
Rats were injected intraperitoneally (i.p.) with either TRH, amph., bovine thyroidstimulating hormone (TSH), melanocytestimulating hormone release inhibiting factor (MIF-I) or sterile0.970 NaCl alone. All drugs were dissolved in sterile0.970 NaCI, and injection volume was always 2 ml/kg body weight. T R H (lot No. 21-220-AL) was a giftof Abbott Laboratories, North Chicago, IL; MIF-I Clot No. 15487) was purchased from U.S. Biochemical Corporation, Cleveland, OH: and amph. was purchased from Sigma Chemical Co., St. Louis, M O .
37
the hindlimbs to groom the head was included in this category. ( 7 ) G r o o m i n g of the hindquarters: Licking of the hindlimbs and body, or the tail. Use of the forelimbs to groom the hindquarters was included in this category. (8) Chewing or teeth chattering: Rapid, repetitive movement of the jaws not associated with self-biting or biting of other objects (such as the wire mesh floor). (9) Resting: Any resting position with the eyes simultaneously open throughout a 10 sec interval. (10)Sleeping: Any of the resting positions with the eyes closed throughout a 10 sec interval. Forward locomotion and rearing scores are the number of times these behaviors were observed. All other behavioral items received a
2.4. Time samples of behavior taken a't 10 rain in tervals TABLE 1
Between 08:00 and 1 0 : 0 0 h , rats were transferred from the colony room to observation boxes in group cages. The observation cages were devoid of food, water, or other objects. Rats were habituated to observation cages for at least 2 h prior to the first behavioral observation. Three consecutive 10sec behavioral samples were observed every ten rain starting 30 min before and lasting 2 h beyond i.p. injection of T R H (10, 20, 30, 60 mg/kg), MIF-I (60 mg/kg)., TSH (10 IU/ kg), amph. (0.3 or 2 mg/kg) or 0.9% NaC1 (2 ml/kg), and occurrence of the following behavioral items were noted as previously described (Ervin et al., 1977). (1) Forward locomotion: The movement of the animal from one end of the cage to the other. (2) Rearing: The vertical extension of the head, b o d y , and forelimbs, excluding such postures associated with grooming. (3) Circling: Complete 360 ° turn of the animal's b o d y axis. Direction of circling was also noted. (4) Sniffing: Repetitive m o v e m e n t of the snout and nostrils directed toward some cage surface. ( 5 ) H e a d stereotypy: Rapid movement or bobbing of the head n o t directed toward any cage surface. (6) Grooming of the head or forelimbs: Any licking, washing, or scratching of the forelimbs or head. Use of
Effects o f t h y r o t r o p i n - r e l e a s i n g h o r m o n e a n d a m p h e t a m i n e o n arousal f r o m resting a n d sleeping. R a t s were scored for resting or sleeping if t h e y p e r f o r m e d such b e h a v i o r t h r o u g h o u t a I 0 sec behavioral sample. S h o w n is t h e m e d i a n p e r c e n t a g e a n d range of observ a t i o n t i m e in t h e h o u r a f t e r i n j e c t i o n t h r o u g h w h i c h rats o f each g r o u p (n = 8) e i t h e r rested or slept. T r e a t m e n t (i.p.)
Sampling time spent resting or sleeping %
Range
{A) Experiment 1. Three behavioral samples every 10 rain NaCI, 0.9% (vehicle) T R H , 10 m g / k g T R H , 20 m g / k g T R H , 30 m g / k g TRH, 60 mg/kg TSH, I 0 l.U./kg MIF, 60 m g / k g AMPIi, 0.3 m g / k g AMPH, 2 m g / k g
83 61 ,17 28 42 67 78 22 0
a • b b
b b
50--83 33--83 17--72 17--44 6-56 28 - 8 3 67--94 0 56
(B) Experiment 2. Three behavioral samples every 5rain
NaCI, 0.9% (vehicle) TRH, 30 mg/kg AMPH, 2 mg/kg
78 28 b 0 b
53--97 6--44
a P < 0.05 c o m p a r e d to vehicle g r o u p ' s score with M a n n - W h i t n e y U-test. b p < 0.01 c o m p a r e d to vehicle g r o u p ' s score with M a n n - W h i t n e y U-test.
