Thyrotropin releasing hormone increases 5-hydroxytryptamine1 receptors in the limbic brain of the rat

Peptides, Vol. 6, pp. 563-566, 1985. 'z; Ankho International Inc. Printed in the U.S.A.

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Thyrotropin Releasing Hormone Increases 5-Hydroxytryptamine Receptors in the Limbic Brain of the Rat KUNIHIKO

FUNATSU,

SHIGEKI TESHIMA

AND KAZUTOYO

INANAGA

D e p a r t m e n t o f Psychiatry, K u r u m e University School o f Medicine, A s a h i m a c h i 67, K u r u m e 830, Japan R e c e i v e d 8 N o v e m b e r 1984 FUNATSU, K., S. TESHIMA AND K. INANAGA. Thyrotropin releasing hormone increases 5-hydroxytrvptaminel receptors in the limbic brain of the rat. PEPTIDES 6(3) 563-566, 1985.--The effect of TRH on 5-HT, receptors in the rat brain was investigated. A crude membrane preparation was incubated at 37°C for 15 rain with or without TRH prior to [:~H]5-HT binding assay. TRH at 100 nM increased the number of 5-HT~ receptors significantly (approximately 20%) in the limbic forebrain and the hippocampus without altering their affinity. As this concentration of TRH is close to its dissociation constant (2 nM and 51 nM in the limbic forebrain, 11 nM in the hippocampus), this effect is probably of physiological relevance. This finding seems to support a pharmacological finding of others that the anti-convulsion effect of TRH may be related to increased serotonergic transmission. 5-HT, receptor

TRH

Limbic forebrain

Hippocampus

W H I L E thyrotropin releasing hormone (TRH) has stimulatory effects on dopaminergic transmission in the rat [15], it has been also suggested that the hormone modulates the central serotonergic system. Barbeau and Bedard [2] observed that TR H stimulates 5-hydroxytryptamine (5-HT) receptors in the rat spinal cord. The anti-convulsion effect of TRH, measured as the antagonistic effect against the reduction of the electroconvulsive threshold induced by reserpine in mice, seems to be due to the stimulation of central serotonergic transmission, since the effect has been attenuated by methysergide or p-chlorophenylalanine [10]. The coexistence of T R H and 5-HT in the same neuron of the rat medulla oblongata [7] also suggests a possible interaction between these substances. There are several reports on the modulation of central 5-HT~ receptor. 5-HT~ receptors increase in number, but decrease in their affinity in the rat hippocampus after incubation with vasoactive intestinal peptide (VIP) [17]. The increase of 5-HT~ receptors also occurs after the addition of substance P to rat spinal cord homogenate. These findings encouraged us to investigate the effect of TR H on the modulation of 5-HTt receptors in discrete brain regions of the rat.

coronal cuts at the chiasma opticum and at 2 mm further rostral. In the rest of this slab accumbens nuclei, olfactory tubercles, septi and cingulate cortices were included. The cortex rostral of the limbic forebrain was used as the frontal cortex. The median portion of the brain stem which contained raphe nuclie was dissected out according to the method of Deguchi et al. [3]. The tissue collected from 5-6 rats was homogenized in 40 vol w/v 0.3 M sucrose solution with Biotron Ultradisperser and a Teflon homogenizer. The supernatant after centrifugation of the homogenate at 1,100x g was incubated at 37°C for 15 min, and centrifuged at 30,000xg for 20 min. The supernatant was discarded. The pellet was resuspended in 50 mM Tris/HC1 buffer, pH 7.4, and kept at - 6 0 °C until use. After thawing, the membrane preparation was divided into several portions which each contained 5-8 mg protein according to the method of Lowry et al. [9]. Each of the divided membrane preparations was diluted five-fold with Krebs-Ringer solution, bacitracin (20 ~M) was added, and the preparations were incubated at 37°C for 15 min in the presence or absence of TRH under an oxygen atmostphere. The incubation was stopped by cooling the membrane preparation in ice. Samples were centrifuged at 30,000xg for 10 min. The pellet was resuspended with Tris buffer and used for the [:~H]5-HT binding assay. In the binding assay, the membrane preparations were incubated at 25°C for 60 min with various concentrations of [:~H]5-HT (26.3 Ci/mmol, N E N , Boston, MA). Samples contained 2 mM CaCI2, 2 mM MgC1.., an~l 25/zM pargyline in 600 p~l of Tris buffer. Incubation was terminated by filtration of samples over Whatman GF/B glass fiber filters under vac-

