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Keywords:

  • [35S]GTPγS binding;
  • in vivo microdialysis;
  • quantitative autoradiography;
  • serotonin transporter binding

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Serotonin-1A (5-HT1A) receptors in the dorsal raphe nucleus (DRN) function as somatodendritic autoreceptors, and therefore play a critical role in controlling serotonergic cell firing and serotonergic neurotransmission. We hypothesized that a decrease in the capacity of 5-HT1A receptors to activate G proteins was a general mechanism by which 5-HT1A receptors in the DRN are desensitized following chronic administration of selective serotonin reuptake inhibitors (SSRIs). Using in vivo microdialysis, we found that the ability of the 5-HT1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) (0.025 mg/kg, s.c.) to decrease extracellular 5-HT levels in striatum was attenuated following chronic treatment of rats with the SSRIs sertraline or fluoxetine. This apparent desensitization of somatodendritic 5-HT1A autoreceptor function was not accompanied by a decrease in 5-HT1A receptor sites in the coupled, high-affinity agonist state as measured by the binding of [3H]8-OH-DPAT. In marked contrast to what was observed following chronic administration of fluoxetine, 5-HT1A receptor-stimulated [35S]GTPγS binding in the DRN was not altered following chronic sertraline treatment. Thus, desensitization of 5-HT1A somatodendritic autoreceptor function following chronic sertraline administration appears not to be due to a decrease in the capacity 5-HT1A receptors to activate G proteins in the DRN. Our findings suggest that the SSRIs may not be a homogeneous class of antidepressant drug with regard to the mechanism by which the function of somatodendritic 5-HT1A autoreceptors is regulated.

Abbreviations used
(R)-(+)-8-OH-DPAT

(R)-(+)-8-hydroxy-DPAT hydrobromide

[3H]CN-IMI

[3H]cyanoimipramine

5-HT

5-hydroxytryptamine, serotonin

5-HT1A

serotonin-1A

8-OH-DPAT

8-hydroxydipropylaminotetralin hydrobromide

DPCPX

1,3-dipropyl-8-cyclopentylxanthine

SSRI

selective serotonin reuptake inhibitor

There is generally a 2–3 week delay in obtaining therapeutic effectiveness with antidepressants, which has been attributed to compensatory changes in neurotransmitter systems as a result of long-term drug treatment. One example of these compensatory changes is the desensitization of serotonin-1A (5-HT1A) receptors in the dorsal raphe nucleus following chronic administration of selective serotonin reuptake inhibitors (SSRIs) (e.g. Chaput et al. 1986; Kreiss and Lucki 1995; Le Poul et al. 1995). 5-HT1A receptors localized in the dorsal raphe nucleus function as somatodendritic autoreceptors (see Aghajanian et al. 1990), and therefore play a critical role in the control of serotonergic cell firing and serotonergic neurotransmission. Desensitization of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus results in the recovery of the firing rate of serotonergic neurons (e.g. Chaput et al. 1986; Blier et al. 1987; Le Poul et al. 1995), and therefore would be expected to result in an increase in serotonergic neurotransmission.

Electrophysiological and neurochemical studies have shown that chronic treatment with the SSRI fluoxetine desensitizes somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus (Kreiss and Lucki 1995; Le Poul et al. 1995; Czachura and Rasmussen 2000; Newman et al. 2004). Although this desensitization is not accompanied by changes in 5-HT1A receptor agonist or antagonist radioligand binding (Le Poul et al. 1995; Hensler 2002; Castro et al. 2003), we and others have shown that chronic treatment with fluoxetine attenuates 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal raphe nucleus (Hensler 2002; Pejchal et al. 2002; Shen et al. 2002; Castro et al. 2003). Therefore, the desensitization of somatodendritic 5-HT1A autoreceptor function in the dorsal raphe nucleus following chronic fluoxetine administration appears to be due to a decrease in the capacity of 5-HT1A receptors to activate G proteins. Based on these observations, we hypothesized that a reduction in the capacity of 5-HT1A receptors to activate G proteins is a general mechanism by which 5-HT1A receptors in the dorsal raphe nucleus are desensitized following chronic SSRI administration.

