Imipramine-induced increase in 5-HT2C receptor mRNA level in the rat brain

NEllROSCIENC RESERRCM

Neuroscience Research 24 (1996) 189-193

ELSEVIER

Rapid communication

Imipramine-induced increase in 5-HT2C receptor mRNA level in the rat brain Michihisa Tohda, Hiroshi Watanabe* “Division

of Pharmacology,

Research

Institute

for

Wakan-yaku 2630 Sugitani,

(Oriental Toyama

Medicines). Toyama 930-01, Japan

Medical

and Pharmaceutical

University,

Received 16 August 1995; accepted 9 November 1995

Abstract

Repeated oral administration of 20 mg/kg imipramine elevated the level of 5-HT2C mRNA in the rat brain. Hybridization signals in nearly all regions stained by digoxigenin-labeled antisense cRNA probe, such as the hippocampus, choroid plexus, habenular nucleus, and dorsomedial hypothalamic nucleus, were more intense following imipramine treatment. These results suggest that long-term treatment with imipramine stimulates 5-HT2C receptor gene expression. Keywords: 5-HT2C receptor; Antidepressant;

Digoxigenin;

In situ hybridization;

Serotonin receptors are divided into many kinds of subtypes (Humphrey et al., 1993; Hoyer et al., 1994). The 5-HT2C subtype receptor (5-HT2CR), previously designated 5-HTlC subtype receptor, is the first serotonin receptor to be cloned and was originally identified using a Xenopus oocyte system (Julius et al., 1988). 5-HT2CR is widely distributed in the brain, especially in the choroid plexus, and its signaling cascade is coupled with polyphosphoinositide turnover (Hoyer et al., 1994). One of the authors reported previously that some antidepressants inhibited the chloride current induced by 5HT2CR stimulation in Xenopus oocytes injected with mRNA (Tohda et al., 1989; Tohda and Nomura, 1991), and also described the generation of inositol phosphates in 5-HT2CR cDNA-transfected COS-7 cells (Tohda et al., 1995). Thus, 5-HT2CR was suggested to be involved in depression. To determine their therapeutic value, long-lasting and repeated treatment with antidepressants for several weeks are necessary, suggesting that antidepressants act not only pharmacologically but also * Corresponding author,

Tel.:

+8l

764 34 2281; fax: +81 764 34

5056.

Ol68-0102/96/$15.00 SSDI

0 1996 Elsevier Science Ireland

0 168-O IO2(95)00992-3

Ltd.

Rat brain

physiologically. There have been several reports that antidepressants decrease the levels of the classical 5-HT2 receptor (5-HT2AR) (Roth et al., 1990), although conflicting results have also been reported. Since antidepressants act on 5-HT2CR as antagonists and the 5-HT2CR gene is a protooncogene itself (Julius et al., 1989), we expected antidepressants to act through 5HT2C expression. Thus, we examined the influence of antidepressants on 5-HT2CR mRNA expression by in situ hybridization using digoxigenin-labeled cRNA as a probe. To obtain the digoxigenin-labeled antisense RNA probe corresponding to the sequence from the third intracellular loop to the N side of the C terminus (nucleotides 1353-1888), the pBluescript II KS(-) vector containing rat 5-HT2CR cDNA was rearranged since each G protein-coupled receptor has a unique sequence in the third intracellular loop responsible for G protein coupling (Kobilka et al., 1988; Wess et al., 1990; Cotecchia et al., 1992; Wong et al., 1995). The linearized vector, digested by BarnHI at nucleotide 689 of the vector, migrated as a single 6.0 kbp band on agarose gel electrophoresis (Fig. 1, lane 1). Digestion by EcoRI to

All rights reserved

190

M.

M123M45

Tohda, H. Watanabe/Neuroscience

67

Fig. 1. Reconstitution of the vector to obtain the specific probe for 5HTZC mRNA. (1) S-HT2C cDNA/pBluescript KS(-) vector linearized by BarnHI. (2) Digestion of the vector by EcoRI. (3) Digestion of the vector by BInI and BsmI. (4,5) Digestion of the S-HT2C 540 bp fragment/pBluescript KS(-) vector by Him11 (4) or Apal (5). (6,7) Digoxigenin-labeled antisense (7) and sense (6) cRNA probes corresponding to the 540 bp 5-HT2C fragment. M, molecular weight marker, 1 kb DNA ladder (GIBCO) including bands of 506, 1018, 1636 ( ), 2036, 3054, 4072, 5090, 6108 bp, etc.