METHOD Fresh brains obtained from adult male Wistar rats were dissected on ice. The limbic forebrain was dissected out as described by Horn and Phillipson [8]. Briefly, the corpora striata, a major part of the cortex and the corpus callosum were removed from the disc which was obtained by two

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FIG. I. Effect of TRH at various concentrations on [:~H]5-HTbinding. A membrane preparation was divided equally into several portions and incubated with various concentrations of TRH at 37°C for 15 min in bacitracin-containing Krebs-Ringer solution. After the removal of TRH by centrifugation, the resuspended membrane preparation was used for the [:~H]5-HTbinding assay as indicated in the text. The concentration of [:~H]5-HTis 2 nM. Regions examined are the limbic forebrain (O C3), hippocampus (A. . . . A), amygdala ( ~ ' - - - - - - T ) and the brain stem (C~--D). Data are shown as the percent of the control. Each point represents mean+S.E.M, of three (hippocampus, amygdala, brain stem) or four (limbic forebrain) triplicate determinations. Statistical significance of the percent increase was calculated using a one-factor analysis of variance. F values are: F(5,18)=6.89, p<0.01 for the limbic forebrain, F(5,12)=8.36, p<0.01 for the hippocampus. At each concentration of TRH, statistical significance of the increase was calculated using Student's paired t-test. *p<0.05.

uum. After the washing of the f'dters with 2× 10 ml of ice-cold Tris buffer, the radioactivity of the membrane-bound tall] 5-HT trapped on the filter was counted using liquid scintillation spectrophotometry at the efficiency of 40%. Specific [:~H]5-HT binding was defined as the excess over blanks taken in the presence of 10 tzM d-lysergic acid diethylamide (d-LSD). RESULTS

It was confirmed in preliminary experiments that TRH itself does not bind to [:~H]5-HT binding sites. Under our experimental condition, 5-HT, receptors are labeled as previously described [12,13]. The effect of TRH at various concentrations on [3H]5-HT binding is shown in Fig. 1. In the limbic forebrain, 10 nM TRH increased [3H]5-HT binding by a significant 17%. The increases were of similar degree at TRH concentrations up to 100 tzM. Hippocampal [:~H]5-HT binding was also increased. In this region the increase was somewhat dose-dependent. A significant increase was obtained with 100 nM TRH. However ['~H]5-HT binding was not influenced by TRH in the brain stem and the amygdala. The effect of 100 nM TRH on 5-HT, receptors in the limbic forebrain and the hippocampus was further investigated with [:~H]5-HT saturation experiment (Fig. 2). The saturation experiment was carried out for all other regions using 100/zM TRH, because such an interaction on a receptor may be of physiological relevance even at micromolar concentrations [19]. Results were analysed with Scatchard plot. The plot was fitted by non-linear regression in all regions examined, suggesting that multiple 5-HT, receptors were present, and

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[:tH]5-HT binding in the limbic forebrain. The membrane preparation was incubated at 37°C for 15 min in the presence (~;-- -(J) or the absence (& &) of 100 nM TRH in bacitracin-containing Krebs-Ringer solution prior to [:~H]5-HTbinding assay. Nonspecific binding ( O - - O ) was determined using 10/zM d-LSD. Curves were determined with 5 or 7 points. A typical result with 7 points is shown. Points are mean values of triplicate samples. The experiment for the limbic forebrain was repeated four times.

