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The role of dopamine in regulation of thyrotropin-releasing hormone in the striatum and nucleus accumbens of the rat
Neuropeptides (1991) 19, 189-195 @ Longman Group UK Ltd 1991
The Role of Dopamine in Regulation of ThyrotropinReleasing Hormone in the Striatum and Nucleus Accumbens of the Rat E. PRZEGALIfiSKI,
L. JAWORSKA,
Institute of Pharmacology, request to EPI
R. KONARSKA
Polish Academy
and K. GOt!EMBIOWSKA
of Sciences, 12 Smetna Street, Krakdw, Poland (Reprint
Abstract-The effect of a-methyl-p-tyrosine lol-MT), FLA-63, amphetamine, apomorphine and quinpirole on the concentration of thyrotropin-releasing hormone (TRH) in the striatum and nucleus accumbens was studied in rats. It has been found that the TRH content was increased in both those structures after a-MT, an inhibitor of tyrosine hydroxylase which reduced the concentration of both dopamine (DA) and noradrenaline (NA), but not after FLA-63, an inhibitor of DA-P-hydroxylase which decreased the NA level without affecting DA. On the other hand, the indirectly acting dopaminomimetic amphetamine, the non-selective DA receptor agonist apomorphine, and the selective DZ receptor agonist quinpirole reduced the TRH level in the striatum, but not in the nucleus accumbens. Moreover, the decrease in the striatal peptide content induced by DA-mimetics was antagonized by the selective Dn-receptor antagonist sulpiride, but not by the selective D, receptor antagonist SCH 23390. The effect of amphetamine was not modified by the selective aI-adrenoceptor antagonist prazosin. These results indicate that DA and D2 receptors play a significant role in the regulation of the striatal TRH.
Introduction
Thyrotropin-releasing hormone (TRH) is widely distributed throughout the brain, including the hypothalamus and several extrahypothalamic structures (13, 25). As far as the hypothalamic TRH regulates thyrotropin (TSH) secretion from the anterior pituitary (lo), the extrahypothalamic
Date received 10 January 1991 Date accepted 15 February 1991
peptide has a neurotransmitter- or neuromodulator-like function (18, 25, 26). Irrespective of its hypophysiotropic activity, the exogenous TRH induces a variety of behavioural and neurochemical effects (5, 9, 12). Among others, it has been demonstrated that the peptide or its analogs can affect cholinergic (27), serotoninergic (1,17) and dopaminergic (6,8,11.17,20) neurotransmissions. On the other hand, information on the neurotransmitters which may affect extrahypothalamic 189
190
NEUROPEPTIDES
TRH neurons is still sparse. For example, we have recently found that fenfluramine increases the TRH content in the striatum, and that this effect is due to the release of 5-hydroxytryptamine (5-HT) and is mediated by 5-HT receptors (15). An opposite effect, i.e., a decrease in the striatal TRH concentration has been shown after amphetamine, and evidence has been presented that this effect requires an intact dopaminergic neurotransmission within the structure (22). At present we have obtained further evidence that dopamine (DA) and D2 receptors are involved in regulation of the striatal TRH.
Materials and Methods Animals
Male Wistar rats weighing 220-2608, purchased from licenced dealers, were used. The animals were housed at a room temperature (20-21 “C) on a 12:12h 1ight:dark cycle (the light on at 7a.m.) with food (granular standard diet, Bacutil) and water ad libitum. Experimental
protocol
The rats were treated with DL-cr-methyl-p-tyrosine methyl ester hydrochloride (Sigma; a-MT, 200mg/kg i.p.), bis(Cmethyl-l-homopiperazinyl(Lundbeck; FLA-63, thiocarbonyl)disulfide 25mg/kg i.p.), dexamphetamine sulphate (SK&F Lab. Ltd; lOmg/kg i.p.), apomorphine hydrochloride (Sigma; 5mg/kg i.p), quinpirole hydrochloride (Research Biochemicals Inc. ; 5mg/kg i.p.), R( +)-SCH 23390 hydrochloride (Research Biochemical Inc.; O.Zmg/kg i.p.), sulpiride (Sigma; 30 and 100 mg/kg i.p.), prazosin hydrochloride (Pfizer; 4mg s.c.) or vehicle, and were then killed by decapitation at different intervals after administration of the drugs. Their brains were quickly removed and dissected. The striatum and nucleus accumbens were immediately frozen on dry ice and stored at -70°C for TRH and catecholamine determinations. Determination
of TRH
Dried ethanol extracts of the brain structures collected from individual rats were used for determination of the TRH content by a radioimmuno-
assay method (RIA) as described previously (14, 15) using specific anti-TRH antibodies. The average sensitivity of the RIA was 4pg/tube. The interassay variation was 11%; the intraassay variation was 2.2%. The results are given as pg/mg of the wet tissue. Determination
of DA and NA
The DA and the noradrenaline (NA) content was determinated by a high pressure liquid chromatography (HPLC) with an electrochemical detection, according to the procedure described previously (4). The monoamines were quantified by peak height comparisons with the standards run on the day of analysis, with a sensitivity of lo-1OOpg. The results are given as rig/g of wet tissue Statistical analysis
All data are expressed as the mean f SEM. When only one drug was given (time-course effect), data were subjected to a one-way analysis of variance (ANOVA) followed by the Dunnett’s test. When two drugs were given, data were analysed by a 2 x 2 ANOVA followed by the Tukey’s test. Differences were considered to be significant when p < 0.05.