38
G.N. ERVIN ET AL.
score of 1 when they occurred, however often, in a 10-sec interval. Thus the total score of these behavioral items was the number of 10-sec intervals in which that behavior occurred, and the m a x i m u m possible score for a particular behavioral item in 1 h was 18. Resting and sleeping were scored only when t h e y persisted throughout a 10-second interval. The sum of the resting and sleeping scores is an inverse measure of arousal. 2.5. Time samples o f behavior taken at 5 min intervals
Rats were injected i.p. with TRH (30 mg/ kg), amph. (2 mg/kg) or vehicle (0.9% NaC1).
The design of this experiment was identical to the design described in 2.4 with the following exceptions: (1)Behavior was observed for only l h after drugs. (2) Behavior was sampled for 30 sec every five, rather than every 10 min. (3) The occurrence of wet dog shakes, tail elevation and piloerection was also noted. (4) General activity was also measured automatically by counting interruptions of a photocell beam. 2.6. Data analysis
All behavioral data were expressed as medians and ranges; statistical comparisons were performed using the Mann-Whitney Utest (Conover, 1971).
TABLE 2 Effects of thyrotropin-releasing hormone and d-amphetamine on behavioral excitation. Behavioral item
NaC1 0.9%
TRH
d-Amph
10 mg/kg
20 mg/kg
30 mg/kg
60 mg/kg
0.3 mg/kg
2 mg/kg
MIF 60 mg/kg
TSH 10 IU/kg
Forward locomotion
0 (0)
0 (0--1)
0 (0--3)
0.5 (0--2)
0 (0--4)
0 (0--2)
4b (0--9)
0 (0)
0 (0--1)
Rearing
0 (0--1)
0.5 (0--3)
0.5 (0--6)
0 (0--3)
0 (0--2)
1 (0--3)
4.5 b (2--11)
0 (0--3)
0 (0--5)
Sniffing
1.5 (0--5)
1.5 (0--4)
1 (0--4)
3 (0--7)
1.5 (0--4)
7b (0--12)
17 b (13--17)
0 (0--6)
2.5 (0--4)
Circling
0 (0)
0 (0)
0 (0)
0 (0--1)
0 (0--1)
0 (0)
0 (0-6)
0 (0)
0 (0--1)
Head stereotypy
0 (0--1)
0 (0)
0 (0)
0 (0--2)
0 (0--2)
0 (0)
2.5 a (0--6)
0 (0)
0 (0)
Grooming of head and forelimbs
0.5 (0--5)
3a (0--8)
3a (1--7)
3a (0--7)
4b (3--15)
4a (1--9)
1.5 (0-6)
1 (0--4)
0 (0--3)
Grooming of hindquarters Chewing/teeth chattering
0 (0--2) 0.5 (0--5)
1 (0--3) 1.5 (0--2)
0.5 (0--5) 3 (0--6)
0 (0--1) 7.5 b (2--15)
0 (0--2) 9.5 b (8--18)
1.5 (0--5) 0 (0--2)
0 (0--2) 0 (0--3)
0.5 (0--3) 0.5 (0-6)
1 (0--3) 0 (0--1)
Resting
4 (0--12)
3 (0--12)
2.5 (0--7)
3.5 (0--5)
2.5 (0--7)
4 (0--9)
0a (0)
4 (0--14)
7 (3--15)
Sleeping
9 (0--15)
7 (0--12)
6 (0--11)
3b (0--3)
2.5 b (0--10)
0b (0--3)
0b (0)
9 (3--15)
4.5 (0--9)
After being habituated to observation cages for 2.5 h rats were injected intraperitoneally and observed for 30 sec every 10 min for 2 h. Eight rats were in each group. Data shown are the median behavioral scores over the first hour after injection. Numbers in parentheses are the ranges of behavioral scores recorded for that item. Suffixes indicate significant differences from NaCl-injected rats: a p < 0.05; b p < 0.01, Mann-Whitney U-test.
BEHAVIORAL PROFILES OF TRH AND AMPHETAMINE
formed significantly more chewing and teeth chattering, and slept significantly less. TRH did not increase sniffing--a cardinal feature of amph.-induced excitation. TRH resembled amph. only because low amph. (0.3 mg]kg) - - like TRH-- significantly increased grooming (of the head and forelimbs) and decreased sleeping, but not resting. Animals injected with the moderate dose of amph. (2 mg/kg) exhibited significant increases in locomotion, rearing, sniffing, and head stereotypy, and significant decreases in resting as well as in sleeping. In the second hour after injections, rearing, sniffing, head stereotypy and insomnia were still significantly elevated by amph. (2 mg/kg; P < 0.05, data not shown), but only chewing/teeth chattering was still elevated by TRH (60 mg/kg, P < 0.05).