was further analysed to two populations of binding sites according to the graphic method of Rosenthai [161. One reason for the multiple 5-HT, receptors is probably that we used a crude membrane fraction as the receptor preparation. Fillon et al. [4] obtained a single population of high affinity [:~H]5-HT binding sites using a purified synaptosomal membrane preparation, whereas they obtained multiple [:~H]5-HT binding sites using crude membrane preparations. Multiplicity of central 5-HT~ receptors has been reported by other investigators. Gozlan et al. [6] indicate that [:~H]5-HT is not so selective as [:~Hl8-hydroxy-2-(di-n-propylamino)tetralin for labeling of 5-HT, receptors. It was shown by Pedigo et al. [I 11 and Schnellmann et al. 118] that 5-HTt receptors labeled by [3HI5-HT can be classified into subgroups according to the affinity to neuroleptics. The curvilinear Scatchard plots in our result may reflect, therefore. multiple 5-HT, receptors that have been reported by these authors. Results are summarized in Table 1. TRH (100 nM) caused a significant increase of approximately 2(~Voin the number of high and low affinity binding sites in the limbic forebrain and the hippocampus. The number of low affinity [:~H]5-HT binding sites in the frontal cortex increased significantly when 100/xM TRH was applied. None of these increases in the number of binding sites was accompanied by a significant change in the affinity. Neither the number of binding sites nor the affinity of [:~H]5-HT binding was altered in the brain stem, in which raphe nuclei were contained, and the same was true in the amygdala, the region which is richest in 5-HT, receptors and TRH receptors [5].

E F F E C T O F T R H ON 5-HTI R E C E F F O R

565

TABLE 1 EFFECT OF TRH ON 5-HT, RECEPTORS IN BRAIN REGIONS High affinity ['~H]5-HT binding sites Region

control

TRH-added

Low affinity [:'H]5-HT binding sites control

limbic forebrain hippocampus

Kd Bmax Kd Bmax

0.54+0.04 163+17 0.58-+0.10 88_+ 8

0.56_+0.05 195-+21t 0.63-+0.13 108_+ 8*

16.3-+ 468+ 14.3_+ 425_+

frontal cortex hypothalamus

Kd Bmax Kd Bmax Kd Bmax Kd Bmax Kd Bmax Kd Bmax

0.45_+0.08 101-+27 0.51 _+0.11 101 _+12 0.45_+0.15 70_+10 0.45-+0.30 47_+ 17 0.67-+0.11 87+26 0.24_+0.01 57_+ 7

0.49+0.04 109_+27 0.52_+0.10 103-+ 103 4 0.50_+0.18 74_+ 3 0.42-+0.26 44_+ 16 0.70-+0.08 90_+25 0.32+0.06 60_+ 8

9.3-+ 1.2 350 + 45 30.4-+ 7.9 645_+ 42 24.0+ 4.1 610-+ 91 21.7_+ 17.2 423+ 198 27.0-+ 10.7 834+ 154 12.2+ 2.5 302_+ 89

thalamus striatum amygdala brain stem

2.2 63 3.2 17

TRH-added 18.8 + 565-+ 14.6_+ 501_+

4.2 77* 3.0 30*

I 1.3-+ 0.9 428_+ 36* 30.9 + 7.2 691 _+154 27.1 + 3.0 680+ 81 20.7_+ 11.3 465±205 29.2+ 14.9 938_+335 10.2+ 0.4 319_+ 86

The concentrations of TRH examined are 100 nM for the limbic forebrain and the hippocampus, and 100/xM for other regions. The dissociation constant (Kd) and the number of [:~H]5-HT binding sites (Bmax) were determined from Scatchard plots. Kd is expressed in nM, and Bamx in fmoles per mg protein. Data are mean + S.E.M. of 3-5 experiments. In each experiment determinations were made on 3 samples. Statistical significance was calculated using Student's paired t-test. *p<0.05: tp<0.01.