Results a-MT increased the TRH level in the striatum and nucleus accumbens. In the striatum a statistically significant effect (an increase by 60 and 97%, respectively) was observed 12 and 24h after administration of the drug (Table 1). In the nucleus accumbens the effect was weaker (an increase by 28-37%) and reached the level of significance 12-48h after a-MT administration (Table 1). At the same time a-MT reduced the concentration of DA, the maximum effect (reduction by 65% in the striatum and by 85% in the nucleus accumbens) having been observed 4h after its administration (Table 1). In the striatum the concentration of DA reached the control level 48h after administration of a-MT (Table 1)) whereas in the nucleus accumbens the DA content was still significantly reduced (by 27%) even 72 h after its administration (Table 1). u-MT also decreased the level of NA by
DOPAMINE IN REGULATION OFTHYROTROPIN-RELEASING
Table 1
HORMONE IN THE RAT
Time-course effect of a-methyl-p-tyrosine (a-MT; 200mg/kg i.p.) on TRH, dopamine (DA) and
noradrenaline parentheses
(NA) content in the rat striatum and nucleus accumbens.
TRH pdw
12h
6.92 f 0.39 (19) 9.47 + 0.85 (13) 10.48 + l.ll*
24h
(12) 12.96 f 1.56**
Oh 4h
control
72h
Oh 4h 12h 24h 48h 72h
8.87 f 1.06 (10) 7.74 ?I 0.69 (9) 41.53 * 2.05 (20) 45.52 + 3.75 (14) 53.69 + 2.59** (20) 51.41 f 2.63” (18) 50.21 + 3.49* (18) 51.14 f 4.20 (9)
Striatum 9825 * 680 (5) 3474 t 239** (6) 4116 + 894**
144 160* 197** 135 118
100
116
894 82” 87 110
(6) 2863 ? 545** (3) 2972 f 528** (5) 3775 f 1017**
128* 131
35**
Nucleus Accumbens 8488 + 556 (8) 1245 + 265**
132*
% of control
ngk
100
(4) 8030 f 293* (5) 8586 t 358 (4) 10791+611 (4)
137**
is given in
NA % of control
ndg
100
(14)
48h
The number of estimations
DA
% of
Time (4
191
(6) 6169 f 661* (4)
100
970 f 152
100
15**
(8) 767 f 273
79
34**
(6) 391 + 156**
40**
35**
(4) 536 +_ 136*
55%
44**
(6) 844 f 232
87
73*
(6) 900 f 180
93
(4)
*p < 0.05; **p < 0.001 Dunnett’s test following ANOVA
4560% in the nucleus accumbens 12-24h after its administration (Table 1). FLA-63 did not affect the concentration of TRH and dopamine in the striatum (Table 2) or nucleus accumbens (Table 2) of animals killed 4-24h after its administration. On the other hand, FLA-63 decreased the NA content in nucleus accumbens by 66-69% at 4-24h after its administration (Table 2). Amphetamine, apomorphine and quinpirole decreased the TRH level in the striatum (reduction by 26-41% at 2-8h after administration of amphetamine (Fig. 1); reduction by 31-44% at 1-2 h after apomorphine (Fig. 2)) and reduction by 23-24% at 2-4h after quinpirole (Fig. 3)), but not in the nucleus accumbens (Figs 1,2, 3).
OJ
61
.
i
4
a
24
lhrrl
Fig. 1 Time-course effect of amphetamine (lOmg/kg Lp.) on TRH content in the rat striatum and nucleus accumbens. The absolute values of TRH (pg/mg wet tissue): 6.79 f 0.42 (striatum) and 40.57 + 2.57 (nucleus accumbens). ‘p < 0.05; **p < 0,001 (Dunnett’s test following ANOVA). The number of estimations is given in parentheses
192
%
NEUROPEPTIDES o-o
STRIATUM.
A-A
N *CCUMBENS
of
Conlml
SCH (Fig. 5). Prazosin, sulpiride and SCH 23390
administered alone did not affect the striatal TRH level (results not shown). Discussion The data obtained in this report indicate that pharmacological manipulation of the brain DA system leads to modification of the TRH content in the striatum and/or nucleus accumbens in rats. In
251 OT,
, 012
. 4
8
’
24
iksl
Time-course effect of apomorphine (Smg/kg Lp.) on TRH content in the rat striatum and nucleus accumbens. The absolute values of TRH (pg/mg wet tissue): 6.86 + 0.38 (striatum) and 39.45 + 3.78 (nucleus accumbens). **p < 0.001 (Dunnett’s test following ANOVA). The number of estimations is given in parentheses. Fig. 2
The amphetamine-induced reduction in the TRH content in the striatum was blocked by lOOmg/kg of sulpiride, but not by a lower dose (30mg/kg) of the drug or prazosin (Fig. 4). The lower dose of sulpiridine (30mg/kg) antagonized the reduction in the striatal TRH content induced by apomorphine or quinpirole (Fig. 5). The effect of apomorphine or quinpirole was not affected by
Time-course effect of FLA-63 (25 mg/kg i.p.) on TRH, dopamine (DA) and noradrenaline in the rat striatum and nucleus accumbens. The number of estimations is given in parentheses
fh) Oh 4h 8h 24h
Oh 4h 8h 24h
PiYw
% of control
6.58 f 0.40 (5) 7.00 + 0.95 (7) 7.21 + 0.99 (7) 6.18 f 0.81 (7)
100
36.84 + (8) 34.86 f (7) 37.31 f (7) 29.95 f (7)
2.01
100
2.58
95
4.75
101
2.64
81
106 109 94
**p < 0.001 Dunnett’s test following ANOVA
N ACCUME~
% of
% of ndg
Striatum 9065 + 873 (4) 9668 f 697 (4) 9529 + 708 (4) 9360 + 769 (4)
(NA) content
NA
DA
TRH
STPIAWM
Fig. 3 Time-course effect of quinpirole (5mg/kg i.p.) onTRH content in the rat striatum and nucleus accumbens. The absolute values of TRH (pg/mg wet tissue): 6.78 + 0.32 (striatum) and 38.30 + 3.60 (nucleus accumbens). **p < 0.001 (Dunnett’s test following ANOVA). The number of estimations is given in parentheses.