In the present study, we have examined the effect of chronic administration of the SSRI sertraline on 5-HT1A receptor function in the dorsal raphe nucleus. For comparison, experiments in which animals were treated chronically with fluoxetine were also conducted. We used in vivo microdialysis to assess somatodendritic 5-HT1A autoreceptor function, measuring the effect of acute subcutaneous injection of the 5-HT1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) to decrease extracellular serotonin (5-hydroxytryptamine; 5-HT) in striatum. The striatum is highly innervated by serotonergic neurons arising from cell bodies localized in the dorsal raphe nucleus (Molliver 1987), and systemic injection of 8-OH-DPAT decreases extracellular 5-HT in striatum through activation of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus (Kreiss and Lucki 1994). This approach has been used to show that following chronic administration of the SSRI fluoxetine, somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus are desensitized (Kreiss and Lucki 1995). We also determined 5-HT1A receptor function at the level of receptor G protein interaction by measuring [35S]GTPγS binding stimulated by the 5-HT1A receptor agonist 8-OH-DPAT. The high-affinity agonist state of the 5-HT1A receptor, which is coupled to G proteins, was measured by the binding of [3H]8-OH-DPAT (Vergéet al. 1986; Chamberlain et al. 1993).

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

Male Sprague–Dawley rats (150–175 g; Harlan, Indianapolis, IN, USA) were group-housed and maintained on a 14 : 10 h day : night cycle with constant access to food and water. These studies were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Every effort was made to prevent animal suffering and to minimize the number of animals used.

Drug treatment

After an acclimation period of 10 days, animals were treated with sertraline hydrochloride (10 mg/kg/day) or vehicle (50% ethanol) via subcutaneously implanted osmotic minipumps (ALZET osmotic pumps, model 2ML4; Durect, Cupertino, CA, USA) for 21 days. Osmotic minipumps were filled under aseptic conditions with sterile-filtered sertraline solution or vehicle, and then placed overnight in an incubator at 37°C. This served to prime the minipumps so that they had begun to release drug or vehicle before implantation in the animal. On day 21 of treatment, minipumps were surgically removed to terminate drug treatment.

A separate group of animals were killed on day 21 of treatment, and trunk blood collected to determine steady state serum levels of sertraline (Clinical Psychopharmacology Laboratories, University of Texas Health Science Center at San Antonio, TX, USA). In animals treated for 21 days with sertraline (10 mg/kg/day, s.c.), steady state serum levels of sertraline were 131.5 ± 15.1 ng/mL (= 10). These values are within the range that is therapeutically effective in human patients (30–150 ng/mL) (Baumann 1996; Tournel et al. 2001).

For comparison, experiments were conducted in which animals were treated chronically with fluoxetine hydrochloride. Rats were injected intraperitoneally (i.p.) with fluoxetine (10 mg/kg) or saline once a day for 14 days. Fluoxetine (2 mL/kg) was dissolved in water and injected according to body weight. This dose of fluoxetine was chosen from the literature to correspond to a clinically relevant dose (Czachura and Rasmussen 2000). Fresh fluoxetine solution was made each day.

Forty-eight hours after termination of drug treatment, in vivo microdialysis experiments were performed, or animals were killed and brain tissue prepared for quantitative autoradiographic experiments measuring 8-OH-DPAT-stimulated [35S]GTPγS binding, [3H]-8-OH-DPAT binding, and [3H]cyanoimipramine ([3H]CN-IMI) binding.

In vivo microdialysis

Upon termination of drug treatment, a CMA/12 guide cannula (CMA/Microdialysis, North Chelmsford, MA, USA) was stereotaxically implanted in the striatum using the following coordinates according to rat atlas of Paxinos and Watson (1998): anterior-posterior +0.1 mm from bregma, mediolateral +3.0 from midline, and dorsal-ventral −3.0 mm from dura. The guide cannula was secured in place by fast curing orthodontic acrylic resin (Lang Dental, Wheeling, IL, USA) and skull screws. Microdialysis experiments were conducted in freely moving animals 48 h after surgery. Animals were placed in cylindrical plastic containers with a counterbalance arm holding a liquid swivel and spring tether (Instech Solomon, Plymouth Meeting, PA, USA), and acclimated to the experimental apparatus for 30 min prior to insertion of the dialysis probe into the guide cannula. Dialysis probes (3 mm membrane length, 0.5 mm o.d., CMA/12; CMA/Microdialysis) were continuously perfused with sterile perfusion fluid (147 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, and 0.85 mM MgCl2; CMA/Microdialysis) at a rate of 0.8 μL/min. The first baseline samples were collected 2 h after probe insertion. Samples were collected at 20-min intervals into microcentrifuge tubes containing 5 μL 3 mM EDTA, pH 6. After the collection of four baseline samples, animals were injected with either saline or the 5-HT1A receptor agonist 8-OH-DPAT (0.01–0.025 mg/kg, s.c.). An additional 12 samples were collected to complete the experiment. At the end of the experiment, animals were killed, brains removed and sectioned for histological determination of probe placement.