DISCUSSION T h e r e are s e v e r a l r e p o r t s on in vitro i n t e r a c t i o n involving T R H r e c e p t o r s or 5-HT r e c e p t o r s [1, 14, 17, 19]. H o w e v e r t h e r e is no report so far on the m o d u l a t i o n o f 5-HT, r e c e p t o r by T R H in the brain. In our results, the m o d u l a t i o n o f 5-HT~ r e c e p t o r s o c c u r r e d with n a n o m o l a r c o n c e n t r a t i o n s o f T R H e x c l u s i v e l y in the limbic brain and the h i p p o c a m p u s . Dissociation c o n s t a n t s o f T R H r e c e p t o r s are 2 n M and 51 nM in the limbic f o r e b r a i n and 11 n M in the h i p p o c a m p u s [5]. T h e r e f o r e the i n c r e a s e in the n u m b e r o f 5-HT1 r e c e p t o r s c a u s e d by 100 n M T R H in t h e s e regions is likely to b e o f physiological r e l e v a n c e . T R H s e e m s to act in a facilitating the s e r o t o n e r g i c t r a n s m i s s i o n w h i c h is m e d i a t e d via 5-HTI

r e c e p t o r s in t h e s e regions. This finding s u p p o r t s the pharmacological o b s e r v a t i o n o f Nagai et al. [10] that the antic o n v u l s i o n effect o f T R H may be related to the stimulation o f the s e r o t o n e r g i c s y s t e m . T h e m e c h a n i s m o f the modulation is not clear. W h e t h e r the m o d u l a t i o n o c c u r s t h r o u g h a direct i n t e r a c t i o n o f T R H r e c e p t o r s and 5-HT~ r e c e p t o r s o r t h r o u g h an indirect m e c h a n i s m is yet to be clarified. ACKNOWLEDGEMENT The authors are grateful to Dr. H. Uchimura, Director of Hizen National Mental Hospital, Kanzaki/Saga, Japan and Dr. Ehrenfried Mehl, Max-Planck-lnstitut for Psychiatrie, Martinsried, F.R.G., for helpful discussions and critical suggestions.

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4. Fillon, G. M. B., J.-C. Rousselle, M.-P. Fillon, D. M. Beaudoin, M. R. Goiny, J.-M. Demiau and J. J. Jacob. High-affinity binding of [:~H]5-hydroxytryptamine to brain synaptosomal membranes: comparison with [:~H]lysergic acid diethylamide binding. Mol Pharmacol 14: 50-59, 1977. 5. Funatsu, K., S. Teshima and K. Inanaga. Various types of TRH receptors in discrete brain regions and the pituitary of the rat. J Neurochem, in press. 6. Gozlan, H., S. El Mestikawy, L. Pichat, J. Glowinski and M. Hamon. Identification of presynatpic serotonin autoreceptors using a new ligand: all-PAT. Nature 305: 140-142, 1983.

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FUNATSU,

TESHIMA AND INANAGA

14. 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. 15. Plotnikoff, N. P., A. J. Prange, Jr., G. R. Breese, M. S. Anderson and 1. C. Wilson. Thyrotropin-releasing hormone: Enhancement of dopa activity by a hypothalamic hormone. Science 178: 417-418, 1972. 16. Rosenthak H. E. A graphic method for the determination and presentation of binding parameters in a complex system. Anal Bioehem 20: 525-532, 1967. 17. Rostene, W. H., C. T. Fischette, T. C. Rainbow and B. S. McEwen. Modulation by vasoactive intestinal peptide of serotoninl receptors in the dorsal hippocampus of the rat brain: An autoradiographic study. Neurosei Lett 37: 143-148, 1983. 18. Schnellmann, R. G., S. J. Waters and D. L. Nelson. [:lH]5-hydroxytryptamine binding sites: Species and tissue variation. J Neuroehem 42: 65-70, 1984. 19. Sharif, N. A. and D. R. Burr. Micromolar substance P reduces spinal receptor binding for thyrotropin-releasing hormone-Possible relevance to neuropeptide coexistence? Neurosci Lett 43: 245-251, 1983.