Modulation of dopamine receptors by thyrotropin-releasing hormone in the rat brain

Peptides, Vol. 8, pp. 31%325. ©Pergamon Journals Ltd., 1987. Printed in the U.S.A.

0196-9781/87 $3.00 + .00

Modulation of Dopamine Receptors by Thyrotropin-Releasing Hormone in the Rat Brain KUNIHIKO

S. F U N A T S U 1 A N D K A Z U T O Y O

INANAGA

Department o f Psychiatry, Kurume University School o f Medicine, Kurume 830, Japan R e c e i v e d 28 J u l y 1986 FUNATSU, K. S. AND K. INANAGA. Modulation of dopamine receptors by thyrotropin-releasing hormone in the rat brain. PEPTIDES 8(2) 319-325, 1987.--Nanomolar concentration of thyrotropin-releasing hormone (TRH) in vitro caused a significant reduction of ['~H]apomorphine binding sites (70% of the control) in the rat striatum and the limbic forebrain. [:3H]Spiperone binding was not affected by TRH. On the other hand, dopamine and apomorphine displaced [3H]TRH binding partially, suggesting the presence of a TRH receptor subpopulation that has a high affinity for dopamine agonist. Most of the neuroleptics displaced [3H]TRH binding dose-dependently in the micromolar range. (-)-Sulpiride had no affinity to TRH receptors. These findings suggest that one of the important roles of TRH as a neuromodulator is to modulate receptors for classical neurotransmitters, and this receptor-receptor interaction may be of importance in explaining the well known stimulating effects of TRH on the dopaminergic system. [:3H]Apomorphine binding [3H]Spiperone binding Receptor-receptor interaction

TRH receptors

APART from its neuroendocrine activity, there are reports indicating that thyrotropin-releasing hormone (TRH) [13,25] and TRH receptors [2, 7, 23, 24, 26, 35, 40] are widely distributed throughout the central nervous system. Altered TRH concentrations in post-mortem brain from patients with neuropsychiatric diseases [21,38] suggest that the hormone plays an important role in the human brain. However, whether TRH acts as a neurotransmitter has remained obscure. The recent finding that TRH, like other neuropeptides, coexists with classical neurotransmitters in neurons [10] could mean that it is a neuromodulator. It is well known that TRH facilitates the action of the central dopaminergic system. Plotnikoffet al. [28] found that TRH potentiates the behavioral effects of L-dopa in mice. Similar results have been reported by others [5,19]. One neurochemical finding is that TRH stimulates dopamine eftlux in several regions of the rat brain [5, 9, 12, 22, 34] and from the bovine retina [29]. It seems likely, therefore, that TRH evokes some kinds of behavioral changes in animals by stimulating release of dopamine. However this dopamine-releasing action of TRH may not be based on a simple mechanism, but rather complicated [29]. It is not known whether TRH modulates the dopaminergic system at the receptors. If TRH stimulates dopaminergic neurotransmission by affecting dopamine receptors, the following two possibilities are to be considered: (1) TRH may

Neuroleptics

prevent the action of presynaptic dopamine autoreceptors in such a way as to increase dopamine release. (2) TRH may stimulate postsynaptic O2L dopamine receptors directly, or TRH may somehow increase the sensitivity of postsynaptic D2L receptors. Involvement of D2L receptors should not be ignored, since some central effects of TRH in mice are enhanced by neuroleptics, potent antagonists for O2L receptors [20]. We then attempted to investigate whether TRH affects [3H]apomorphine binding or [aH]spiperone binding and, for comparison, whether dopaminergic agonists or antagonists affect [3H]TRH binding by performing radioligand binding studies. A possible interaction of dopamine receptors with TRH has been reported [29]. Additionally, substances such as 5-hydroxytryptamine (5-HT) [41], substance-P [32], and benzodiazepines [33,36] have affinity to TRH receptors, while receptors for such transmitters as acetylcholine [27] or 5-HT(5-HT1 receptor) [7] are increased by TRH. Therefore it must be fully discussed if the findings have any specificity and relevance. [3H]Apomorphine and [3H]spiperone were chosen since [3H]apomorphine binding sites (D3 dopamine sites, i.e., DIH sites, plus D4 sites, i.e., DzH sites according to the recent concept [14, 31, 43]) may involve presynaptic dopamine antoreceptors [18,30], though this has not yet been verified [4], and since a majority of dopaminergic [aH]spiperone binding sites (D2L receptors) represent postsynaptic dopamine recep-

1Requests for reprints should be addressed to Dr. K. S. Funatsu. Until October 1987: Physiologisch-Chemisches Institut, Universit/it Tiibingen, Hoppe-Seyler-Strasse 1, D-7400 Tiibingen, Federal Republic of Germany. Permanent address: Department of Psychiatry, Kurume University School of Medicine, Asahimachi 67, Kurume 830, Japan.