Analysis of dialysate samples

The amount of 5-HT in dialysate samples was determined by HPLC with electrochemical detection using a BAS 502 microbore isocratic liquid chromatographic system with an Epsilon e5 single channel electrochemical detector, and refrigerated autosampler (Bioanalytical Systems, West Lafayette, IN, USA). The composition of the mobile phase was 8% acetonitrile, 100 mM sodium acetate, 1.25 mM octylsulfonic acid, and 0.5 mM EDTA, adjusted to a pH 5.0. Samples (20 μL) were injected directly onto a Unijet microbore column (Bioanalytical Systems, West Lafayette, IN, USA) (3 μm ODS, 100 × 1 mm). The flow rate through the system was 90 μL/min, and the detector was set at +650 mV relative to the Ag/AgCl electrode. Dialysate 5-HT values were calculated by reference to standard curves run daily. The retention time of 5-HT was 5–6 min. Our detection limit for 5-HT was 1 fmole/10 μL or 0.176 pg. This sensitivity was sufficient to measure baseline levels of 5-HT without the need to add a 5-HT reuptake inhibitor to the perfusion medium.

Tissue preparation for quantitative autoradiography

Rat brains were rapidly removed and frozen on powdered dry ice. Brains were stored at −80°C until sectioning. Coronal sections of 20-μm thickness were cut at −17°C in a cryostat microtome at the level of the hippocampus (plates 32–34) and dorsal raphe nucleus (plates 50–52) according to the atlas of the rat brain (Paxinos and Watson 1998). Sections were thaw-mounted onto gelatin-coated glass slides, desiccated at 4°C for 18 h under vacuum and then stored at −80°C.

Quantitative autoradiography of [35S]GTPγS binding

Autoradiography of 5-HT1A receptor-stimulated [35S]GTPγS binding was performed as previously described (Hensler and Durgam 2001; Hensler 2002) with some modifications. Slide-mounted sections at the level of the dorsal raphe nucleus (plates 50–52) (Paxinos and Watson 1998) were removed from −80°C and equilibrated in HEPES buffer (50 mM, pH 7.4), supplemented with 3 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, and 0.2 mM dithiothreitol for 10 min at 25°C. Sections were then pre-incubated in HEPES buffer containing GDP (2 mM) and the adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 μM) dissolved in 0.02% dimethyl sulfoxide for 15 min at 25°C. Sections were then incubated in the pre-warmed supplemented HEPES buffer containing GDP (2 mM), DPCPX (1 μM), and 40 pM [35S]GTPγS, either in the absence or in the presence of 8-OH-DPAT (1 nM–10 μM) for 60 min at 25°C. Basal [35S]GTPγS binding was defined in the absence of 8-OH-DPAT. Non-specific [35S]GTPγS binding was defined in the absence of 8-OH-DPAT and in the presence of 10 μM GTPγS. The incubation was stopped by two washes for 5 min each in ice-cold 50 mM HEPES buffer, pH 7.4, followed by a brief immersion in ice-cold de-ionized water. Sections were dried on a slide-warmer and exposed to Kodak Biomax MR film (Amersham, Piscataway, NJ, USA) for 48 h to generate autoradiograms.