release the insert from the vector gave a single band at 3.0 kb since both vector and insert were the same size (Fig. 1, lane 2). The 5-HT2CR cDNA/vector was digested by XhoI and BlnI at nucleotides 740 of the vector and 1888 of the 5-HT2CR cDNA, respectively. The 5’ cohesive terminus was treated with the large fragment of DNA polymerase I (Klenow fragment) to make a blunt end and was selfligated. The vector was further digested by Sac1 and BsmI at nucleotides 657 of vector and 1353 of 5-HTZCR cDNA, respectively. The 3’ cohesive terminus was treated with T4 DNA polymerase and was selfligated. The reconstituted vector, which was used to make a probe, was cut by HincII at nucleotide 1368 of 5-HT2C cDNA for T3 RNA polymerase reaction to make antisense RNA or by EcoO1091 at nucleotide 749 of the vector for T7 RNA polymerase reaction to make sense RNA. The reactions to make these probes were performed essentially as described previously (Sambrook et al., 1989), except for the use of the digoxigenin-conjugated UTP to label the probe. After incubation for 60 min at 37°C the reaction mixtures were further incubated with 0.1 mg/ml DNase I for 15 min, and then ethanol precipitated in the presence of 0.4 M LiCl and 25 mM EDTA. Both antisense (T3) and sense (T7) cRNA probes, which were labeled with digoxigenin, had apparent lengths of about 500 bp (Fig. 1, lanes 6 and 7). The resultant cRNA was placed into hydration buffer (80 mM NaHCOs, 60 mM Na&Os, 6 mM dithiothreitol) and incubated at 60°C to generate fragments of about 200-300 bp. The reaction was terminated by adding 0.3 M sodium acetate and 250 wg

Research

24 (19%)

189-193

tRNA, then precipitated with ethanol. The pellet was redissolved in sterile water and stored at -80°C. Imipramine or vehicle (water) was administered orally at 15:00 h for 4 days to male Wistar rats (8 weeks old, 220-250 g, Japan SLC Inc., Hamamatsu, Japan). One hour after the last administration, the rats were killed by decaptation. Their brains were removed, immediately frozen with powdered dry ice, and stored for 1 day at -80°C. Frozen brain sections (16 Frn) were cut using a cryostat, mounted onto gelatin-coated slides, and airdried. Before hybridization, sections were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, followed by 0.5 &ml proteinase K treatment and acetylation. The sections were dehydrated through a graded ethanol series, placed in chloroform to remove fat, treated with 100% ethanol and dried. After prehybridization, the sections were hybridized overnight with digoxigenin-labeled probe in a hybridization buffer consisting of 5 x standard saline citrate (SSC: 150 mM NaCl, 17 mM sodium citrate, pH 7.0), 50% formamide, 2.7 x Denhardt’s solution, 10 mM EDTA, 20 mM dithiothreitol, 0.25 mg/ml tRNA, and 10% dextran sulfate at 55°C. The hybridized sections were washed with 2 x SSC at 55”C, treated with 50 &ml RNase A for 30 min, washed with 50% formamide/ x SSC at room temperature, dehydrated in ethanol and dried. 5HT2CR mRNA hybridized with the digoxigenin-labeled probe was detected immunohistochemically using an alkaline phosphatase-conjugated antidigoxigenin antibody with 450 j&ml nitroblue tetrazolium and 175 &ml X-phosphate as substrates. Gray scale images of stained sections were scanned and saved on a computer using Photoshop. Quantitative analysis of the staining densities was performed using an NIH Image analysis system. When pictures of stained sections were incorporated, the light intensity of the microscope (Olympus AX-80) was controlled such that the staining density of the regions between the hippocampal fissure and CA1 areas in the hippocampus, as background, was 40-50 by NIH Image analysis which shows values ranging from 0 (white) to 255 (black). All other factors in the system were held constant throughout scanning. Data are shown as the ratio compared with the density degree of background as 100. Statistical analysis was carried out by Student’s r-test. The digoxigenin-labeled antisense probe showed strong hybridization with the choroid plexus of the lateral and third ventricles (Fig. 2A,E) and also hybridized in the hippocampus, medial and lateral habenular nucleus (Fig. 2A,E) amygdala and piriform cortex (data not shown). These findings are in good agreement with previous results obtained using a radiolabeled probe (Mengod et al., 1990). When the sense probe was used under the same conditions, the sections showed only faint signals (data not shown). The effects of repeated administration of imipramine on 5-HT2C mRNA ex-

M.

