Psychopharmacology

Psychopharmacology 52, 145-149 (1977)

9 by Springer-Verlag 1977

Antagonism of the Analeptic Activity of Thyrotropin-Releasing Hormone (TRH) by Agents which Enhance GABA Transmission JERRY COTT* and JORGEN ENGEL** Department of Pharmacology, University of G6teborg, S-400 33 G6teborg, Sweden

Abstract. Administration of 10 mg/kg TRH to mice was found to reduce the sleep and hypothermia induced by 4.7 g/kg ethanol. However, TRH did not reduce the sleep of mice that were given ?-hydroxybutyric acid (GHBA), baclophen, or aminooxyacetic acid (AOAA) in combination with 3 g/kg ethanol. TRH also failed to reverse the hypothermia induced by the combination of ethanol and baclophen or GHBA, and the characteristic neurological effects of TRH e.g. tremor, increased muscle tone, and increased respiratory rate were reduced. In addition, TRHinduced locomotor stimulation was prevented by pretreatment with small doses of the GABA-ergic agents, and while 30 mg/kg TRH reduced the hypothermia produced by large doses of the GABA-ergic drugs, it did not antagonize the locomotor retardation produced by baclophen or GHBA. A hypothesis that the analeptic effects of TRH may be mediated via an inhibition of GABA systems is discussed. Key words." TRH - GABA - Sleep - Locomotor activity.

Thyrotropin-releasing hormone (TRH) has recently received much attention for its extrapituitary effects in the central nervous system. Evidence for behavioral effects of T R H was first provided by the finding that T R H potentiated L-Dopa-induced excitation in pargyline pretreated animals after hypophysectomy (Plotnikoff et al., 1972). It was subsequently found that TRH produced tremor, increased respiratory rate and muscle tone, and reduced the sedation or sleep and the hypothermia produced by several different types of sedative agents including ethanol (Prange et al., * Present address: Department of Pharmacology, University of Ibadan, Ibadan, Nigeria ** To whom offprint requests should be sent

1974; Breese et al., 1974a and b, 1975; Brown and Vale, 1975; Cohn, 1975; Horita and Carino, 1975; Cott et al., 1976a). Up to the present, attempts to define the mechanism of these effects have not been conclusive. While it appears that catecholamine systems are not involved (Breese et al., 1975; Horita and Carino, 1975; Cott et al., 1976a) there is some evidence to indicate that cholinergic systems may be involved in some of the effects (Breese et al., 1975; Cott et al., 1976a). The present investigation was conducted in order to examine the role of GABA-containing pathways in mediating the analeptic effects of T R H by using drugs which, in various ways, are thought to enhance the functional activity of GABA systems (herein referred to as "GABA-ergic" drugs). The GABA-ergic drugs used in the present study were: aminooxyacetic acid (AOAA) which elevates brain GABA levels by inhibiting GABA-transaminase (Wallach, 1961); 7-hydroxybutyric acid (GHBA), a GABA derivative which appears to mimic the action of GABA (Aghajanian and Roth, 1970; And~n and Stock, 1973; Stock et al., 1973) and baclophen, a GABA derivative which, biochemically and behaviorally, appears to have many GABA-ergic properties (Fuxe et al., 1975a and b; Cott et al., 1976b; K/i/iri/iinen, 1976; And6n and Wachtel, 1977).

METHODS Subjects and Drugs. Female mice of the N.M.R.I. strain, weighing approximately 20 g, were used. Animals were fed ad libitum and were housed under a 12 h light/dark cycle at approximately 25~ The following drugs were used: Ethanol 99.5 ~ ; baclophen (fl-[-4chlorophenyl]-7-aminobutyric acid, Lioresal | Ciba-Geigy, M61ndal, Sweden); 7-hydroxybutyric acid (GHBA, sodium form, Sigma Chemical Co., St. Louis, Mo.); aminooxyacetic acid hemichloride (AOAA, Sigma); pyroGlu-His-Pro-NH2 (TRH, Abbott Laboratories, North Chicago, Ill.). Ethanol was given intraperitoneally (i.p.) as a 15 % v/v solution with saline. All other drugs were dis-