3. Results 3.1. Time samples o f behavior taken at 10minute intervals
Prior to i.p. injections, all rats behaved similarly - - t h e y rested or slept, but displayed few other behaviors (data not shown). Injections of TRH (10-60 mg/kg i.p.) and amph. (0.3 and 2.0 mg/kg i.p.) produced arousal, but injections of vehicle, MIF and TSH did not (table 1A). After vehicle, MIF and TSH, rats quickly resumed resting or sleeping. TRH and amph. produced different pat~ terns of behavioral arousal (table 2). After TRH, rats groomed the head and forelimbs significantly more. In addition, after higher doses of TRH (30 and 60 mg/kg), rats per-
35
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39
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Fig. 1. Behavioral profiles o f TRI-I and a m p h e t a m i n e . As in e x p e r i m e n t 1, animals were a l l o w e d t o h a b i t u a t e t o cages f o r 2.5 h p r i o r t o (i.p.) injections. AnimaLs were observed f o r 30 see every 5 rain f o r 1 h. Data s h o w n are m e d i a n scores f o r 8 rats over the entire o b s e r v a t i o n period. P h o t o c e l l score given is actual c o u n t divided b y 10. With the e x c e p t i o n o f scores f o r l o c o m o t i o n and rearing ( w h i c h are actual f r e q u e n c y o f behavior), m a x i m a ] score possible f o r a p a r t i c u l a r behaviors] i t e m is 36. * P < 0.05; * * P < 0.01 c o m p a r e d t o vehic|e (0.9% NaC|)
&coup's score with Mann-Whitney U-test.
40 3.2. Time samples o f behavior taken at 5 min intervals
Both TRH (30 mg/kg) and amph. (2 mg/ kg) elicited significant increases in photocell counts when compared with vehicle-treated controls (fig. 1), though the amph. effect was significantly greater than the TRH effect (P < 0.01). As in experiment 1, TRH and amph. produced arousal (table 1B) but different patterns of behavioral activation (fig. 1). Amph. elicited large increases in forward locomotion, rearing, sniffing and head stereotypy, but TRH did not. However, increasing the frequency of behavioral sampling did increase the sensitivity of our technique, because it was demonstrated that TRH does, in fact, increase forward locomotion to a slight degree (though significantly less than amph., P < 0.01). TRH elicited large increases in grooming (of head and forelimbs), teeth chattering, wet dog shakes, tail elevation, and piloerection, but amph. did not. Both amph. and TRH produced insomnia, but only amph. significantly decreased resting behavior.
4. Discussion Both TRH and amph. produced arousal, but vehicle, TSH and MIF did not (table 1). The low dose of amph. produced arousal much like TRH; rats were more wakeful, but still restful. After the higher dose of amph., rats neither slept nor rested (table 2 and fig. 1). Electroencephalographically, TRH arousal and amph. arousal are similar (Itil et al,, 1975). However, EEG arousal and behavioral arousal are separable (Jones et al., 1978; Bradley and Elks, 1957), and the behavioral arousal produced by TRH and amph. are strikingly different. The most pronounced behavioral effect of TRH was grooming of the head and forepaws. Grooming is the most frequently observed behavior of laboratory rats during wakefulness (Bolles, 1960). Thus, frequent grooming after