319

320

FUNATSU AND INANAGA

tors that are closely related to the schizophrenic disorders. Experiments were made mostly using the striatum and the limbic forebrain that are abundant in these dopamine receptors. METHOD

['~H]TRH Binding Fresh brains of adult male Wistar rats were used. The receptor-containing particulate fraction for [aH]TRH binding was prepared as we described previously [6]. Briefly the tissue collected from 5-6 rats was homogenized in 0.3 M sucrose solution. The homogenate was centrifuged at 1,100×g for 10 min, and the pellet was discarded. The supernatant was incubated at 37°C for 20 min and centrifuged at 30,000×g for 20 min. The supernatant was discarded, and the pellet (P2) was exposed twice to osmotic shock to break up the cells. Finally the membrane preparation was suspended in 50 mM Tris-HC1 buffer (pH 7.4). In the [3H]TRH binding assay, the membrane preparation was incubated with [3H]TRH (100 Ci/mmol, New England Nuclear, Boston, MA) and displacers in ice for 3 hr. Samples contained 0.02% ascorbic acid, 1 mM EDTA and 0.05% bovine serum albumin in 600/xl of Tris buffer. In the experiment with (-)-sulpiride, the incubation in the presence of 100 mM NaCI, 2 mM CaC12, 2 mM MgC12 and 100/zM MnCI2 was also performed. Incubation was stopped by filtrating 500 /,1 of the sample over Whatman GF/C glassfiber filter. The filter was rinsed twice with 10 ml of ice-cold Tris buffer, and the radioactivity trapped on the filter was counted by liquid scintillation spectrophotometry at the efficiency of 40%. Ten /zM TRH was used to determine nonspecific binding. Samples contained 0.2-0.5 mg of protein according to the method of Lowry et al. [16]. Determinations were made on triplicate samples. Standard deviations were less than 5% of the mean values.

[ZH]Apomorphine Binding The membrane fraction for [3H]apomorphine binding was prepared by homogenizing the tissue in 40 volumes (wt./vol.) of 50 mM Tris-HCl buffer (pH 7.4) that contained 0.01% ascorbic acid and 1 mM EDTA. The homogenate was incubated at 37°C for 15 min and centrifuged at 30,000×g for 15 min. The supernatant was discarded and the pellet was suspended either with Tris buffer or with Krebs-Ringer solution. When suspended with Tris buffer, TRH was added to half of the samples, and the samples were subjected to the binding. When suspended with Krebs-Ringer solution, TRH was added to half of the samples, and the samples were incubated at 37°C for 15 min under an oxygen atmosphere. TRH was then removed by centrifuging samples at 30,000×g for 15 min, and the pellet was resuspended with fresh 50 mM Tris buffer and subjected to the binding. [3H]Apomorphine binding was performed according to the method of Leysen and Gommeren [15] with slight modifications. Briefly, membrane preparation was incubated with [3H]apomorphine (27 Ci/mmol, New England Nuclear) and displacers at 25°C for 45 min in 600/zl of 50 mM Tris buffer that contained 0.01% ascorbic acid and 1 mM EDTA. Separation and counting of the membrane-bound [3H]apomorphine was performed as in [3H]TRH binding, but using GF/B filters. Nonspecific binding was determined using 10 /zM (+)-butaclamol.

[3H]Spiperone Binding Tissue was homogenized in 40 volumes (wt./vol.) of Tris buffer, incubated at 37°C for 15 min and centrifuged at 30,000 × g for 15 min. The pellet was suspended with Tris buffer. Membrane preparation was then incubated with [3H]spiperone (25 Ci/mmol, New England Nuclear) in 600/~1 of 50 mM Tris buffer at 25°C for 1 hr in the presence or absence of TRH. Samples contained 2 mM CaCI2, 2 mM MgCIz, 100 /~M MnCI2, 250 nM cinanserin and 100 nM pyrilamine. Separation and counting of the membrane-bound [3H]spiperone was performed as in [3H]apomorphine binding.