146 solved in 0.9 ~ NaC1, and were administered i.p. in a volume of 10 ml/kg. The doses given refer to the forms mentioned above. Sleeping Time Studies. Sleeping time was measured as the time from which righting reflex was lost after drug treatments until it was regained. For these studies ethanoI (4.7 g/kg) alone was administered or ethanol (3 g/kg) was administered immediately after GHBA (300 mg/kg), 10 rain after baclophen (10 mg/kg), or 60 rain after AOAA (40 mg/kg). A lower dose of ethanol was used in conjunction with the GABA-ergic drugs since previous results (Cott et al., 1976b) demonstrated that these agents greatly potentiate the depressant properties of ethanol. Experimental groups received TRH (10 mg/kg) within 5 min after the loss of the righting reflex. Control animals received saline injections. Rectal temperatures were determined in separate groups of similarly treated mice 40 rain after ethanol administration. Arab• temperature was approximately 26~C. Locomotor Activity Studies. Locomotor activity was measured using the "M/P 40 Fc Electronic Motility Meter" (Motron Products, Stockholm) (for a detailed description of the apparatus, see Melberg et al., 1976). Every 10th interruption of the photocell beams registered one count. Baclophen (2.5 or 20 mg/kg) was given 20 min before; AOAA (35 or 75mg/kg) 90rain before; and GHBA (100 or 500 mg/kg) and TRH (5 or 30 mg/kg) immediately before placing the mice (in groups of 3) into the activity chambers. Control animals received saline injections. Activity was recorded for 45 min. Temperature in activity boxes was approximately 28 ~C. Animals were only used once. Rectal temperatures were determined in separate groups of similarly treated mice according to the schedule in Table 2. Ambient temperature was approximately 26~C. Statistics. All statistical comparisons were made using a 2-tailed Student's t-test or Dunnett's t-test (when comparing several experimental groups with control).

Psychopharmacology 52 (1977) Table 1. Effect of GABA-ergic drugs on ethanol-induced sleep and hypothermia Treatment

Sleeping time (min _+ S.E.M.)

Ethanol Ethanol + TRH Ethanol + GHBA Ethanol + GHBA +TRH Ethanol + baclophen Ethanol + baclophen -t- TRH E t h a n o l + AOAA Ethanol + AOAA +TRH"

Rectal temperature (~ • S.E.M.)

68 • 33 • 54 •

7 (10) 7** (10) 3 (20)

34.5 _+ 0.1 35.0 _+ 0.1" 34.4 • 0.2

(12) (20) (15)

50 • 70 •

3 4

(20) (22)

34.6_+ 0.2 34.3 • 0.3

(14) (14)

68 • 132 •

5 6

(23) (12)

34.2_+ 0.2 33.8 _+ 0.1

(13) (12)

111 • 13

(11)

34.6 • 0.1"** (11)

Due to the relatively short duration of the analeptic action of TRH (Cott et al., 1976a) and the long duration of ethanol + AOAA sleep, this group received a supplementary injection of TRH 80 min after the first dose * When compared with corresponding group not receiving TRH, P < 0.02 ** When compared with corresponding group not receiving TRH, P < 0.005 *** When compared with corresponding group not receiving TRH, P < 0.001 Numbers in parentheses refer to number of animals. Ethanol, when not combined with GABA-ergic drugs was given in a dose of 4.7 g/kg; when given with GABA-ergic drugs, the dose given was 3.0 g/kg. The other drugs were given in the following doses : TRH 10 mg/kg; GHBA 300 mg/kg; baclophen 10 mg/kg; AOAA 40 rag/ kg

RESULTS

Effect of GABA-ergic Drugs on TRH Antagonism of Ethanol-Induced Sleep and Hypothermia. In agreement with previous findings (Breese et al., 1974a and b; Cottet al., 1976 a) TRH significantly reduced ethanolinduced sleep and hypothermia (Table 1). However, when animals received the combination of 3 g/kg ethanol (a dose which, by itself, causes no sedation, but rather stimulation) and baclophen (10 mg/kg), GHBA (300 mg/kg), or the GABA transaminase inhibitor AOAA (40 mg/kg), they no longer responded to the TRH injection, i.e., they sleep just as long as non-TRH-treated animals (Table 1). Similarly, pretreatment with GHBA or baclophen prevented the TRH antagonism of hypothermia. Pretreatment with AOAA, however, did not (Table 1). Normal temperature of untreated control animals was 36.2 + 0.1~ n = 19. In addition, GHBA and baclophen pretreatment reduced the neurological effects of TRH, e.g., tremor, and increased muscle tone and respiratory rate, as measured by gross observation. Effects of TRH and GABA-ergic Agents on Locomotor Activity. In these studies TRH administration was

Table 2. Effect of OABA-ergic drugs on TRH-induced locomotor stimulation Treatment

Dose (mg/kg)

Locomotor activity (counts/45 rain • S.E.M.)