Quantitative autoradiography of [3H]8-OH-DPAT binding

Autoradiography of the binding of [3H]8-OH-DPAT to 5-HT1A receptors was performed as described with slight modification (Hensler et al. 1991; Rossi et al. 2006). Briefly, slide-mounted sections at the level of the dorsal raphe nucleus (plates 50–52) (Paxinos and Watson 1998) were thawed and desiccated at 4°C for 1 h. Sections were pre-incubated for 30 min at 30°C in assay buffer (170 mM Tris–HCl, pH 7.6), and then incubated in assay buffer containing 2 nM [3H]8-OH-DPAT for 60 min at 25°C. Non-specific binding was defined by incubating adjacent sections in the presence of 10 μM WAY 100635. Incubation was terminated by two washes for 5 min each in ice-cold 170 mM Tris–HCl buffer, pH 7.6, followed by a dip in ice-cold de-ionized water. Sections were dried on a slide warmer and exposed to Kodak BioMax MR Film (Amersham) for a period of 9 weeks to generate autoradiograms.

Quantitative autoradiography of [3H]cyanoimipramine binding

The binding of [3H]CN-IMI to serotonin reuptake sites was performed as previously described (Kovachich et al. 1988; Gould et al. 2006). Briefly, slide-mounted sections at the level of the dorsal hippocampus (plates 32–34) (Paxinos and Watson 1998) were pre-incubated in 50 mM Tris–HCl, 150 mM NaCl buffer, pH 7.4 at 4°C for 1 h, and then incubated with [3H]CN-IMI (1 nM) in 50 mM Tris–HCl, 150 mM NaCl buffer, pH 7.4 at 4°C for 24 h. The concentration of [3H]CN-IMI used is approximately 8× its KD value (Kovachich et al. 1988) so the values obtained approximate Bmax values. Non-specific binding, which was determined in adjacent sections and defined in the presence of 1 μM paroxetine, was no more than 5% of total binding in any brain region examined. Sections were washed in the same buffer, rinsed in cold water, and dried on a slide warmer. Autoradiograms were produced by apposing the slide-mounted tissue sections to Kodak Biomax MR film (Amersham) for 4 weeks.

Image analysis

Analysis of the digitized autoradiograms was performed using the image analysis program NIH Image, version 1.47 (NIH, Bethesda, MD, USA). Tissue sections were stained with thionin and the brain areas identified using the atlas of the rat brain (Paxinos and Watson 1998). Autoradiograms of [3H]8-OH-DPAT and [3H]CN-IMI binding were quantified by the use of simultaneously exposed [3H] standards (ART-123; American Radiochemicals, St Louis, MO, USA), which had been calibrated using brain-mash sections according to the method of Geary and colleagues (Geary and Wooten 1983; Geary et al. 1985). The amount of ligand bound was determined by converting optical density measurements to femtomoles per milligram of protein. Specific binding was calculated by subtracting non-specific binding from total binding on adjacent sections. Autoradiograms of 8-OH-DPAT-stimulated [35S]GTPγS binding were quantified by the use of simultaneously exposed [14C] standards (ARC-146; American Radiochemicals). Standard curves, fit to pixel data obtained from [14C] standards and tissue equivalent values (nCi/g) provided by American Radiochemicals, were used to transform the actual regional densitometric values into relative radioactivity measures. Non-specific binding of [35S]GTPγS was subtracted from basal binding and from binding in the presence of 8-OH-DPAT. Specific 8-OH-DPAT-stimulated binding was expressed as percentage above basal.

Data analysis

Data from the in vivo microdialysis experiments are expressed in percentage baseline values. For each animal, the three samples prior to injection of 8-OH-DPAT or saline were averaged to derive a baseline value against which the remaining sample values were compared. Analysis of these transformed data were conducted using two-way anova with repeated measures over time. F values reaching significance (< 0.05) were evaluated further by post hoc analysis using Student–Newman–Keuls post hoc test (Statistica, version 4.1; StatSoft, Tulsa, OK, USA). Individual dose–response curves for 8-OH-DPAT-stimulated [35S]GTPγS binding were fit by non-linear regression using KaleidaGraph software (version 4.0.1; Synergy Software, Reading, PA, USA) to the model: E = Emax/(1 + EC50/[A])n, where E is the response at the 8-OH-DPAT concentration [A], Emax is the maximal response, EC50 is the concentration of drug that yields a half-maximal response, and n is the slope factor. Statistical comparisons for all radioligand binding data were made using an unpaired t-test (GraphPad Prism 4; GraphPad Software Inc., San Diego, CA, USA).

