Differential Regulation of 5-HT1A Receptor-G Protein Interactions in Brain Following Chronic Antidepressant Administration Julie G

Differential Regulation of 5-HT1A Receptor-G Protein Interactions in Brain Following Chronic Antidepressant Administration Julie G. Hensler, Ph.D. Changes in 5-HT1A receptor function or sensitivity the dorsal and median raphe following chronic SSRI following chronic antidepressant treatment may involve treatment appears to be due to a reduced capacity of the changes in receptor-G protein interaction. We have 5-HT1A receptor to activate G protein. By contrast, no examined the effect of chronic administration of the SSRI significant change in postsynaptic 5-HT1A receptor- fluoxetine or the tricyclic antidepressant amitriptyline on stimulated [35S]GTP␥S binding was observed in any of the 35 ␥ 5-HT1A receptor-stimulated [ S]GTP S binding in forebrain areas examined following chronic antidepressant serotonergic cell body areas, and cortical and limbic treatment. Thus, changes in postsynaptic 5-HT1A receptor- structures using quantitative autoradiography. Treatment mediated responses reported to follow chronic SSRI or of rats with fluoxetine, but not amitriptyline, resulted in an tricyclic antidepressant administration most likely occur 35 ␥ attenuation of 5-HT1A receptor-stimulated [ S]GTP S distal to receptor-G protein interaction, perhaps at the level binding in the dorsal and median raphe nuclei. The binding of effector, or involving changes in neuronal function at the 3 of the antagonist radioligand [ H]MPPF to 5-HT1A receptor system or circuit level. sites was not altered, suggesting that the observed changes [Neuropsychopharmacology 26:565–573, 2002] 35 ␥ in 5-HT1A receptor-stimulated [ S]GTP S binding were © 2002 American College of Neuropsychopharmacology. not due to changes in receptor number. Thus, the Published by Elsevier Science Inc. desensitization of somatodendritic 5-HT1A autoreceptors in 35 KEY WORDS: [ S]GTPgammaS; Quantitative in particular has been implicated in affective disorders autoradiography; 5-HT1A receptor; 8-OH-DPAT; such as anxiety and depression. Adaptive changes in Fluoxetine; Amitriptyline; [3H]MPPF the serotonergic system are believed to underlie the Of the multiple types of receptors for serotonin (5-hydroxy- therapeutic effectiveness of the azapirone anxiolytics and a variety of antidepressant drugs. Thus, studies of tryptamine; 5-HT) present in brain, the 5-HT1A receptor the regulation of 5-HT1A receptor function may have important implications for our understanding the role of this receptor in the mechanism of action of these From the Department of Pharmacology, University of Texas therapeutic agents. Health Science Center-San Antonio, San Antonio, Texas. Chronic administration of selective serotonin re- Address correspondence to: J.G. Hensler, Ph.D. Department of uptake inhibitors (SSRIs) results in the desensitization Pharmacology, MC 7764, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, Tel.: (210) 567- of 5-HT1A somatodendritic autoreceptor function in the 4236, Fax: (210) 567-4303, E-mail: [email protected] dorsal raphe (see Blier and de Montigny 1994; Kreiss Received July 25, 2001; revised October 11, 2001; accepted Octo- and Lucki 1995; Le Poul et al. 2000). Chronic SSRI treat- ber 17, 2001. Online publication: 10/18/01 at www.acnp.org/citations/ ment also results in the desensitization of physiological Npp101801188. responses mediated by postsynaptic 5-HT1A receptors NEUROPSYCHOPHARMACOLOGY 2002–VOL. 26, NO. 5 © 2002 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/02/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(01)00395-5 566 J.G. Hensler NEUROPSYCHOPHARMACOLOGY 2002–VOL. 26, NO. 5 (Goodwin et al. 1987; Hensler et al. 1991; Li et al. 1996). phy to study the regulation of 5-HT1A receptor-G pro- Electrophysiological and neurochemical studies indi- tein interaction in brain following repeated antidepres- cate, however, that in hippocampus the sensitivity of sant administration. postsynaptic 5-HT1A receptor-mediated responses is not changed (Blier and de Montigny 1983; Chaput et al. 1986; Varrault et al. 1991; Le Poul et al. 2000). METHODS Unlike the SSRIs, chronic administration of tricyclic Animals antidepressants does not result in the desensitization of the 5-HT1A somatodendritic autoreceptor (Blier and de Male Sprague-Dawley rats (250–300 g; Harlan, India- Montigny 1980; Kreiss and Lucki 1995). Although some napolis, IN) were group-housed and maintained on a investigators have reported an attenuation of behav- 14:10 h day:night cycle with constant access to food and ioral and physiological responses mediated by postsyn- water. These studies were carried out in accordance aptic 5-HT1A receptors (Goodwin et al. 1987; Lesch et al. with the Guide for the Care and Use of Laboratory Animals 1990), this has not been a consistent observation (Lucki as adopted and promulgated by the National Institutes and Frazer 1982; Wozniak et al. 1987; Hensler et al. of Health. 