Regional Dissection of the Brain Dissection of the limbic forebrain, in which the nucleus accumbens, olfactory tubercle, septum and cingulate cortex are included as the limbic structure, was performed according to Horn and Phillipson [11].

Materials TRH (tartrate) was the generous gift of Takeda Chemical Industries (Osaka, Japan), and timiperone was donated by Daiichi Seiyaku (Tokyo, Japan). Other chemicals were purchased from commercial sources. RESULTS TO investigate the effect of TRH on [:~H]apomorphine and [3H]spiperone binding, 100 nM of TRH was used preliminarily. This concentration was chosen because dissociation constants (Kd) of central TRH receptors reported so far are between 2-130 nM when investigated with [:~H]TRH (see the Discussion section). Effect of TRH on [:~H]apomorphine binding was investigated with two different procedures. When 100 nM TRH was added to the samples at binding, the amount of the bound [3H]apomorphine reduced significantly to 71% that of the control (Fig. IA). When the effect of TRH was examined by adding 100 nM of the hormone to samples prior to the binding, [3H]apomorphine bound to the TRH-pretreated membrane decreased significantly to 70% that of the control, indicating that the effect of TRH remains even after the removal of free TRH (Fig. 1B, C). However TRH (100 nM-100 p.M) had no effect on [3H]spiperone binding when added to the samples at binding (Fig. ID, E). The effect of TRH at various concentrations on [3H]apomorphine binding was then investigated by adding TRH at the binding. Figure 2 shows that the TRH-effect can be observed even at the low nanomolar range of TRH, but becomes evident at 100 nM. Therefore further investigation was made of [3H]apomorphine saturation by adding I00 nM TRH to samples at the binding. As shown in Fig. 3, specific [:~H]apomorphine binding was saturable in the range of [:~H]apomorphine concentrations examined. The Scatchard plot fitted to the linear regression suggested the presence of a single population of binding sites. Binding parameters were Kd=0.7_+0.2 nM (mean-+SD, n=4), Bmax=30_+4 fmol/mg protein for the control, and Kd=0.6-+0.2 nM, Bmax= 19_+3 fmol/mg protein for the TRH-added group. The range of the concentrations of ['~H]apomorphine may not be wide enough. Because of the remarkable increase of nonspecific binding at higher concentrations, we took determinations at concentrations lower than 1.2 nM into account. The decrease of ['~H]apomorphine binding by TRH was constantly between

TRH MODULATION

OF DOPAMINE RECEPTORS

(A)

321

(B)

(C)

(DI

120 -

I00

~= so

I00 I-

~

80

= 60 f 40

~ 60 - 40

20

T

o

0

(E)

120

I Ctrl

20 0

TRHatE._1 binding J

CtrlTRHbefoL~r e binding I

I

J limbi¢ forebrain

Ctrl T R ~

Ctrl TRH~fo~re binding J

I

Ctrl TRH~at

binding I

I

I

binding I

I

I

limbic forebrain

striaturn

J

striatum

FIG. 1. Effect of TRH on [3H]apomorphine binding (A,B,C) and [:~H]spiperone binding (D,E). For A, D and E, effect of TRH was examined by adding 100 nM TRH to the sample at [3H]apomorphine binding (A) or ['~H]spiperone binding (D,E). For B and C, 100 nM TRH was added to the membrane preparation that was suspended with Krebs-Ringer solution, and incubated at 37°C for 15 min under an oxygen atmosphere. TRH was then removed by centrifugation, and the pellet was resuspended with fresh 50 mM Tris buffer and subjected to [aH]apomorphine binding. For A, B, C, the concentration of [:~H]apomorphine used is 0.8 nM. Controls obtained 1,000-1,500 dpm as total binding. Specific binding occupied 40-45% of the total binding. For D and E, concentration of [:3H]spiperone used is 0.4 nM. Control obtained approximately 2,000 dpm. Specific binding occupied 75-80% of the total binding. Values are means_SD of 4--5 triplicate experiments and shown as a percent of the control. *p<0.05, **p<0.01 vs. control (Student's paired t-test).