Materials

Sertraline hydrochloride, fluoxetine hydrochloride, and paroxetine hydrochloride were purchased from Shanco International Inc. (Hazlet, NJ, USA). [35S]GTPγS (1250 Ci/mmol) was purchased from PerkinElmer Life Sciences Inc. (Boston, MA, USA). [3H]8-OH-DPAT (210 Ci/mmol) was purchased from GE Healthcare (Piscataway, NJ, USA). [3H]CN-IMI (60 Ci/mmol) was purchased from American Radiochemicals. (R)-(+)-8-hydroxy-DPAT hydrobromide [(R)-(+)-8-OH-DPAT], 8-OH-DPAT, and DPCPX were purchased from Tocris (Ellisville, MO, USA). WAY 100635 maleate and GDP (disodium salt) were purchased from Sigma/RBI (St Louis, MO, USA). GTPγS (tetralithium salt) was purchased from Roche (Indianapolis, IN, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In vivo microdialysis studies

In drug naïve animals, acute subcutaneous injection of the 5-HT1A receptor agonist 8-OH-DPAT resulted in a dose-dependent decrease in extracellular levels of 5-HT when compared with saline injection (Fig. 1). Two-way anova with repeated measures revealed a significant effect of dose [F(2,13) = 68.5, < 0.001], time [F(14,182) = 12.8, < 0.001], and dose × time interaction [F(28,182) = 5.3, < 0.001]. Post hoc analysis revealed significant decreases in extracellular 5-HT at both doses of 8-OH-DPAT (0.01 and 0.025 mg/kg, s.c.) when compared with saline. Systemic injection of 8-OH-DPAT (0.01 mg/kg, s.c.) resulted in a moderate decrease in extracellular 5-HT in the striatum, 20–40% below baseline values. The higher dose of 8-OH-DPAT (0.025 mg/kg, s.c.) resulted in a greater decrease in extracellular 5-HT, 40–60% below baseline values. Because the higher dose of 8-OH-DPAT had a more robust effect to decrease striatal extracellular 5-HT levels, this dose of 8-OH-DPAT was used in subsequent experiments to determine the effect of chronic administration of the SSRIs sertraline or fluoxetine on somatodendritic 5-HT1A autoreceptor function.

image

Figure 1.  Acute systemic injection of the 5-HT1A receptor agonist 8-OH-DPAT produces a dose-dependent decrease in extracellular 5-HT in striatum. Dialysate samples were collected at 20-min intervals following a 2-h period of equilibration. Saline or 8-OH-DPAT (0.01–0.025 mg/kg, s.c.) was injected (arrow) after the collection of three baseline samples. Baseline 5-HT values: saline-injected =27.7 ± 2.1 fmole/sample (= 4); 8-OH-DPAT-injected (0.01 mg/kg) =31.4 ± 4.0 fmole/sample (= 6); 8-OH-DPAT-injected (0.025 mg/kg) = 21.1 ± 2.7 fmole/sample (= 5). Plotted are mean ± SEM. Saline, = 4; 8-OH-DPAT (0.01 mg/kg), = 6; 8-OH-DPAT (0.025 mg/kg), = 5. *< 0.05, post hoc Student–Newman–Keuls test.

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Chronic administration of fluoxetine resulted in a significant increase in basal 5-HT levels in the striatum 48 h after termination of treatment [baseline 5-HT levels: saline-treated = 22.1 ± 2.7 fmole/sample; fluoxetine-treated = 60.9 ±11.7 fmole/sample (< 0.05, unpaired Student’s t-test)]. By contrast, basal 5-HT levels in the striatum were not altered 48 h after termination of sertraline treatment (baseline 5-HT levels: vehicle-treated = 25.5 ± 5.2 fmole/sample; sertraline-treated = 21.3 ± 10.3 fmole/sample).