1991; Gartside et al. 1992). Electrophysiological studies indicate that chronic treatment with tricyclic antide- Drug Treatment pressants results in the sensitization of postsynaptic neu- rons in hippocampus to 5-HT1A receptor agonists (de Rats were injected intraperitoneally (i.p.) with saline Montigny and Aghajanian 1978; Chaput et al. 1991). (n ϭ 12), fluoxetine (10 mg/kg) (n ϭ 8), or amitriptyline These discrepant observations regarding the regula- (10 mg/kg) (n ϭ 8) once a day for 14 days. Amitrip- ␷ ϭ ␷ ϭ tion of postsynaptic 5-HT1A receptor sensitivity follow- tyline ( 1 ml/kg) and fluoxetine ( 2 ml/kg) were ing chronic SSRI or tricyclic antidepressant administra- dissolved in water and injected according to body tion may be due to the effects of these drug treatments weight. These doses of amitriptyline and fluoxetine on complex neuronal circuits, or differences in the regu- were chosen from the literature to correspond to clini- lation of 5-HT1A receptor function in specific brain re- cally relevant doses (de Montigny and Aghajanian gions. In general, changes in 5-HT1A receptor number 1978; Czachura and Rasmussen 2000). Fresh fluoxetine have not been observed following chronic administra- and amitriptyline solutions were made each day. Ani- tion of antidepressants (Wieland et al. 1993; Hensler et mals were injected at the same time each day, specifi- al. 1991; Le Poul et al. 2000; Hervàs et al. 2001). Because cally between 11:00 A.M. and 1:00 P.M. Animals were changes in the sensitivity of 5-HT1A receptor-mediated sacrificed 48 h after the last injection. responses do not appear to be mediated by changes in 5-HT receptor binding, the basis for changes in 5-HT 1A 1A Tissue Preparation receptor function or sensitivity may involve changes in the capacity of the 5-HT1A receptor to activate G pro- Rat brains were rapidly removed and frozen on pow- tein. Receptor-stimulated [35S]GTP␥S binding is a direct dered dry ice. Brains were stored at –80ЊC until section- assay of receptor activation of G proteins, as it mea- ing. Coronal sections of 20 ␮m thickness were cut at sures the exchange of GDP for [35S]GTP␥S. [35S]GTP␥S Ϫ17ЊC in a cryostat microtome at the level of the lateral autoradiography allows the demonstration of receptor-G septum, dorsal hippocampus or dorsal raphe according protein interaction with neuroanatomical resolution. We to the atlas of the rat brain of Paxinos and Watson have used this approach previously to examine regional (1986). Sections were thaw-mounted onto gelatin- Њ differences in the regulation of the 5-HT1A receptor coated glass slides, desiccated at 4 C for 18 h under vac- at the level of receptor-G protein interaction following uum and then stored at Ϫ80ЊC until use. chronic agonist administration (Hensler and Durgam 2001). [35S]GTP␥S Autoradiography In the current study we have examined the effect of repeated administration of the SSRI fluoxetine or the Autoradiography of (Ϯ)8-OH-DPAT-stimulated [35S]GTP␥S tricyclic antidepressant amitriptyline on 5-HT1A recep- binding in brain sections was performed as previously tor-stimulated [35S]GTP␥S binding. Using quantitative described (Hensler and Durgam 2001). Because both autoradiography, this analysis was performed for post- MgCl2 and GDP affect the interaction of the agonist with 35 ␥ synaptic 5-HT1A receptors in forebrain areas, which the receptor and G protein, as well as basal [ S]GTP S serve as terminal field areas of serotonergic innerva- binding, we have determined in preliminary experi- tion, and presynaptic 5-HT1A receptors located on the ments the optimal concentrations of MgCl2 and GDP to soma and dendrites of serotonergic cell bodies in the maximize agonist-stimulated [35S]GTP␥S binding. The dorsal and median raphe nuclei. The present study is largest increases in 8-OH-DPAT-stimulated [35S]GTP␥S the first to report the use of [35S]GTP␥S autoradiogra- binding above basal were obtained with 2 mM GDP NEUROPSYCHOPHARMACOLOGY 2002–VOL. 26, NO. 5 Regulation of 5-HT1A Receptor-G Protein Interactions 567 35 ␥ and 3 mM MgCl2 in the assay buffer. Slide-mounted OH-DPAT-stimulated [ S]GTP S binding were quanti- sections were thawed and desiccated at 4ЊC for 2 h, and fied by the use of simultaneously exposed [14C] stan- then equilibrated in HEPES buffer (50 mM, pH 7.4), dards (ARC-146, American Radiochemicals, St. Louis, supplemented with 3 mM MgCl2, 0.2 mM EGTA, 100 MO). Standard curves were fit to pixel data obtained mM NaCl, and 0.2 mM dithiothreitol for 10 min at 30ЊC. from [14C] standards and tissue equivalent values (nCi/g) Sections were pre-incubated in HEPES buffer contain- provided by American Radiochemicals, and were used ing GDP (2 mM) for 10 min at 30ЊC, and then incubated to transform the actual regional densitometric values in HEPES buffer containing GDP (2 mM) and 80 pM into relative radioactivity measures. Nonspecific bind- [35S]GTP␥S, either in the absence or in the presence of ing of [35S]GTP␥S was subtracted from basal binding (Ϯ)8-OH-DPAT (1 ␮M), for 45 min at 30ЊC.

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