G.N. ERVIN ET AL. TRH may have simply been due to increased wakefulness. Grooming was also frequent after low amph., but not after the higher dose of amph. which elicited behaviors which would clearly compete with grooming (table 2 and fig. 1; Ayhan and Randrup, 1973). The higher dose of amph. markedly increased the frequency of forward locomotion and rearing, but low amph. and TRH did not. Although we did not detect any increase in locomotion after TRH when behavior was observed at 10-minute intervals, we did detect a very slight increase in locomotion when behavior was observed at five-minute intervals. Thus the sensitivity of time-sampling methods is clearly dependent on observation frequency. Many groups have emphasized locomotor hyperactivity after peripheral or intracerebral administration of TRH, but the data were general 'activity' measurements from photocells or electronic devices (Miyamoto and Nagawa, 1977; Vogel et al., 1979; Heal and Green, 1979) and these devices measure certain behaviors in addition to locomotion (Krsiak et al., 1970; Ljungberg, 1978). Sniffing was a cardinal characteristic of amph.-induced excitation. Although low amph. was like TRH in many respects (e.g., both increased grooming and neither markedly increased locomotion or rearing), low amph. elicited sniffing but TRH did not (table 2). After TRH, but not after amph., we observed chewing and teeth chattering, tail elevation, wet-dog shakes .and piloerection. The higher d o s e of amph., 2 mg/kg, was threshold for stereotypies because head bobbing was slightly increased. After higher doses of amph., stereotypies (e.g., biting, licking and stereotyped sniffing) become prominent and decrease forward locomotion and rearing (Fog, 1972; Kelly et al., 1975; Ervin et al., 1981). TRH does not elicit such stereotyped behavior (Vogel et al., 1979; Miyamoto and Nagawa, 1977). Although TRH, like amph., produces arousal, this cannot explain its potency in many pharmacological tests. For example, TRH (1 mg/kg) is as effective as amph. (3
BEHAVIORAL PROFILES OF TRH AND AMPHETAMINE
mg/kg) in reversing pentobarbital sedation in mice (Prange et al., 1975), but amph. (3 mg/ kg) produces intense arousal in otherwise untreated rats while TRH (1 mg/kg) does not (Miyamoto and Nagawa, 1977). Also, TRH and amph. both potentiate L-DOPA-induced excitation (Plotnikoff et al., 1974; N. Plotnikoff, personal communication), but TRH is as effective as more arousing doses of amph. Moreover, TRH, unlike amph., is effective after immediate arousal effects have concluded. The potentiation of L-DOPA-induced excitation is also affected by substances, such as MIF and TSH, which alone do n o t produce arousal (tables 1 and 2). Rats apparently perceive T R H and amph. arousal differently. Jones et al. (1978) demon° strated that both T R H and amph. act as cues in a two-lever operant discrimination task, but generalization between T R H (10, 20 or 30 mg/kg, i.p.)and amph. (0.8 or 1.6 mg/kg, i.p.) does not occur. Other reported differences between T R H and amph. are: (1) T R H reverses ethanol-induced sedation; amph. either exerts no effect or enhances it (Breese et al., 1974); (2) T R H possesses considerable activity in an anti-conflict activity whereas arnph, exerts no such effect (Vogel et al., 1980), and (3) T R H reverses oxotremorineinduced hypothermia whereas amph. does not. Several other differences between T R H and amph. have been reported, and these have been reviewed (Manberg et al., 1979), Amph. exerts its major behavioral effects through the presynaptic release of catecholamines (especially DA), and TRH has been shown to cause release of DA in nucleus accumbens as well as in nucleus caudatus in vitro (Miyamoto et al., 1979). Kerwin and Pycock (1979) have reported that TRH releases [3H IDA from slices of the nucleus accumbens but not from nucleus caudatus. The present results and failure of others to observe amph.like stereotypies after TRH administration (Vogel et al., 1979; Miyamoto and Nagawa, 1977) suggest that TRH does n o t exert arnph.like neurochemical effects in vivo. Despite the numerous pharmacological
41