Table 2
Time
o-o A-A
control
ngk
control
100 107 105 103
Nucleus Accumbens 100 8634 +_477 (8) 108 9354 + 1156 (6) 96 8246 f 633 (6) 97 8352 + 801 (6)
970 + 152 (8) 298 + 68”’ (6) 330 f 106** (5) 302 + 67** (6)
100 31** 34** 31**
DOPAMINE IN REGULATION OFTHYROTROPIN-RELEASING
HORMONE IN THE RAT
FLA-63, an inhibitor of DA+-hydroxylase (3) which decreases the NA content not affecting the DA one, changed the TRH concentration in neither the striatum nor the nucleus accumbens. In this respect our results differ from those reported by other authors. Actually, Kardon et al. (7) found that a-MT at the dosage as high as 200 + 3 x 150mg/kg did not induce any changes in the TRH content in the hypothalamus and several extrahypothalamic areas in rats. However, the latter authors did not measure the peptide level in the striatum or nucleus accumbens. Spindel et al. (1981) found that a-MT did not modify the striatal TRH concentration, but they administered the tyrosine hydroxylase inhibitor 4h prior to the sacrifice of the animals, whereas we have demonstrated that a significant increase in the peptide content in both the striatum and the nucleus accumbens takes place not earlier than 12 h after a-MT administration. It is also noteworthy that Spindel et al. (22) reported no changes in the striatal TRH following 6-hydroxydopamine (6OH-DA)-induced lesion of the substantia nigra, which may suggest that the nigro-striatal DA input is not involved in regulation of the peptide level in the striatum. On the other hand, Winokur et al. (24) found that intracisternal injection of 6-OH-DA induced a dramatic increase in the TRH content in the posterior cortex and a marked increase in the forebrain, but not in other brain structures in rats. However, the above effects were shown only after a subtoxic dose of 6-OH-DA (1000 @rat) and it is not clear whether it is due to destruction of DA and/or NA nerve terminals or to a non-specific effect of the neurotoxin. Nevertheless, our finding that a-MT increases the striatal TRH content may indicate that a similar effect observed after cerebral hemisection (22) results from the loss of the nigrostriatal DA input. The involvement of DA in regulation of the extrahypothalamic TRH is further suggested by the results that show that DA stimulants distinctly reduced the TRH content in the striatum, but not in the nucleus accumbens. Moreover, some of our data indicate that this effect is mediated by DZ receptors. Actually, the striatal TRH was diminished not only by the indirectly acting amphetamine, but also by the non-selective DA receptor agonist apomorphine and the selective DZ receptor
!Fjlij IillIll0 SAL SAL SUc?o) SAL AMW AMPH
SAL SAL SdlOd SAL AM31 AMi=tl
SAL SAL PRA SAL kWHMil31
Fig. 4 Effect of sulpiride (SUL; 30 or lOOmg/kg i.p.) and prazosin (PRA; 4mg/kg i.p.) on the decrease in TRH content induced by amphetamine (AMPH; lOmg/kg i.p.) in the rat striatum. The animals were killed 2h after AMPH. SUL and PRA were administered 1 h before AMPH. The absolute values of TRH (pg/mg wet tissue) in three control (SAL+SAL treated) groups were 6.61 + 0.31. 6.70 + 0.25, 6.75 + 0.22, respectively. **p < 0.01 vs control group; ttp < 0.01 vs AMPH group (Tukey’s test following ANOVA 2x2). The number of estimations is given in parentheses.
fact, a-MT, an inhibitor of tyrosine hydroxylase (21), elevated the TRH concentration in both those structures, whereas the dopaminomimetic drugs amphetamine, apomorphine and quinpirole reduced the peptide level in the striatum, but not in the nucleus accumbens. The effect of a-MT, which inhibits synthesis of both DA and NA, seems to result from the reduction in DA neurotransmission, since
Fig. 5 Effect of sulpiride (SUL; 30mg/kgi.p.) and SCH 23390 (SCH: 0.2mglkg i.p.) on the decrease in TRH content in the rat striatum after treatment with apomorphine (APO; 5mg/kg i.p.) or quinpirole (QUINP; Smg/kg ip.). The animals were killed 1 or 2h after APO or QUINP administration, respectively. SUL and SCH were administered 1 h before APO or QUINP. The absolute values of TRH (pg/mg wet tissue) in four control (SAL+SAL treated) groups were 6.61 + 0.31, 6.76 + 0.12, 6.44 + 0.26, 6.75 + 0.13, respectively. **p < 0.01 vs control group; tp < 0.05 vs APO group; ttp < 0.01 vs QUINP group (Tukey’s test following ANOVA 2x2). The number of estimations is given in parentheses.