II0

6O

dp~ [

~

9o

®

c

80

~

70

o

E

60

~

5o

~

40 o

\

40

"

I

~ 400

O.

~

,,

0

30 -

o

~

[

//

I

I

l

I

-9

-8

-7

-6

Iog[TRH(M)]

FIG. 2. Effect of TRH at various concentrations on [3H]apomorphine binding. A membrane preparation was divided equally into several portions and incubated with TRH for [:~H]apomorphine binding. Concentration of ['~H]apomorphine was 0.6 nM. Data are mean_+SE of 3 determinations for the striatum (©) and unique values for the limbic forebrain (A) and shown as the percent of the control. Determinations were made on 3 samples. Statistical significance of the decrease in ['~H]apomorphine binding was calculated using Student's paired t-test. *p<0.05, **p<0.01.

20

0 0

10

20

30

40

B (fmol / mg protein) FIG. 3. Representative Scatchard plot of [:~H]apomorphine binding in the striatum, derived from the specific binding saturation curve (inset). One hundred nM TRH was added to half of the samples at the binding (A) and compared to the control (©). Values presented in the inset are means of triplicate samples of a representative experiment. The experiment was repeated four times with similar results. For binding parameters, see text.

322

F U N A T S U AND INANAGA

3 b

5

I""-.

(%) "0

80

•1-

60

~,

4o

rt" I-

4

2

v

100

=

0 .0

E

._~

~

v

o 0

m

20 0-

20

10

I

z/,,t'

I

-8

I

-7

I

-6

I

-5

0

10

log [displacer (M)]

A

a~

80

o-.- ~ , / /

O

--'~.i,~..~~

//.

i"m,

40

I"~,

50

I

60

70

FIG. 5. Representative Scatchard plot of [3H]TRH binding in the striatum, derived from the specific binding saturation curve. Ten /zM dopamine was added to half of the samples at the binding (A) and compared to the control (O). Values presented in the inset are means of triplicate samples of a representative experiment. The experiment was repeated four times with similar results. For binding parameters, see text.

_~ . - - - ~

. . . . . . .

I----

~\\\

\N

~O t-

-r n~

I

30

[+H]TRH bound (frnol / mg protein)

FIG. 4. Displacement of specific [3H]TRH binding by dopamine (O) and apomorphine (V) in the striatum. Membrane preparation was incubated at 0°C for 3 hr with 5 nM [3H]TRH and displacers in 50 mM Tris buffer containing 0.02% ascorbic acid, 1 mM EDTA and 0.05% bovine serum albumin. Total binding was approximately 1300 dpm. Nonspecific binding, determined with l0/~M TRH, was approximately 600 dpm. Data shown is the mean of 2-3 triplicate experiments.

100

I 20

I

0

-4

",

60

\

"%'-0..

40

\\o..

"r

20 0

/

I

I

-8

-7

I

log [ d i s p l a c e r

I

~--(~

-6

-5

-4

(M)]

FIG. 6. Displacement of specific [3H]TRH binding by TRH and neuroleptics in the striatum. Concentration of [aH]TRH used is 5 nM. Specific binding were 7001,000 dpm in each experiment, and occupied approximately 60% of the total binding. O - - - - O : TRH; O O: (+)-butaclamol; • • : chlorpromazine; A.--.A: spiperone; O - - - - © : timiperone; • ,': (-)-butaclamol; I . - - . I : (-)-sulpiride in the presence of Na+; [] IS]: (-)-sulpiride in the presence of ascorbic acid and EDTA.

60-70% at 0.8-1.2 nM. If the affinity was lowered, the decrease would have been greater at lower [3H]apomorphine concentrations. Therefore it seems likely that Bmax of the TRH-added group was reduced significantly (p<0.01, Student's paired t-test) to 63% that of the control. Decrease in Bmax rather than decrease in affinity indicates a noncompetitive type of inhibition. One possible interpretation of these results is that some TRH receptors are present near

[3H]apomorphine binding sites and that these receptors (binding sites) influence each other, resulting in a decrease in Bmax of [3H]apomorphine binding sites. To examine this possibility, a [3H]TRH binding study was performed. Affinity of dopamine agonists and antagonists to striatal TRH receptors was investigated. [3H]TRH was displaced with dopamine and apomorphine by approximately 25% (Fig. 4). Similar results were obtained in the limbic forebrain. We