effects of TRH which have been reported, the physiological roles of central TRH have yet to be established. References Ayhan, I.H. and A. Randrup, 1973, Behavioral and pharmacological studies on morphine-induced excitation of rats. Possible relation to brain catecholamines, Psyehopharmaeologia 29, 317. Boler, J., F. Enzmann, K. Folkers, C.Y. Bowers and A.V. Schally, 1969, The identity of chemical and hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl-proline-amide, Biochem. Biophys. Res. Commun. 37,705. Bolles, R.C., 1960, Grooming behavior in the rat, J. Comp. Physiol. Psych. 53, 306. Bradley, P.B. and J. Elks, 1957, The effect of some drugs on the electrical activity of the brain, Brain 80, 77. Breese, G.R., J.M. Cott, B.R. Cooper, A.J. Prange, Jr. and M.A. Lipton, 1974, Antagonism of ethanol narcosis by thyrotropin-releasing hormone, Life Sci. 14, 1053. Breese, G.R., J.M. Cott, B.R. Cooper, A.J. Prange, Jr., M.A. Lipton and N.P. Plotnikoff, 1975, Effects of thyrotropin-releasing hormone (TRH) on t h e actions of pentobarbital and other centrally acting drugs, J. Pharmacol. Exp. Ther. 193, 11. Brownstein, M.T., M. Palkovits, J~I. Saavedra, R.M. Bassiri and R.D. Utiger, 1974, Thyrotropin-releasing hormone in specific nuclei of rat brain, Science 195, 267. Burgus, R., T.F. Dunn, D. Desiderio and R. Guillemin, 1969, Molecular structure of the hypothalamic thyrotropin-releasing factor (TRF) of ovine origin, demonstration o f the pyroglytamyl-histidyl-prolinamide sequence by mass spectrometry, C.R. Acad. Sci. (Paris) 263, 1870. Conover, W.J., 1971, Practical Nonparametric Statistics (Wiley, New York). Costall, B., G.H. Siv-Chun, G. Metcalf and R.J. Naylot, 1979, A study of the changes in motor behavior caused by TRH on intracerebral injection, European J. Pharmacol. 53, 143. Ervin, G.N., L.S. Birkemo, C.B. Nemeroff and A.J. Prange, Jr., 1981, Neurotensin blocks certain amphetamine-induced behaviors, Nature (in press). Ervin, G.N., J.S. Fink, R.C. Young and G.P. Smith, 1977, Different behavioral responses t o L-DOPA after anterolateral o r posterolateral hypothalamic injections of 6-OHDA, Brain Res. 132,507. Fog, R., 1972, On stereotypy and catalepsy: Studies on the effects of amphetamines and neurolepties in rats, Acta Neurol. Scand. 48, 7.
42 Guillemin, R., 1978, Biochemical and physiological correlates of hypothalamic peptides. The endocrinology of the neuron, in: The Hypothalamus, eds. R.J. Baldessarini and J.B. Martin (Raven Press, New York) p. 155. Heal, D.T. and A.R. Green, 1979, Administration of thyrotropin-releasing hormone (TRH) to rats releases dopamine in nucleus accumbens but not nucleus eaudatus, Neuropharmacoh 18, 23. Itil, T.M., C.D. Patterson, N. Polvar, A. Bigelow and B. Bergey, 1975, Clinical and CNS effects of oral and I.V. thyrotropin-releasinghormone in depressed patients, Dis. Nerv. Syst. 36, 529. Jackson, I.M.D. and S. Reichlin, 1974, Thyrotropinreleasing hormone (TRH): Distribution in hypothalamic and extrahypothalamic brain tissues of mammalian and submammalian chordates, Endocrin. 95,854. Johansson, O. and T. H6kfelt, 1980, Thyrotropin releasing hormone, somatostatin and enkephalin: Distribution studies using immunohistochemical techniques, J. Histochem. Cytochem. 28,364. Jones, C.N., L.D. Grant and A.J. Prange, Jr., 1978, Stimulus properties of thyrotropin-releasing hormone, Psychopharmacol. 59,217. Kelly, P.H., P.W. Seviour and S.D. Iversen, 1975, Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum, Brain Res. 94,507. Ljungberg, T., 1978, Reliability of two 'activity boxes' commonly used to assess drug-induced behavioral changes, Pharmacol. Biochem. Behav. 8,191. Kerwin, R.W. and C.J. Pycock, 1979, Thyrotropinreleasing hormone stimulates release of [3H]dopamine from slices of rat nucleus accumbens in vitro, Br. J. Pharmacol. 67,323. Krsiak, M., H. Steinberg and I.P. Stolerman, 1970, Uses and limitations of photocell activity cages for assessing effects of drugs, Psychopharmacologia 17, 258. Manberg, P.J., C.B. Nemeroff and A.J. Prange, Jr., 1979, Thyrotropin-releasing hormone and amphetamine: A comparison of pharmacological profiles in animals, Prog. Neuropsychopharmacol. 