193
194 agonist quinpirole (23). Furthermore, we also found that the effect of all the three DA stimulants was blocked by sulpiride, a selective DZ receptor antagonist (23), whereas the effect of apomorphine and quinpirole was not affected by the selective D1 receptor antagonist SCH 23390 (23). At the same time, the lack of antagonism of prazosin, a selective blocker of al-adrenoceptors (2), to the amphetamine-induced reduction in the striatal TRH indicates that NA or, strictly speaking, al-adrenoceptors are not involved in the effect of the latter drug which also releases NA besides DA. Interestingly, our results obtained with amphetamine are in firm agreement with those of Spindel et al. (22), who also found that the drug reduced the striatal TRH concentration and that its effect was blocked by a-MT, 6-OH-DA and the DA receptor antagonist haloperidol. The reason why DA stimulants do not affect - in contrast to the striatum - the TRH level in the nucleus accumbens, i.e., in the structure in which - like in the striatum - a-MT increased the peptide concentration, is not clear. A possible explanation is that stimulation of DA receptors in the nucleus accumbens, but not in the striatum, initiates a mechanism(s) which counteract the effect of DA stimulants. On the other hand, our finding that the a-MT-induced elevation in the TRH content in the nucleus accumbens is much lower than in the striatum - despite a more profound and longer lasting depletion of the DA level in the former structure - may also indicate that the peptide in the nucleus accumbens is less sensitive to DA regulation. Modification of the TRH content in brain structures may result from changes in the synaptic release or intracellular metabolism and synthesis of the peptide. In this respect it is noteworthy that DA and apomorphine have been found to increase the release of TRH from hypothalamic synaptosomes, this effect being blocked by DA receptor antagonists (19). In the light of these results the DA stimulant-induced reduction in the striatal TRH content demonstrated in the present study may be due to the increased release of the peptide. However, it remains to be found out whether DA stimulants increase TRH release in the striatum. Further studies are also necessary to explain the mechanism responsible for the increase in the
NEUROPEPTIDES
TRH content in the striatum and nucleus accumbens after a-MT. In conclusion, our results demonstrate that DA and D2 receptors participate in regulation of TRH in the striatum, as inhibition of the amine synthesis augments the peptide concentration in this structure, whereas indirect and direct DA agonists produce an opposite effect, which can be blocked by the selective DZ receptor antagonist. On the other hand, the role of DA in regulation of TRH in the nucleus accumbens is more ambiquous, since the peptide content in that structure was increased after a-MT, but was not modified by DA stimulants. At the same time our results provide further evidence that the striatum is the extra-hypothalamic structure in which TRH is most sensitive to pharmacological manipulations (14, 15, 16).
Acknowledgements This study was supported Academy of Sciences.
by Grant 06.03.3.4 of the Polish
References 1. Agarwal, R. A., Rastogi, R. B. and Singhal, R. L. (1977). Enhancement of locomotor activity and catecholamine and 5-hydroxytryptamine metabolism by thyrotropin releasing hormone. Neuroendocrinology 23: 236-247. 2. Cambridge, D., Davey, M. J. and Massingham, R. (1977). Prazosin, a selective antagonist of postsynaptic cy-adrenoceptors. Br. J. Pharmac. 59: 514-515P. 3. Corrodi, H., Fuxe, K., Hamberger, B., and Ljungdahl, A. (1970). Studies on central and peripheral noradrenaline neurons using a new dopamine-P-hydroxylase inhibitor. Eur. J. Pharm. 12: 145-155. 4. Golembiowska, K. (1990). Ipsapirone, a new anxiolytic drug, stimulates catecholamine turnover in various regions of the rat brain. Pol. J. Pharmacol. Pharm. 42: 143-150. 5. Horita, A., Kalivas, P. W. and Simasko, S. M. (1983). Thyrotropin releasing hormone (TRH): possible physiological functions not related to the neuroendocrine system. Rev. Pure Appl. Pharmacol. Sci. 4: 111-137. 6. Kalivas, P. W., Stanley, D. and Prange, Jr, A. J. (1987). Interaction between thyrotropin-releasing hormone and the mesolimbic dopamine system. Neuropharmacology 26: 33-38. 7. Kardon, F., Marcus, R. J., Winokur, A. and Utiger, R. D. (1977). Thyrotropin-releasing hormone content of rat brain and hypothalamus: results of endocrine and pharmacologic treatments. Endocrinology 100: 1604-1609. 8. Kenvin, R. W. and Pycock, C. J. (1979). Thyrotropin-releasing hormone stimulates release of [3H]dopamine from
DOPAMINE
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IN REGULATION
OFTHYROTROPIN-RELEASING