Saline

-

461 +_ 39

TRH TRH

(8)

5 30

612 +_ 53* (8) 637 _+ 59* (9)

GHBA GHBA

150 500

502 _+ 29 (6) l l • 4 (6)

GHBA + TRH GHBA+TRH

150 and 5 500 and 30

502 _+ 30 7_+ 2

(6) (6) (7) (6)

Baclophen Baclophen

2.5 20

357 + 46 33 • 9

Baclophen + TRH Baelophen + TRH

2.5 and 5 20 and 30

396 _+ 40 (7) 23 _+ 9 (6)

AOAA AOAA

35 75

334 --t- 57 (4) 73 • 15 (9)

AOAA + TRH AOAA + TRH

35 and 5 75 and 30

369 • 36 (4) 168 _+ 39* (9)

* When compared with corresponding group not receiving TRH, P < 0.05 Numbers in parentheses refer to number of observations

J. Cott and J. Engel: Antagonism of TRH by GABA-ergic Drugs

147

Table 3. Effect of TRH on the hypothermia induced by GABAergic drugs

(Fuxe et al., 1975a and b; And~n and Wachtel, 1977; Cott et al., 1976b) and GHBA (Roth and Suhr, 1970; Roth et al., 1973; Stock et al., 1973; And6n and Stock, 1973), abolish the sleep-reducing effects of TRH in ethanol-treated mice. In addition, the GABA transaminase (GABA-T) inhibitor, AOAA, which has been found to elevate brain GABA levels (Wallach, 1961) also antagonized the ability of TRH to reduce ethanolinduced sleep. Furthermore, the antihypothermic effects of 10 mg/kg TRH were not apparent after administration of the combination of ethanol and baclophen or GHBA. The present findings also indicate that TRH-induced locomotor stimulation can be prevented by prior treatment with GABA-ergic agents, and that T R H does not antagonize the locomotor retardation produced by large doses of baclophen or GHBA. This finding is in contrast to the marked antagonism by TRH of a wide variety of other types of sedative agents (Breese et al., 1975; Horita and Carino, 1975; Cott et al., 1976a). Furthermore, the neurological signs produced by TRH administration, e.g., tremor, increased respiratory rate and muscle tone, can be reduced by these GABA-ergic drugs. It should be noted here that recent reports regarding the electrophysiological effects of baclophen indicate that this GABA-derivative has actions that cannot be explained by a simple mimicking of the endogenous transmitter (Curtis et al., 1974; Davies and Watkins, 1974; Haefely, 1976) e.g., baclophen's inhibition of single cell firing isnot antagonized by the GABA-antagonist, bicuculline. However, biochemically and behaviorally, baclophen has a profile remarkably similar to GHBA and to GABA itself (Fuxe et al., 1975a and b; Cott et al., 1976b; Kfifirifiinen, 1976; And6n and Wachtel, 1977). These data suggest that agents which are thought to activate GABA systems in brain may directly or indirectly reduce or eliminate the behavioral effects of TRH. Therefore, it is tempting to suggest that GABA pathways may mediate many of the actions of TRH, as opposed to a secondary influence, at best, of acetylcholine (Breese et al., 1975; Cott et al., 1976a). The finding in the present series of experiments that AOAA does not produce as complete an antagonism of TRH as do GHBA and baclophen (drugs which probably act postsynaptically) could be explained by a TRH-induced inhibition of certain GABA neurons. In this case, high levels of GABA, resulting from inhibition of GABA-T by AOAA might not be sufficient to activate GABA transmission if TRH were reducing its release. Secondly, the highest dose of AOAA used in these studies (75 mg/kg) might not facilitate GABA transmission to the same degree as GHBA or baclophen. Larger doses were not

Treatment

Dose (mg/kg)

Injection time (minutes before measurement)

Rectal temperature (~ _+ S.E.M.)

Saline TRH GHBA GHBA + TRH Baclophen Baclophen + TRH AOAA AOAA + TRH

-

35 35 35 35and35 45 45and 35 90 90and35

37.0 37.2 32.4 34.8 33.2 35.8 33.7 36.1

30 500 500 and 30 20 20 and 30 75 75and 30

_+ 0.1 (12) _+ 0.I (15) _+ 0.3 (15) _+ 0.2"(15) _+ 0.4 (15) _+ 0.2"(15) + 0.5 (14) _+ 0.4*(23)

* When compared with corresponding group not receiving TRH, P < 0.001 Numbers in parentheses refer to number of animals

found to produce a small but significant locomotor stimulation at 2 different doses (Table 2). Pretreatment with low doses of the GABA-ergic agents (those which do no significantly affect control activity) resulted in an inhibition of the locomotor stimulation produced by the low dose (5 mg/kg) of TRH. Similarly, the locomotor depression caused by high doses of GHBA and baclophen were not antagonized by the high dose (30 mg/kg) of TRH. The locomotor depression caused by the high dose of AOAA, however, was antagonized to a small degree by the high dose of TRH (Table 2).