TRH M O D U L A T I O N OF DOPAMINE RECEPTORS TABLE 1 DISPLACEMENT OF [aHITRH BINDING BY 10/~M (+)-BUTACLAMOL IN VARIOUS BRAIN REGIONS [3H]TRH B I N D I N G

Region

(% of control)

Striatum Limbic forebrain Frontal cortex Hypothalamus Hippocampus Amygdala

44 65 66 54 73 82

-4-+_ _+ _+ -+

4 4 6 16 5 9

Determination was made using 5 nM [3H]TRH. Values are means _+ SD of two triplicate experiments. Mean concentration of TRH receptors per 1 mg protein in the control were 17.5 fmol for the striatum, 16.0 fmol for the limbic forebrain, 14.4 fmol for the frontal cortex, 26.2 fmol for hypothalamus, 19.1 fmol for hippocampus and 31.4 fmol for amygdala.

then assumed the presence of a TRH receptor subpopulation (TRH-dopamine sites), and the dopamine agonists seem to have a moderate affinity for these sites, displacing [3H]TRH at higher nanomolar concentrations. [3H]TRH saturation was performed in the presence or the absence of l0 ~M dopamine in the striatum. Scatchard analysis indicated a single population of TRH receptors (Fig. 5). Dopamine reduced Bmax by 22% (p<0.05, n=4, Student's paired t-test) without altering the affinity (for control Kd--11.8_+4.6 nM, Bmax=37.5_+ 12.4 fmol/mg protein; for the dopamine-added group Kd=ll.3-+4.3 nM, Bmax=29.3-+10.5 fmol/mg protein, mean-+SD. [3H]TRH binding was displaced by micromolar concentrations of neuroleptics dose-dependently (Fig. 6). One exception was (-)-sulpiride, a selective antagonist for D2L receptors, that showed no displacement of [3H]TRH. The displacement by (-)-sulpiride was repeated in the presence of Na ÷, Ca +÷, Mg ++ and Mn ÷+ instead of EDTA plus ascorbic acid, since the binding of (-)-sulpiride to D2L receptors is highly dependent on these ions [42]. However [aH]TRH binding was scarcely (10%) displaced even in this assay condition. Binding of (+)-butaclamol to TRH receptors was investigated in various brain regions. As shown in Table 1, ['~H]TRH binding was displaced by 10 tzM (+)-butaclamol to 40-80% that of control in all regions investigated. DISCUSSION

We have classified rat central TRH receptors into three groups according to the affinity using ['~H]TRH [6]. High affinity TRH receptors have Kd of 2 nM whereas intermediate affinity receptors and low affinity receptors have Kd of approximately 10 and 70 nM respectively. Kd values obtained by other investigators using [3H]TRH are 5 and 130 nM [23] or 50 nM and 5 #M [2] in the rat brain and 36 nM in the sheep brain [3]. Therefore the inhibitory effect of 100 nM TRH on [3H]apomorphine binding in the present study is consistent with the idea that TRH and dopamine autoreceptors may have some functional interactions. The effect may be irreversible, as it remains after the possible dissociation of TRH from receptors by dilution (Fig. 1B, C). It seems likely that some [3H]apomorphine binding sites and some TRH re-