We assessed the effect of systemic injection of 8-OH-DPAT to inhibit 5-HT release in striatum following chronic treatment of animals with fluoxetine or saline (Fig. 2a). Two-way anova with repeated measures revealed a significant effect of treatment [F(1,8) = 22.9, < 0.001], time [F(14,112) = 17.3, < 0.001], and a significant treatment × time interaction [F(14,112) = 3.4, < 0.001]. Post hoc analysis revealed that the effect of acute injection of 8-OH-DPAT (0.025 mg/kg, s.c.) to decrease extracellular 5-HT levels in the striatum was markedly attenuated in fluoxetine-treated versus saline-treated animals. Our data indicate that chronic fluoxetine treatment results in the desensitization of somatodendritic 5-HT1A autoreceptor function in the dorsal raphe nucleus, in agreement with previous studies (Kreiss and Lucki 1995; Le Poul et al. 1995; Czachura and Rasmussen 2000; Newman et al. 2004).

image

Figure 2.  Chronic administration of fluoxetine or sertraline attenuated the effect of acute injection of 8-OH-DPAT (0.025 mg/kg, s.c.) on extracellular 5-HT in striatum. Microdialysis experiments were conducted 48 h after termination of drug treatment. Dialysate samples were collected at 20-min intervals following a 2-h period of equilibration. (a) Fluoxetine (10 mg/kg) or saline was administered to rats by i.p. injection for 14 days. Plotted are mean ± SEM; = 5 per experimental group. *< 0.05, post hoc Student–Newman–Keuls test. (b) Sertraline (10 mg/kg/day) or vehicle (50% ethanol) was administered to rats by osmotic minipump for 21 days. Plotted are mean ± SEM; = 9 per experimental group. *< 0.05, post hoc Student–Newman–Keuls test.

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We assessed the effect of systemic injection of 8-OH-DPAT to inhibit 5-HT release in striatum following chronic treatment of animals with sertraline or vehicle (Fig. 2b). Two-way anova with repeated measures revealed a significant effect of treatment [F(1,16) = 23.2, < 0.001], time [F(14, 224) = 25.6, < 0.001], and a significant treatment × time interaction [F(14,224) = 4.4, < 0.001]. Post hoc analysis revealed that the effect of acute injection of 8-OH-DPAT (0.025 mg/kg, s.c.) to decrease extracellular 5-HT levels in the striatum was markedly attenuated in sertraline-treated versus vehicle-treated animals. Our data indicate that chronic sertraline treatment results in the desensitization of somatodendritic 5-HT1A autoreceptor function in the dorsal raphe nucleus.

5-HT1A receptor binding in the dorsal raphe nucleus

We measured the binding of the agonist radioligand [3H]8-OH-DPAT to 5-HT1A receptor sites in the dorsal raphe nucleus. There was no change in [3H]8-OH-DPAT binding in this serotonergic cell body area following chronic administration of fluoxetine [saline-treated: 467.7 ± 22.5 fmol/mg protein (= 6); fluoxetine-treated: 438.2 ± 17.5 fmol/mg protein (= 7), > 0.05] or sertraline [vehicle-treated: 461 ± 29.7 fmol/mg protein (= 7); sertraline-treated: 494 ± 17.9 fmol/mg protein (= 7), > 0.05]. These data suggest that the desensitization of somatodendritic 5-HT1A autoreceptors observed in the in vivo microdialysis experiments following chronic fluoxetine or sertraline treatment (see Fig. 2) is not accompanied by a decrease in 5-HT1A receptors in the coupled, high-affinity agonist state.

5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal raphe nucleus

In agreement with numerous previous studies (Hensler 2002; Pejchal et al. 2002; Shen et al. 2002; Castro et al. 2003), we found that chronic administration of fluoxetine resulted in an attenuation of 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal raphe nucleus. Although the potency of the 5-HT1A receptor agonist 8-OH-DPAT (1 nM–10 μM) to stimulate [35S]GTPγS binding was not altered (EC50) [saline-treated: 30.5 ± 8.0 nM (= 6); fluoxetine-treated: 27 ±8.7 nM (= 7)], the efficacy of 8-OH-DPAT was significantly reduced (Emax) [saline-treated: 44.2 ± 5.5% above basal (= 6); fluoxetine-treated: 31.1 ± 4.5% above basal (= 7), < 0.05].