Tohda. H. Watanabe/Neuroscience

Research

24 (1996)

189-193

Fig. 2. Effects of repeated treatment with imipramine on S-HTZC mRNA expression in the rat brain. lmipramine (20 mg/kg) or vehicle (water) was administered once a day for 4 days. (A-D) Imipramine-treated rats; (E-H) water-administered rats. (A,E) Hippocampus and lateral ventricles; nucleus; (D,H) dorsomedial hypothalamic nucleus. (B,F ‘) lateral ventricles; (C,G) third ventricles and habenular

191

192

h4. Tohda, H. Watanabe/Neuroscience

pression were examined. The results showed that imipramine treatment caused an elevation of the 5-HT2CR mRNA levels (Fig. 2 and Table l), although a single administration of imipramine showed only a slight, not significant, increase in the level of 5-HT2C mRNA (data not shown). In almost all regions labeled by the antisense probe including the hippocampus (Fig. 2A,E), choroid plexus of the lateral ventricle (Fig. 2B,F), lateral habenular nucleus (Fig. 2C,G) and dorsomedial hypothalamic nucleus (Fig. 2D,H), stronger signals were detected in the imipramine-treated rat brain (Fig. 2A-D) than in vehicle-treated controls (Fig. 2E-H). Quantitative analysis of the staining intensities supported the results described above - in control (vehicletreated) rat brains, the choroid plexus showed the strongest hybridization signals and repeated treatment with imipramine significantly increased the 5-HT2C mRNA levels in the choroid plexus, habenular nucleus, CA3 region of the hippocampus and dorsomedial hypothalamic nucleus. Previously, we showed that some antidepressants including imipramine may act as 5-HT2C antagonists (Tohda et al., 1989; Tohda and Nomura, 1991). On the other hand, repeated administration of antidepressants is necessary for their therapeutic effects. These characteristics of antidepressants, i.e., their acute 5HT2C antagonistic effect and the necessity for chronic treatment, suggest that long-term occupation of 5HTZCR by antidepressants may stimulate 5-HT2CR gene expression to compensate for the receptor function. It has been reported that 5-HT2CR acts as a transformation factor in itself, suggesting that increased 5HTZCR stimulates expression of other genes such as those encoding anti-stress proteins. Recent reports that neuroleptics such as haloperidol and fluphenazine increase the levels of expression of the immediate-early Table 1 Effects of repeated treatment expression in rat brain

Blank Choroid plexus of lateral ventricle CA3 region of hippocampus Medial habenular nucleus Lateral habenular nucleus Dorsomedial hypothalamic nucleus

with

imipramine

on 5-HT2C

mRNA

Vehicle

Imipramine

loo l 1.2 181.0 f 10.4

100 * 2.2 236.0 f 3.6*’

153.6 f 3.1

165.8

146.8 + 4.3 133.2 l 1.4 124.4 & 2.6

163.8 zk 4.5* 153.3 f 3.1** 146.2 zt 5.0**

l

2.1*

lmipramine (20 mg/kg) or vehicle (water) was administered once a day for 4 days. The staining densities were quantified using an NIH Image scanner. Values represent the means * S.E.M. percent of the blank (the area not labeled by 5-HTZC probe) of four individual experiments (4 rats). Differences were analyzed for significance by Student’s f-test. *P < 0.05, **P < 0.01 compared with vehicle treatment.

Research

24 (19%)

189-193

genes (IEG) fus, jun and ~$268 (Dragunow et al., 1990; Nguyen et al., 1992; Simpson and Morris, 1994) have raised interest regarding the relationship among the therapeutic effects, IEG expression induced by such neuroleptics and their functional significance including de novo protein synthesis mediated through IEG expression. Repeated administration of antidepressants has been shown to decrease the levels of classical 5-HT2 receptors (5-HT2A) (Roth et al., 1990), and it will be of interest to investigate the functional significance of the enhancement of 5-HT2C gene expression induced by long-term antidepressant treatment, and the relationship between antidepressants and 5-HT2C receptormediated biological functions such as eating and neuronal excitation (Tectt et al., 1995). Acknowledgment