323 ceptors adjoin on the membrane because the effect of TRH is possibly noncompetitive and irreversible, and because a subpopulation of TRH receptors (TRH-dopamine sites) showed a high affinity to dopamine agonists. As approximately 50% of [3H]apomorphine binding sites (D1, sites) may be dopamine autoreceptors [18,31], some type of interaction between these receptors is suggested. We speculate that TRH increases the efflux of dopamine [5, 12, 34] by inhibiting the dopamine autoreceptors on the nerve ending, and this action causes the potentiation of the L-dopa-induced hyperactivity of the rat [5, 19, 28]. However additional studies are required to verify if DIH sites really reflect dopamine autoreceptors [4]. The interaction of TRH receptors and D2L receptors is, however, not unequivocal. [3H]Spiperone was not displaced by TRH. The order of affinity of neuroleptics to TRH receptors is different from that to D2L receptors. ICs0 values for TRH receptors are in the micromolar range, whereas they are in the nanomolar range for D2L receptors. Affinity of chlorpromazine to DZL receptors is approximately 100-fold weaker than that of spiperone [30]. Neuroleptics have stereospecificity for the binding to D2L receptors. (+)-Butaclamol is 100-10,000 times more potent than (-)-butaclamol in binding to D~L receptors [30]. However these affinity differences are not evidently observed at TRH receptors. (-)-Sulpiride, a potent antagonist for D2L receptors with a high selectivity [42], scarcely displaced [3H]TRH binding even in the presence of Na ÷ and other ions. Furthermore displacement of [3H]TRH binding by (+)-butaclamol was observed in all regions investigated, though D2L receptors are present preferentially in the striatum or the nucleus accumbens in the limbic forebrain [30]. These findings suggest that the binding of neuroleptics to TRH receptors is nonspecific and that there is no functional relation between TRH receptors and D2L receptors. Effects of TRH on the dopaminergic system were studied by others also. Reading [29] reported, using bovine retina, that dopamine has a nanomolar affinity to retinal TRH receptors, that spiperone has a micromolar affinity, but that ( - ) sulpiride is virtually inactive. [3H]Spiperone binding was not displaced by TRH. Reading therefore concluded that TRH probably regulates dopamine release through D3 (D1H) sites on the retinal membrane. Simasco and Weiland [37] observed that TRH does not affect [3H]spiperone binding in either the rat nucleus accumbens or the striatum even after administration for 7 consecutive days. These results are in good agreement with our findings. Our present findings suggest the possible interaction of some TRH receptors and some [3H]apomorphine binding sites, and this receptorreceptor interaction may be related to the mechanism of action of TRH to stimulate the dopaminergic system. It remains to be studied which type of TRH receptors is relevant as TRH-dopamine sites, as central TRH receptors are heterogenous and there may be at least three types according to the affinity [6]. Similar receptor-receptor interaction was observed between TRH receptors and 5-HT~ receptors [7,41], and these findings are in agreement with the known effect of TRH on the serotonergic system [8,17]. It seems likely that one of the actions of TRH is to modulate receptors for classical transmitters. A hypothesis has been proposed in this respect [1]. However the mode of receptor modulation may not be uniform. In the present study, the reduction of [3H]apomorphine binding sites appeared to be irreversible, whereas Simasco and Horita [36] observed using a method which was

324

FUNATSU AND INANAGA

essentially the same as ours that the binding of chlordiazepoxide on T R H receptors is reversible. It has not b e e n clear w h e t h e r T R H releases dopamine exclusively in the nucleus a c c u m b e n s or in the striatum as well [5, 12, 34]. In addition to the T R H depleting effect of d-amphetamine in the rat striatum [39], our present findings suggest the possible interaction of dopamine and T R H in

both regions. A question is why the contents of T R H and T R H receptors are small in the striatum, whereas dopamine and d o p a m i n e receptors are rich, if it is true. F u r t h e r studies should be m a d e to determine whether the decrease of [3H]apomorphine binding sites really reflects a decrease in presynaptic autoreceptors.

ACKNOWLEDGEMENTS The authors are grateful to Prof. Syogoro Nishi, Department of Physiology, Kurume University and Dr. Ehrenfried Mehl, GenTechnologisches Zentrum, Max-Planck-Institut for Biochemie, Martinsried/Munich, F.R.G., for helpful discussions and critical suggestions. The technical assistance of Ms. Hatsuko Shimokawa is greatly acknowledged.