To determine whether chronic sertraline treatment alters the capacity of 5-HT1A receptors in the dorsal raphe to activate G proteins, we measured the binding of [35S]GTPγS stimulated by (R)-(+)-8-OH-DPAT (1 nM–10 μM) (Fig. 3). Surprisingly, chronic treatment of rats with sertraline did not alter the potency (EC50) [vehicle-treated: 56.7 ± 6.8 nM (= 7); sertraline-treated: 50.8 ± 7.0 nM (= 7)] or efficacy (Emax) [vehicle-treated: 58.1 ± 4.4% above basal (= 7); sertraline-treated: 57.3 ± 5.0% above basal (= 7)] of (R)-(+)-8-OH-DPAT to stimulate [35S]GTPγS binding. These data suggest that the desensitization of somatodendritic 5HT1A autoreceptors observed in the in vivo microdialysis experiments following chronic sertraline treatment (see Fig. 2) is not accompanied by a decrease in the capacity of 5-HT1A receptors to activate G proteins in the dorsal raphe nucleus. Thus, the desensitization of 5-HT1A somatodendritic autoreceptors following sertraline administration appears not to be at the level of receptor-G protein interaction.

image

Figure 3.  Chronic administration of sertraline does not alter 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal raphe nucleus. Rats were administered vehicle or sertraline (10 mg/kg/day) for 21 days. [35S]GTPγS was binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1 nM-10 μM). Specific [35S]GTPγS binding is expressed as percentage above basal. Shown are the mean ± SEM; = 7 per experimental group.

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Serotonin transporter binding following chronic sertraline treatment

Chronic treatment of rats with sertraline results in the down-regulation of serotonin transporter binding sites throughout the forebrain (Benmansour et al. 1999; Gould et al. 2006). In view of the lack of effect of sertraline treatment on 5-HT1A receptor-stimulated [35S]GTPγS binding (see Fig. 3), quantitative autoradiographic experiments measuring the binding of [3H]CN-IMI to serotonin transporter sites were conducted in tissue from the same animals. In sections of the dorsal hippocampus taken from the same animals in which 5-HT1A receptor binding and 5-HT1A receptor-stimulated [35S]GTPγS binding were determined in the dorsal raphe nucleus, we found a marked decrease in the binding of [3H]CN-IMI to serotonin transporter sites throughout the forebrain (Fig. 4a). Data for hippocampal regions are shown in Fig. 4b. Thus, the lack of effect of chronic sertraline treatment on 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal raphe nucleus was not because of ineffective sertraline treatment.

image

Figure 4.  Chronic administration of sertraline results in down-regulation of serotonin transporter sites in brain. Panel (a): Autoradiograms of total and non-specific binding of [3H]CN-IMI to serotonin reuptake sites in sections taken at the level of the dorsal hippocampus from animals treated with vehicle or sertraline. Panel (b): Specific binding of [3H]CN-IMI to serotonin transporter sites in hippocampus, expressed as fmol/mg protein. Shown are the mean ± SEM.; = 7 per experimental group. *< 0.001 when compared with vehicle-treated controls.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Based on our previous work and that of others, we hypothesized that a reduction in the capacity of 5-HT1A receptors to activate G proteins is a general mechanism by which 5-HT1A receptors in the dorsal raphe nucleus are desensitized following chronic SSRI administration. As expected, chronic treatment of rats with the SSRI sertraline results in attenuation of the effect of 8-OH-DPAT to decrease extracellular levels of 5-HT in the striatum, suggesting that the sensitivity of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus is reduced. In a separate group of animals, chronic administration of sertraline did not alter 5-HT1A receptor sites in the coupled, high-affinity agonist state as measured by the binding of [3H]8-OH-DPAT, or 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal raphe. The binding of [3H]CN-IMI to serotonin transporter sites, however, was markedly decreased throughout the forebrain following chronic sertraline administration, an indication that the sertraline treatment was effective. Taken together our data indicate that the desensitization of somatodendritic 5-HT1A autoreceptor function following chronic sertraline administration was not a result of a decrease in the capacity of 5-HT1A receptors to activate G proteins, in marked contrast to what was observed following chronic fluoxetine administration.