Effect of TRH on the Hypothermia Induced by GABAergic Drugs. Large doses of the GABA-ergic agents used in the present studies induced a significant (3-5~ reduction of rectal temperature (Table 3). While the administration of a large dose of TRH did not increase rectal temperature in saline-treated mice, it resulted in an antagonism of the hypothermia induced by all 3 GABA-ergic drugs (Table 3). On the other hand, the gross neurological effects produced by 30 mg/kg TRH appeared to be reduced by all of the GABA-ergic agents.

DISCUSSION The results of the present series of experiments are in agreement with earlier reports (Breese et al., 1974a and b; Cott et al., 1976a) that TRH, which causes little behavioral stimulation per se, produces a marked analeptic effect in ethanol-sedated animals. However, pretreatment with agents which are thought to mimic at least some of the effect of GABA such as baclophen

148 attempted, however, due to the well d o c u m e n t e d toxic side-effects o f this c o m p o u n d . In our hands 75 m g / k g A O A A p r o d u c e d a 33~o mortality within 60 rain (unpublished observations). A final explanation for the incomplete blockage o f T R H by A O A A is provided by previous reports that A O A A also inhibits the activity o f glutamate decarboxylase ( G A D ) , the enzyme which is responsible for G A B A synthesis ( W o o d and Peesker, 1973), and that newly synthesized G A B A m a y initially be m o r e i m p o r t a n t for an increased functional activity o f G A B A systems than simply increasing brain levels o f G A B A ( W o o d and Peesker, 1973; Collins, 1972). This initial inhibition o f G A D has been p r o p o s e d to be the cause o f the convulsions and death p r o d u c e d by very large doses o f A O A A ( W o o d and Peesker, 1973). This latter h y p o t h esis could explain the serendipitous finding during the present s t u d y that 30 m g / k g T R H administered 30 rain, rather than 60 rain, after injection of 75 m g / k g A O A A , results in a 93 % as o p p o s e d to a 33 % mortality rate (unpublished observation). The finding that T R H administration results in an enhanced release o f 3Hd o p a m i n e (DA) f r o m striatal synaptosomes (Horst and Spirt, 1974) is also consistent with the above hypothesis. Since G A B A - c a r r y i n g neurons originating in the caudate are reported to exert an inhibitory influence u p o n nigrostriatal D A neurons (Roberts, 1974; Carlsson, 1975; Hornykiewicz et al., 1976; D r a y and Straughan, 1976), inhibition o f the striato-nigral G A B A n e u r o n s by T R H might well result in an enhanced release o f D A due to disinhibition. F u r t h e r m o r e , the only other c o m p o u n d which has been f o u n d to closely resemble the characteristic behavioral effects o f T R H is intracisternally~ administered tubocurarine (Cott et al., 1976a). These same behavioral effects have recently been reported in cats receiving intraventricular injections o f tubocurarine by Beleslin and Samardzic (1976). Other recent studies have concluded that the m e c h a n i s m o f the behavioral stimulant effects o f intraventricularly administered tubocurarine are due to a n t a g o n i s m o f G A B A at the G A B A receptor (Hill et al., 1973; M c K e n z i e and Viik, 1974). In addition, m a n y reports indicating the presence of substantial a m o u n t s o f T R H in brain nerve endings outside the h y p o t h a t a m u s , especially the recent finding by H6kfelt et al. (1975) o f T R H in the nucleus accumbens suggests that e n d o g e n o u s T R H could be involved in the n o r m a l m o d u l a t i o n o f l o c o m o t o r activity. The high concentrations o f G A B A in the basal ganglia (Fahn, 1976) thus m a k e it anatomically possible for these systems to interact, as suggested by the present findings. Studies designed to determine the effects o f exogenously-administered T R H on G A B A metabolism would reveal the plausibility o f this hypothesis.

Psychopharmacology 52 (1977)

Acknowledgements'. This study was supported by grants no. 4247, 04P-4932, and 04R-4790K from the Swedish Medical Research Council. We are indebted to Abbott Laboratories, North Chicago, Illinois, for generous supply of TRH and to Ciba-Geigy, M61ndal, Sweden, for supply of baclophen.

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Received August 31, 1976; Final Version December 6, 1976