REFERENECES 1. Agnati, L. F., K. Fuxe, M. Zoli, C. Rondanin and S.-O. Ogren. New vistas on synaptic plasticity: The receptor mosaic hypothesis of the engram. Med Biol 60: 183-190, 1982. 2. Burt, D. R. and S. H. Snyder. Thyrotropin releasing hormone (TRH): apparent receptor binding in rat brain membranes. Brain Res 93: 309-328, 1975. 3. Burt, D. R. and R. L. Taylor. Binding site for thyrotropinreleasing hormone in sheep nucleus accumbens resemble pituitary receptors. Endocrinology 106: 1416-1423, 1980. 4. Creese, I., D. R. Sibley, M. W. Hamblin and S. E. Leff. The classification of dopamine receptors: Relationship to radioligand binding. Annu Rev Neurosci 6: 43-71, 1983. 5. Fukuda, N., M. Miyamoto, S. Narumi, Y. Nagai, T. Shima and Y. Nagawa. Thyrotropin-releasing hormone (TRH): Enhancement of dopamine-dependent circling behavior and its own circling-inducing effect in unilateral striatal lesioned animals. Folia Pharmacol Jpn 75: 251-270, 1979. 6. Funatsu, K., S. Teshima and K. Inanaga. Various types of thyrotropin-releasing hormone receptors in discrete brain regions and the pituitary of the rat. J Neurochem 45: 390-397, 1985. 7. Funatsu, K., S. Teshima and K. Inanaga. Thyrotropin releasing hormone increases 5-hydroxytryptaminel receptors in the limbic brain of the rat. Peptides 6: 563-566, 1985. 8. Green, A. R. and D. G. Grahame-Smith. TRH potentiates behavioral changes following increased brain 5-hydroxytryptamine accumulation in rats. Nature 251: 524-526, 1974. 526, 1974. 9. Heal, D. J. and A. R. Green. Administration of thyrotropin releasing hormone (TRH) to rats releases dopamine in n. accumbens but not n. caudatus. Neuropharmacology 18: 23-31, 1979. 10. H6kfelt, T., O. Johansson, A. Ljungdahl, J. M. Lundberg and M. Schultzberg. Peptidergic neurons. Nature 284: 515-521, 1980. 11. Horn, A. S. and O. T. Phillipson. A noradrenaline sensitive adenylate cyclase in the rat limbic forebrain: Preparation, properties and the effects of agonists, adrenolytics and neuroleptic drugs. E u r J Pharmacol 37: 1-11, 1976. 12. Kerwin, R. W. and C. J. Pycock. Thyrotropin releasing hormone stimulates release of [aH]dopamine from slices of rat nucleus accumbens in vitro. Br J Pharmacol 67: 323-325, 1979. 13. Kubek, M. J., M. A. Lorincz and J. F. Wilber. The identification of thyrotropin releasing hormone (TRH) in hypothalamic and extrahypothalamic loci of the human nervous system. Brain Res 126: 196-200, 1977.

14. Left, S. E. and I. Creese. Interaction of dopaminergic agonists and antagonists with dopaminergic Da binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the DI dopamine receptor. Mol Pharmacol 27: 184-192, 1985. 15. Leysen, J. E. and W. Gommeren. Optimal conditions for [3H]apomorphine binding and anomalous equilibrium binding of [3H]apomorphine and [3H]spiperone to rat striatal membranes: Involvement of surface phenomena versus multiple binding sites. J Neurochem 36: 201-219, 1981. 16. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951. 17. Manaker, S., T. M. Engber, P. B. Knight and A. Winokur. Intraventricular 5,7-dihydroxytryptamine increases thyrotropin-releasing hormone content in regions of rat brain. J Neurochem 45: 1315-1318, 1985. 18. Meltzer, H. Y. Dopamine autoreceptor stimulation: Clinical significance. Pharmaeol Biochern Behav 17: Suppl 1, 1-10, 1982. 19. Miyamoto, M. and Y. Nagawa. Mesolimbic involvement in the locomotor stimulant action of thyrotropin-releasing hormone (TRH) in rats. Eur J Pharrnacol 44: 143-152, 1977. 20. Mora, S., A. Loizzo and V. G. Longo. Central effects of thyrotropin-releasing factor (TRF): Interaction with some antipsychotic drugs. Pharmacol Biochem Behav 4: 27%282, 1976. 21. Nemeroff, C. B., W. W. Youngblood, P. J. Manberg, A. J. Prange, Jr. and J. S. Kizer. Regional brain concentrations of neuropeptides in Huntington's chorea and schizophrenia. Science 221: 972-974, 1983. 22. Nielsen, J. A. and K. E. Moore. Thyrotropin-releasing hormone and its analog MK-771 increases the cerebroventricular perfusate content of dihydroxyphenylacetic acid. J Neurochem 43: 593-596, 1984. 23. Ogawa, N., Y. Yamawaki, H. Kuroda, I. Nukina, Z. Ota, M. Fujino and N. Yanaihara. Characteristics of thyrotropinreleasing hormone (TRH) receptors in the rat brain. Peptides 3: 66%677, 1982. 24. Parker, C. R., Jr. and A. Capdevila. Thyrotropin-releasing hormone (TRH) binding sites in adult human brain: Localization and characterization. Peptides 5: 701-706, 1984. 25. Parker, C. R., Jr. and J. C. Porter. Regional localization and subcellular compartmentalization of thyrotropin-releasing hormone in adult human brain. J Neurochem 41: 1614--1622, 1983.