We are grateful to Professor Chiyoko Inagaki and Dr. Kyoko Omori (Kansai Medical University) and to Professor Yasushi Kuraishi (Toyama Medical and Pharmaceutical University) for helpful discussion and important advice. This work was supported by a Grantin-Aid from the Uehara Foundation in Japan. References Cotecchia, S., Ostrowski, J., Kjelsberg, M.A., Caron, M.G. and Letlcowitz, R.J. (1992) Discrete amino acid sequences of the aladrenergic receptor determine the selectivity of coupling to phophatidylinositol hydrolysis. J. Biol. Chem., 267: 1633-1639. Dragunow, M., Robertson, G.S., Faull, R.M.L., Robertson, H.A. and Jansen, K. (1990) D2 dopamine receptor antagonists induce fos and related proteins in rat striatal neurons. Neuroscience, 37: 287-294. Hoyer, D., Clarke, D.E., Fozard, J.R., Harting, P.R., Martin, G.R., Mylecharane, E.J., Saxena, P.R. and Humphrey, P.P.A. (1994) International union of pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol. Rev., 46: 157-203. Humphrey, P.P.A., Hatting, P. and Hoyer, D. (1993) A proposed new nomenclature for 5-HT receptors. Trend. Pharmacol. Sci., 14: 233-236. Julius, D., MacDermott, A.B., Axel, R. and Jessell, T.M. (1988) Molecular characterization of a functional cDNA encoding the serotonin 1C receptor. Science, 241: 558-564. Julius, D., Livelli, T.J., Jessell, T.M. and Axel, R. (1989) Ectopic expression of the serotonin IC receptor and the triggering of malignant transformation. Science, 2441 lO57- 1062. Kobilka, B.K., Kobilka, T.S., Daniel, K., Regan, J.W., Caron, M.G. and Lefkowitz, R.J. (1988) Chimeric 012-, fi2-adrenergic receptors: Delineation of domains involved in effector coupling and ligand binding specificity. Science, 240: 13 IO- I3 15. Mengod, G., Nguyen, H., Le. H., Waeber, C., Lubber& H. and Palacios J.M. (1990) The distribution and cellular localization of the serotonin IC receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution. Neuroscience, 35: 577-591. Nguyen, T.V., Kosofsky, B.E., Birbaum, R., Cohen, B.M. and Hymans, SE. (1992) Differential expression of c-fis and zifZ68 in rat striatum after haloperidol, clozapine and amphetamine. Proc. Natl. Acad. USA, 89: 4270-4274.

h4. Tohda,

H. Watanabe/Neuroscience

Roth, B.L., Hamblin, M. and Ciaranello, R.D. (1990) Regulation of S-HT2 and 5-HTIC serotonin receptor levels. Neuropsychopharmacology, 3: 427-433. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Synthesis of RNA probes by in vitro transcription of double-stranded DNA templates by bacteriophage DNA-dependent RNA polymerases. In: Molecular Cloning a Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory Press, pp. 10.27- 10.37. Simpson, C.S. and Morris, B.J. (1994) Haloperidol and fluphenazine induce junE gene expression in rat striatum and nucleus accumbens. J. Neurochem., 63: 1955-1961. Tectt, L.H., Sun, L.M., Akana, S.F., Strack, A.M., Lowenstein D.H., Dallman, M.F. and Julius, D. (1995) Eating disorder and epilepsy in mice lacking S-HT2C serotonin receptors. Nature, 374: 542-546. Tohda, M. and Nomura. Y. (1991) Biphasic effects of mianserin and

Research

24 (19%)

189-193

193

desipramine on serotonin-evoked current and Cl-efIIux in Xenopus oocytes. Eur. J. Pharmacol., 200: 305-310. Tohda, M., Takasu, T. and Nomura, Y. (1989) Effects of antidepressants on serotonin-evoked current in Xenopus oocytes injected with rat brain mRNA. Eur. J. Pharmacol., 166: 57-63. Tohda, M., Tohda, C., Oda, H. and Nomura, Y. (1995) Inhibitory effects of botulinum toxin on 5-HT-induced inositol phosphate formation in 5-HTIC cDNA transfected cells. Neurosci. Lett., 190: I-4. Wess, J., Bonner, T.I., Dorje, F. and Brann, M.R. (1990) Delineation of muscarinic receptor domains conferring selectivity of coupling to guanine nucleotide-binding proteins and second messengers. Mol. Pharmacol., 38: 517-523. Wang, S.K.F., Parker, E.M. and Ross, E.M. (1995) Chimeric muscarinic cholinergic: P-adrenergic receptors that activate GS in response to muscarinic agonists, J. Biol. Chem., 265: 6219-6224.