slices of rat nucleus accumbens in vitro. Br. J. Pharmac. 67: 323-328. Manberg, P. J., Nemeroff, C. B. and Prange, Jr. A. J. (1979). Thyrotropin-releasing hormone and amphetamine: a comparison of pharmacological profiles in animals. Prog. Neuro-Psychopharmacol. 3: 303-314. Martin, J. B.. Reichlin. S. and Brown, G. M. (1977). In: Davis, F.A Co. (ed) Clinical Neuroendocrinology. Philadelphia, Pa., p. 201-228. Miyamoto. H., Narumi, S., Nagai, Y., Shima. T. and Nagawa. Y. (1979). Thyrotropin-releasing hormone: hyperactivity and mesolimbic dopamine system in rats. Japan J. Pharmacol. 29: 335347. Nemeroff. C. B., Loosen, P. T.. Bissette, G.. Manberg, P. Y., Wilson, I. C.. Lipton, M. A. and Prange, Jr. A. J. (1979). Pharmaco-behavioural effects on thyrotropin-releasing hormone and neurotensin. Psychoneuroendocrinology 3: 279-310. Oliver, C. 1Eskay, R. L., Ben-Jonathan, N. and Porter, J. C. (1974). Distribution and concentration of TRH in the rat brain. Endocrinology 95: 540-546. Przegalinski. E. and Jaworska, L. (1990). The effect of repeated administration of antidepressant drugs on the thyrotropin-releasing hormone (TRH) content of rat brain structures. Psychoneuroendocrinology 15: 147-153. Przegaiiriski, E.. Jaworska, L. and Golembiowska, K. (1990). The effect of fenfluramine on the thyrotropin-releasing hormone (TRH) content in the rat brain structures and lumbar spinal cord. Neuropeptides 15: 195-200. Przegalinski, E.. Jaworska, L. and Konarska, R. (1990). Repeated treatment with amitriptyline or electroconvulsive shock does not affect thyrotropin-releasing hormone receptors in discrete rat brain structures. Neurosci. Lett. 115: 86-91. Rastogi. R. B.. Singhal. R. L. and Lapierre. Y. D. (1981). Effects of MK-771. a novel TRH analog, on brain dopa-
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IN THE RAT
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minergic and serotonergic systems. Eur. J. Pharmacol. 73: 307-312. 18. Renaud. L. P. and Martin, J. B. (1975). Thyrotropin releasing hormine (TRH): depressant action on central neuronal activity. Brain Res. 86: 150-154. 19. Schaeffer, J. M., Axelrod. J. and Brownstein, M. J. (1977). Regional differences in dopamine-mediated release of TRH-like material from synaptosomes. Brain Res. 138: 571-574. 20. Sharp, T.. Bennett, G. W. and Marsden. C. A. (1982). Thyrotropin-releasing hormone analogues increase dopamine release from slices of rat brain. J. Neurochem. 39: 1763-1766. 21. Spector. S., Sjoerdsma, A. and Undenfriend, S. (1965). Blockade of endogenous norepinerphine synthesis by a-methyl-tyrosine, an inhibitor of tyrosine hydroxylase. J. Pharmacol. Exp. Ther. 147: 86-95. 22. Spindel. E. R., Pettibone, D. J. and Wurtman, R. J. (1981). Thyrotropin-releasing hormone (TRH) content of rat striatum: modifications by drugs and lesions. Brain Res. 216: 323-331. 23. Stoof. J. C. and Kebabian, J. W. (1984). Two dopamine receptors: biochemistry, physiology and pharmacology. Life Sci. 35: 2281-2296. 24. Winokur, A., Kreider, M. S., Dugan, J. and Utiger, R. D. (1978). The effects of 6-hydroxydopamine on thyrotropinreleasing hormone in rat brain. Brain Res. 152: 203-208. 25. Winokur. A. and Utiger, R. D. (1974). Thyrotropin releasing hormone: regional distribution in rat brain. Science 185: 265-267. 26. Yarbrough, G. (1979). On the neuropharmacology of thyrotropin releasing hormone (TRH). Prog. Neurobiol. 12: 291-312. 27. Yarbrough, G. G. (1983). Thyrotropin releasing hormone and CNS cholinergic neurons. Life Sci. 33: 111-118.
The Role of Dopamine in Regulation of ThyrotropinReleasing Hormone in the Striatum and Nucleus Accumbens of the Rat E. PRZEGALIfiSKI,
L. JAWORSKA,
Institute of Pharmacology, request to EPI
R. KONARSKA
Polish Academy
and K. GOt!EMBIOWSKA
of Sciences, 12 Smetna Street, Krakdw, Poland (Reprint
Abstract-The effect of a-methyl-p-tyrosine lol-MT), FLA-63, amphetamine, apomorphine and quinpirole on the concentration of thyrotropin-releasing hormone (TRH) in the striatum and nucleus accumbens was studied in rats. It has been found that the TRH content was increased in both those structures after a-MT, an inhibitor of tyrosine hydroxylase which reduced the concentration of both dopamine (DA) and noradrenaline (NA), but not after FLA-63, an inhibitor of DA-P-hydroxylase which decreased the NA level without affecting DA. On the other hand, the indirectly acting dopaminomimetic amphetamine, the non-selective DA receptor agonist apomorphine, and the selective DZ receptor agonist quinpirole reduced the TRH level in the striatum, but not in the nucleus accumbens. Moreover, the decrease in the striatal peptide content induced by DA-mimetics was antagonized by the selective Dn-receptor antagonist sulpiride, but not by the selective D, receptor antagonist SCH 23390. The effect of amphetamine was not modified by the selective aI-adrenoceptor antagonist prazosin. These results indicate that DA and D2 receptors play a significant role in the regulation of the striatal TRH.
Introduction
Thyrotropin-releasing hormone (TRH) is widely distributed throughout the brain, including the hypothalamus and several extrahypothalamic structures (13, 25). As far as the hypothalamic TRH regulates thyrotropin (TSH) secretion from the anterior pituitary (lo), the extrahypothalamic
Date received 10 January 1991 Date accepted 15 February 1991
peptide has a neurotransmitter- or neuromodulator-like function (18, 25, 26). Irrespective of its hypophysiotropic activity, the exogenous TRH induces a variety of behavioural and neurochemical effects (5, 9, 12). Among others, it has been demonstrated that the peptide or its analogs can affect cholinergic (27), serotoninergic (1,17) and dopaminergic (6,8,11.17,20) neurotransmissions. On the other hand, information on the neurotransmitters which may affect extrahypothalamic 189
190
NEUROPEPTIDES
TRH neurons is still sparse. For example, we have recently found that fenfluramine increases the TRH content in the striatum, and that this effect is due to the release of 5-hydroxytryptamine (5-HT) and is mediated by 5-HT receptors (15). An opposite effect, i.e., a decrease in the striatal TRH concentration has been shown after amphetamine, and evidence has been presented that this effect requires an intact dopaminergic neurotransmission within the structure (22). At present we have obtained further evidence that dopamine (DA) and D2 receptors are involved in regulation of the striatal TRH.