TRH MODULATION OF DOPAMINE RECEPTORS 26. Pazos, A., C. Roser and J. M. Palacios. Thyrotropin-releasing hormone receptor binding sites: Autoradiographic distribution in the rat and guinea pig brain. J Neurochem 45: 1448-1463, 1985. 27. Pirola, C. J., M. S. Balda, S. Finkielman and V. E. Nahmod. Thyrotropin-releasing hormone increases the number of muscarinic receptors in the lateral septal area of the rat brain. Brain Res 273: 387-391, 1983. 28. Plotnikoff, N. P., A. J. Prange, Jr., G. R. Breese. M. S. Anderson and I. C. Wilson. Thyrotropin-releasing hormone: Enhancement of dopa activity by a hypothalamic hormone. Science 178: 417-418, 1972. 29. Reading, H. W. Dopamine receptors in bovine retina and their interaction with thyrotropin-releasing hormone. J Neurochem 41: 1587-1595, 1983. 30. Seeman, P. Brain dopamine receptors. Pharmacol Rev 32: 229-313, 1981. 31. Seeman, P., G. Ulpian, D. Grigoriadis, I. Pri-Bar and O. Buchman. Conversion of dopamine D1 receptors from high to low affinity for dopamine. Biochem Pharmacol 34: 151-154, 1985. 32. Sharif, N. A. and D. R. Burt. Micromolar substance P reduces spinal receptor binding for thyrotropin-releasing hormone-Possible relevance to neuropeptide coexistence? Neurosci Lett 43: 245-251, 1983. 33. Sharif, N. A. and D. R. Burt. Modulation of receptors for thyrotropin-releasing hormone by benzodiazepines: Brain regional differences. J Neurochem 43: 742-746, 1984. 34. Sharp, T., G. W. Bennett and C. A. Marsden. Thyrotropinreleasing hormone analogues increase dopamine release from slices of rat brain. J Neurochern 39: 1763-1766, 1982.

325 35. Simasco, S. M. and A. Horita. Characterization and distribution of [aH]-(3MeHis2)-thyrotropin releasing hormone receptors in rat brain. Life Sci 30: 1793-1799, 1982. 36. Simasco, S. and A. Horita. Chlordiazepoxide displaces thyrotropin-releasing hormone (TRH) binding. Eur J Pharmacol 98: 419-423, 1984. 37. Simasco, S. M. and G. A. Weiland. Effect of neurotensin, substance P and TRH on the regulation of dopamine receptors in rat brain. Eur J Pharmacol 106: 653-656, 1985. 38. Spindel, E. R., R. J. Wurtman and E. R. Bird. Increased TRH content of the basal ganglia in Huntington's disease. N Engl J Med 303: 1235-1236, 1980. 39. Spindel, E. R., D. J. Pettibone and R. J. Wurtman. Thyrotropin-releasing hormone (TRH) content of rat striatum: Modification by drugs. Brain Res 216: 323-331, 1981. 40. Taylor, R. L. and D. R. Butt. Species differences in the brain regional distribution of receptor binding for thyrotropin-releasing hormone. J Neurochem 38: 1649-1656, 1982. 41. Teshima, S., K. Funatsu, H. Satoh and K. Inanaga. 5-Hydroxytryptamine and (+)-lysergic acid diethylamide displace [3]thyrotropin-releasing hormone at its binding sites in the rat limbic forebrain. Regul Pept 14: 293-299, 1986. 42. Theodorou, A. E., P. Jenner and C. D. Marsden. Cation specificity of aH-sulpiride binding involves alteration in the number of striatal binding sites. Life Sci 32: 1243-1254, 1983. 43. Urwyler, S. and R. Markstein. Identification of dopamine " D 3 " and "D4" binding sites, labelled with [aH]2-amino-6,7-dihydroxy-l,2,3,4-tetrahydronaphthalene, as high agonist affinity states of the D1 and D2 dopamine receptors, respectively. J Neurochem 46: 1058-1067, 1986.