3,303. Miyamoto, M. and Y. Nagawa, 1977, Meso!imbic involvement in the locomotor stimulant action of thyrotropin-releasing hormone (TRH) in rats, European J. Pharmacol. 44,143. Miyamoto, M., S. Narumi, Y. Nagai, T. Shuma and Y. Nagawa, 1979, Thyrotropin-releasing hormone: Hyperactivity and mesolimbic dopamine system in rats, Jap. J. Pharmacol. 29, 335. Morley, J.E., 1979, Extrahypothalamic thyrotropinreleasing hormone (TRH) -- Its distribution and its functions, Life Sci. 25, 1539. Nemeroff, C.B., P.T. Loosen, G. Bissette, P.J. Man-
G.N. ERVIN ET AL. berg, I.C. Wilson, M.A. Lipton and A.J. Prange, Jr., 1978, Pharmaco-behavioral effects of hypothalamic peptides in animals and man: Focus on thyrotropin-releasing hormone (TRH) and neurotensin, Psychoneuroendocrinol. 3, 279. Plotnikoff, N.P., A.J. Prange, Jr., G.R. Breese, M.S. Anderson and I.C. Wilson, 1974, Effects of thyrotropin-releasing hormone on DOPA response in normal, hypophysectomized, and thyroidectomized animals, in: The Thyroid Axis, Drugs and Behavior. ed. A.J. Prange, Jr. (Raven Press, New York) p. 103. Prange, A.J., Jr., G.R. Breese, J.M. Cott, B.R. Martin, B.R. Cooper, I.C. Wilson and N.P. Plotnikoff, 1974, Thyrotropin releasing hormone: Antagonism of pentobarbital in rodents, Life Sci. 14. 447. Prange, A.J., Jr., G.R. Breese, G.D. Jahnke, B.R. Martin, B.R. Cooper, J.M. Cott, I.C. Wilson, L.B. Alltop, M.A. Lipton, G. Bh~sette, C.B. Nemeroff and P.T. Loosen, 1975, Modification of pentobarbital effects by natural and synthetic polypeptides: Dissociation of brain and pituitary effects, Life Sci. 16, 1907. Prange, A.J., Jr., C.B. Nemeroff, M.A. Lipton, G.R. Breese and I.C. Wilson, 1978, Peptides and the central nervous system, in: Handbook of Psychopharmacology, Vol. 13, eds. L.L. Iversen, S.D. Iversen and S.H. Snyder (Plenum Press, New York) p. 1. Prange, A.J., Jr., C.B. Nemeroff, P.T. Loosen, G. Bissette, A.J. Osbahr III, I.C. Wilson and M.A. Lipton, 1979, Behavioral effects of thyrotropin-releasing hormone in animals and man: A review, in: Central Nervous System Effects of Hypothalamic Hormones and Other Peptides, ed. R. Collu (Raven Press, New York), p. 75. Renaud, L.P., 1977, TRH, LHRH and somatostatin: Distribution and physiological action in neural tissue, in: Society for Neuroscience Symposia II: Approaches to the Cell Biology of Neurons, eds. W.M. Cowan and J.A. FerendeUi (Soc. Neurosci. Pub., Bethesda) p. 265. Schally, A.V., A. Arimura, L. Debeljuk, T.W. Redding and J.J. Reeves, 1973, Hypothalamic hormones regulating pituitary function; their physiology and biochemistry, in: Hypothalamic Hypophysiotropic Hormones, eds. C. Gual and E. Rosemberg (Excerpta Medica, Amsterdam) p. 1. Schenkel-Hulliger, L., E.P. Koella, A. Hartmann and L. Maitre, 1974, Tremorogenic effects of thyrotropin releasing hormone in rats, Experientia 30, 1168. Vogel, R.A., B.R. Cooper, T.S. Barlow, A.J. Prange, Jr., R.A. Mueller and G.R. Breese, 1979, Effects of thyrotropin-releasing hormone on locomotor activity operant performance, and ingestive behavior, J. Pharmacol. Exp. Ther. 208,161. Vogel, R.A., G.D. Frye, J.H. Wilson, C.M. Kuhn, R.B.
BEHAVIORAL PROFILES OF TRH AND AMPHETAMINE Mailman, R.A. MueUer and G.R. Breese, 1980, Attenuation of the effect of punishment by thyrotropin-releasing hormone: Comparisons with chlordiazepoxide, J. Pharmacol. Exp. Ther. 212, 153. Wei, E., S. Siegel, H. Loh and E.L. Way, 1975, Thyro-
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tropin-releasing hormone and shaking behavior in rat, Nature 253, 739. Yarbrough, G.C., 1976, Studies on the neuropharmacology of thyrotropin-releasing hormone (TRH), Prog. Neurobiol. 12,291.