Materials and Methods Animals
Male Wistar rats weighing 220-2608, purchased from licenced dealers, were used. The animals were housed at a room temperature (20-21 “C) on a 12:12h 1ight:dark cycle (the light on at 7a.m.) with food (granular standard diet, Bacutil) and water ad libitum. Experimental
protocol
The rats were treated with DL-cr-methyl-p-tyrosine methyl ester hydrochloride (Sigma; a-MT, 200mg/kg i.p.), bis(Cmethyl-l-homopiperazinyl(Lundbeck; FLA-63, thiocarbonyl)disulfide 25mg/kg i.p.), dexamphetamine sulphate (SK&F Lab. Ltd; lOmg/kg i.p.), apomorphine hydrochloride (Sigma; 5mg/kg i.p), quinpirole hydrochloride (Research Biochemicals Inc. ; 5mg/kg i.p.), R( +)-SCH 23390 hydrochloride (Research Biochemical Inc.; O.Zmg/kg i.p.), sulpiride (Sigma; 30 and 100 mg/kg i.p.), prazosin hydrochloride (Pfizer; 4mg s.c.) or vehicle, and were then killed by decapitation at different intervals after administration of the drugs. Their brains were quickly removed and dissected. The striatum and nucleus accumbens were immediately frozen on dry ice and stored at -70°C for TRH and catecholamine determinations. Determination
of TRH
Dried ethanol extracts of the brain structures collected from individual rats were used for determination of the TRH content by a radioimmuno-
assay method (RIA) as described previously (14, 15) using specific anti-TRH antibodies. The average sensitivity of the RIA was 4pg/tube. The interassay variation was 11%; the intraassay variation was 2.2%. The results are given as pg/mg of the wet tissue. Determination
of DA and NA
The DA and the noradrenaline (NA) content was determinated by a high pressure liquid chromatography (HPLC) with an electrochemical detection, according to the procedure described previously (4). The monoamines were quantified by peak height comparisons with the standards run on the day of analysis, with a sensitivity of lo-1OOpg. The results are given as rig/g of wet tissue Statistical analysis
All data are expressed as the mean f SEM. When only one drug was given (time-course effect), data were subjected to a one-way analysis of variance (ANOVA) followed by the Dunnett’s test. When two drugs were given, data were analysed by a 2 x 2 ANOVA followed by the Tukey’s test. Differences were considered to be significant when p < 0.05.
Results a-MT increased the TRH level in the striatum and nucleus accumbens. In the striatum a statistically significant effect (an increase by 60 and 97%, respectively) was observed 12 and 24h after administration of the drug (Table 1). In the nucleus accumbens the effect was weaker (an increase by 28-37%) and reached the level of significance 12-48h after a-MT administration (Table 1). At the same time a-MT reduced the concentration of DA, the maximum effect (reduction by 65% in the striatum and by 85% in the nucleus accumbens) having been observed 4h after its administration (Table 1). In the striatum the concentration of DA reached the control level 48h after administration of a-MT (Table 1)) whereas in the nucleus accumbens the DA content was still significantly reduced (by 27%) even 72 h after its administration (Table 1). u-MT also decreased the level of NA by
DOPAMINE IN REGULATION OFTHYROTROPIN-RELEASING
Table 1
HORMONE IN THE RAT
Time-course effect of a-methyl-p-tyrosine (a-MT; 200mg/kg i.p.) on TRH, dopamine (DA) and
noradrenaline parentheses
(NA) content in the rat striatum and nucleus accumbens.
TRH pdw
12h
6.92 f 0.39 (19) 9.47 + 0.85 (13) 10.48 + l.ll*
24h
(12) 12.96 f 1.56**
Oh 4h
control
72h
Oh 4h 12h 24h 48h 72h
8.87 f 1.06 (10) 7.74 ?I 0.69 (9) 41.53 * 2.05 (20) 45.52 + 3.75 (14) 53.69 + 2.59** (20) 51.41 f 2.63” (18) 50.21 + 3.49* (18) 51.14 f 4.20 (9)
Striatum 9825 * 680 (5) 3474 t 239** (6) 4116 + 894**
144 160* 197** 135 118
100
116
894 82” 87 110
(6) 2863 ? 545** (3) 2972 f 528** (5) 3775 f 1017**
128* 131
35**
Nucleus Accumbens 8488 + 556 (8) 1245 + 265**
132*
% of control
ngk
100
(4) 8030 f 293* (5) 8586 t 358 (4) 10791+611 (4)
137**
is given in
NA % of control
ndg
100
(14)
48h
The number of estimations
DA
% of
Time (4
191
(6) 6169 f 661* (4)
100
970 f 152
100
15**
(8) 767 f 273
79
34**
(6) 391 + 156**
40**
35**
(4) 536 +_ 136*
55%
44**
(6) 844 f 232
87
73*
(6) 900 f 180
93
(4)
*p < 0.05; **p < 0.001 Dunnett’s test following ANOVA
4560% in the nucleus accumbens 12-24h after its administration (Table 1). FLA-63 did not affect the concentration of TRH and dopamine in the striatum (Table 2) or nucleus accumbens (Table 2) of animals killed 4-24h after its administration. On the other hand, FLA-63 decreased the NA content in nucleus accumbens by 66-69% at 4-24h after its administration (Table 2). Amphetamine, apomorphine and quinpirole decreased the TRH level in the striatum (reduction by 26-41% at 2-8h after administration of amphetamine (Fig. 1); reduction by 31-44% at 1-2 h after apomorphine (Fig. 2)) and reduction by 23-24% at 2-4h after quinpirole (Fig. 3)), but not in the nucleus accumbens (Figs 1,2, 3).
OJ
61
.
i
4
a
24
lhrrl
Fig. 1 Time-course effect of amphetamine (lOmg/kg Lp.) on TRH content in the rat striatum and nucleus accumbens. The absolute values of TRH (pg/mg wet tissue): 6.79 f 0.42 (striatum) and 40.57 + 2.57 (nucleus accumbens). ‘p < 0.05; **p < 0,001 (Dunnett’s test following ANOVA). The number of estimations is given in parentheses
192
%
NEUROPEPTIDES o-o
STRIATUM.
A-A
N *CCUMBENS
of
Conlml
SCH (Fig. 5). Prazosin, sulpiride and SCH 23390
administered alone did not affect the striatal TRH level (results not shown). Discussion The data obtained in this report indicate that pharmacological manipulation of the brain DA system leads to modification of the TRH content in the striatum and/or nucleus accumbens in rats. In
251 OT,
, 012
. 4
8
’
24
iksl
Time-course effect of apomorphine (Smg/kg Lp.) on TRH content in the rat striatum and nucleus accumbens. The absolute values of TRH (pg/mg wet tissue): 6.86 + 0.38 (striatum) and 39.45 + 3.78 (nucleus accumbens). **p < 0.001 (Dunnett’s test following ANOVA). The number of estimations is given in parentheses. Fig. 2
The amphetamine-induced reduction in the TRH content in the striatum was blocked by lOOmg/kg of sulpiride, but not by a lower dose (30mg/kg) of the drug or prazosin (Fig. 4). The lower dose of sulpiridine (30mg/kg) antagonized the reduction in the striatal TRH content induced by apomorphine or quinpirole (Fig. 5). The effect of apomorphine or quinpirole was not affected by
Time-course effect of FLA-63 (25 mg/kg i.p.) on TRH, dopamine (DA) and noradrenaline in the rat striatum and nucleus accumbens. The number of estimations is given in parentheses
fh) Oh 4h 8h 24h
Oh 4h 8h 24h
PiYw
% of control
6.58 f 0.40 (5) 7.00 + 0.95 (7) 7.21 + 0.99 (7) 6.18 f 0.81 (7)
100
36.84 + (8) 34.86 f (7) 37.31 f (7) 29.95 f (7)
2.01
100
2.58
95
4.75
101
2.64
81
106 109 94
**p < 0.001 Dunnett’s test following ANOVA
N ACCUME~
% of
% of ndg
Striatum 9065 + 873 (4) 9668 f 697 (4) 9529 + 708 (4) 9360 + 769 (4)
(NA) content
NA
DA
TRH
STPIAWM
Fig. 3 Time-course effect of quinpirole (5mg/kg i.p.) onTRH content in the rat striatum and nucleus accumbens. The absolute values of TRH (pg/mg wet tissue): 6.78 + 0.32 (striatum) and 38.30 + 3.60 (nucleus accumbens). **p < 0.001 (Dunnett’s test following ANOVA). The number of estimations is given in parentheses.
Table 2
Time
o-o A-A
control
ngk
control
100 107 105 103
Nucleus Accumbens 100 8634 +_477 (8) 108 9354 + 1156 (6) 96 8246 f 633 (6) 97 8352 + 801 (6)
970 + 152 (8) 298 + 68”’ (6) 330 f 106** (5) 302 + 67** (6)
100 31** 34** 31**
DOPAMINE IN REGULATION OFTHYROTROPIN-RELEASING
HORMONE IN THE RAT
FLA-63, an inhibitor of DA+-hydroxylase (3) which decreases the NA content not affecting the DA one, changed the TRH concentration in neither the striatum nor the nucleus accumbens. In this respect our results differ from those reported by other authors. Actually, Kardon et al. (7) found that a-MT at the dosage as high as 200 + 3 x 150mg/kg did not induce any changes in the TRH content in the hypothalamus and several extrahypothalamic areas in rats. However, the latter authors did not measure the peptide level in the striatum or nucleus accumbens. Spindel et al. (1981) found that a-MT did not modify the striatal TRH concentration, but they administered the tyrosine hydroxylase inhibitor 4h prior to the sacrifice of the animals, whereas we have demonstrated that a significant increase in the peptide content in both the striatum and the nucleus accumbens takes place not earlier than 12 h after a-MT administration. It is also noteworthy that Spindel et al. (22) reported no changes in the striatal TRH following 6-hydroxydopamine (6OH-DA)-induced lesion of the substantia nigra, which may suggest that the nigro-striatal DA input is not involved in regulation of the peptide level in the striatum. On the other hand, Winokur et al. (24) found that intracisternal injection of 6-OH-DA induced a dramatic increase in the TRH content in the posterior cortex and a marked increase in the forebrain, but not in other brain structures in rats. However, the above effects were shown only after a subtoxic dose of 6-OH-DA (1000 @rat) and it is not clear whether it is due to destruction of DA and/or NA nerve terminals or to a non-specific effect of the neurotoxin. Nevertheless, our finding that a-MT increases the striatal TRH content may indicate that a similar effect observed after cerebral hemisection (22) results from the loss of the nigrostriatal DA input. The involvement of DA in regulation of the extrahypothalamic TRH is further suggested by the results that show that DA stimulants distinctly reduced the TRH content in the striatum, but not in the nucleus accumbens. Moreover, some of our data indicate that this effect is mediated by DZ receptors. Actually, the striatal TRH was diminished not only by the indirectly acting amphetamine, but also by the non-selective DA receptor agonist apomorphine and the selective DZ receptor
!Fjlij IillIll0 SAL SAL SUc?o) SAL AMW AMPH
SAL SAL SdlOd SAL AM31 AMi=tl
SAL SAL PRA SAL kWHMil31
Fig. 4 Effect of sulpiride (SUL; 30 or lOOmg/kg i.p.) and prazosin (PRA; 4mg/kg i.p.) on the decrease in TRH content induced by amphetamine (AMPH; lOmg/kg i.p.) in the rat striatum. The animals were killed 2h after AMPH. SUL and PRA were administered 1 h before AMPH. The absolute values of TRH (pg/mg wet tissue) in three control (SAL+SAL treated) groups were 6.61 + 0.31. 6.70 + 0.25, 6.75 + 0.22, respectively. **p < 0.01 vs control group; ttp < 0.01 vs AMPH group (Tukey’s test following ANOVA 2x2). The number of estimations is given in parentheses.
fact, a-MT, an inhibitor of tyrosine hydroxylase (21), elevated the TRH concentration in both those structures, whereas the dopaminomimetic drugs amphetamine, apomorphine and quinpirole reduced the peptide level in the striatum, but not in the nucleus accumbens. The effect of a-MT, which inhibits synthesis of both DA and NA, seems to result from the reduction in DA neurotransmission, since
Fig. 5 Effect of sulpiride (SUL; 30mg/kgi.p.) and SCH 23390 (SCH: 0.2mglkg i.p.) on the decrease in TRH content in the rat striatum after treatment with apomorphine (APO; 5mg/kg i.p.) or quinpirole (QUINP; Smg/kg ip.). The animals were killed 1 or 2h after APO or QUINP administration, respectively. SUL and SCH were administered 1 h before APO or QUINP. The absolute values of TRH (pg/mg wet tissue) in four control (SAL+SAL treated) groups were 6.61 + 0.31, 6.76 + 0.12, 6.44 + 0.26, 6.75 + 0.13, respectively. **p < 0.01 vs control group; tp < 0.05 vs APO group; ttp < 0.01 vs QUINP group (Tukey’s test following ANOVA 2x2). The number of estimations is given in parentheses.
193
194 agonist quinpirole (23). Furthermore, we also found that the effect of all the three DA stimulants was blocked by sulpiride, a selective DZ receptor antagonist (23), whereas the effect of apomorphine and quinpirole was not affected by the selective D1 receptor antagonist SCH 23390 (23). At the same time, the lack of antagonism of prazosin, a selective blocker of al-adrenoceptors (2), to the amphetamine-induced reduction in the striatal TRH indicates that NA or, strictly speaking, al-adrenoceptors are not involved in the effect of the latter drug which also releases NA besides DA. Interestingly, our results obtained with amphetamine are in firm agreement with those of Spindel et al. (22), who also found that the drug reduced the striatal TRH concentration and that its effect was blocked by a-MT, 6-OH-DA and the DA receptor antagonist haloperidol. The reason why DA stimulants do not affect - in contrast to the striatum - the TRH level in the nucleus accumbens, i.e., in the structure in which - like in the striatum - a-MT increased the peptide concentration, is not clear. A possible explanation is that stimulation of DA receptors in the nucleus accumbens, but not in the striatum, initiates a mechanism(s) which counteract the effect of DA stimulants. On the other hand, our finding that the a-MT-induced elevation in the TRH content in the nucleus accumbens is much lower than in the striatum - despite a more profound and longer lasting depletion of the DA level in the former structure - may also indicate that the peptide in the nucleus accumbens is less sensitive to DA regulation. Modification of the TRH content in brain structures may result from changes in the synaptic release or intracellular metabolism and synthesis of the peptide. In this respect it is noteworthy that DA and apomorphine have been found to increase the release of TRH from hypothalamic synaptosomes, this effect being blocked by DA receptor antagonists (19). In the light of these results the DA stimulant-induced reduction in the striatal TRH content demonstrated in the present study may be due to the increased release of the peptide. However, it remains to be found out whether DA stimulants increase TRH release in the striatum. Further studies are also necessary to explain the mechanism responsible for the increase in the
NEUROPEPTIDES
TRH content in the striatum and nucleus accumbens after a-MT. In conclusion, our results demonstrate that DA and D2 receptors participate in regulation of TRH in the striatum, as inhibition of the amine synthesis augments the peptide concentration in this structure, whereas indirect and direct DA agonists produce an opposite effect, which can be blocked by the selective DZ receptor antagonist. On the other hand, the role of DA in regulation of TRH in the nucleus accumbens is more ambiquous, since the peptide content in that structure was increased after a-MT, but was not modified by DA stimulants. At the same time our results provide further evidence that the striatum is the extra-hypothalamic structure in which TRH is most sensitive to pharmacological manipulations (14, 15, 16).
Acknowledgements This study was supported Academy of Sciences.
by Grant 06.03.3.4 of the Polish
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