The firing activity of serotonergic neurons in the dorsal raphe nucleus, which is inhibited by acute administration of the SSRI fluoxetine, recovers during the course of chronic administration (e.g. 14–21 days). The gradual recovery of activity of serotonergic neurons is attributed to desensitization of somatodendritic 5-HT1A autoreceptors, as evidenced by decreased responsiveness of serotonergic cells to the 5-HT1A receptor agonist 8-OH-DPAT (Le Poul et al. 1995, 2000; Czachura and Rasmussen 2000). The ability of acute systemic injection of 8-OH-DPAT to decrease extracellular levels of 5-HT in forebrain areas, i.e. striatum, hippocampus, and frontal cortex, is attenuated following chronic administration of fluoxetine (Kreiss and Lucki 1995; Invernizzi et al. 1996; present study), as is the ability of acute systemic injection of fluoxetine to decrease extracellular levels of 5-HT in frontal cortex (Hervás et al. 2001). Taken together, these studies indicate that chronic administration of fluoxetine results in desensitization of somatodendritic 5-HT1A autoreceptor function.

In the present study, chronic administration of fluoxetine resulted in a significant increase in basal 5-HT levels in the striatum 48 h after termination of treatment. This is in keeping with earlier findings from several investigators (Kreiss and Lucki 1995; Invernizzi et al. 1996; Hervás et al. 2001) and is most likely a result of 5-HT reuptake blockade due to the presence of the active metabolite norfluoxetine (Czachura and Rasmussen 2000). Importantly, the ability of the 5-HT1A receptor agonist 8-OH-DPAT to modulate 5-HT release is not altered in the presence of elevated baseline 5-HT levels because of the acute presence of an SSRI, either systemically or in the perfusion medium (Kreiss et al. 1993; Rutter and Auerbach 1993), or as a result of chronic desipramine administration (Kreiss and Lucki 1995). Moreover, somatodendritic 5-HT1A autoreceptor desensitization following chronic SSRI administration is observed regardless of elevated basal levels of extracellular 5-HT (e.g. Bel and Artigas 1993; Kreiss and Lucki 1995; Invernizzi et al. 1996; Cremers et al. 2000).

Desensitization of somatodendritic 5-HT1A autoreceptor function observed following chronic fluoxetine administration is not accompanied by changes in 5-HT1A receptor agonist or antagonist radioligand binding in the dorsal raphe nucleus (Le Poul et al. 1995; Hensler 2002; Castro et al. 2003; present study). We and others have shown that chronic treatment with fluoxetine attenuates 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal raphe nucleus (Hensler 2002; Pejchal et al. 2002; Shen et al. 2002; Castro et al. 2003). In the current study, the efficacy, but not potency, of 8-OH-DPAT to stimulate [35S]GTPγS binding was decreased. Therefore, the desensitization of somatodendritic 5-HT1A autoreceptor function in the dorsal raphe nucleus following chronic fluoxetine administration appears to be due to a decrease in the capacity of 5-HT1A receptors to activate G proteins.

Following chronic administration of the SSRI sertraline, the effect of 8-OH-DPAT to decrease extracellular levels of 5-HT in the striatum was attenuated, an indication that the ability of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus to decrease 5-HT release in the striatum is reduced. In mice, 8-OH-DPAT induced hypothermia, a response mediated by 5-HT1A autoreceptors (Goodwin and Green 1985; Goodwin et al. 1985), is attenuated following repeated administration of sertraline (Maj and Moryl 1992). To the best of our knowledge there are no other reports of the effect of chronic administration of sertraline on somatodendritic 5-HT1A autoreceptor function.

The desensitization of somatodendritic 5-HT1A autoreceptor function in the dorsal raphe nucleus following chronic sertraline treatment was not accompanied by a change in the number of 5-HT1A receptor sites in the coupled, high affinity agonist state as measured by [3H]8-OH-DPAT binding. These results are perhaps not unexpected based on previous studies that found no decrease in the binding of [3H]8-OH-DPAT in the dorsal raphe nucleus following chronic administration of the SSRIs citalopram or fluoxetine (Hensler et al. 1991; Le Poul et al. 1995; Castro et al. 2003). Thus, the attenuation of somatodendritic 5-HT1A autoreceptor function in the dorsal raphe following chronic administration of this class of antidepressant drug appears not to be due to a decrease in the number of 5-HT1A receptors coupled to G proteins.

The potency and efficacy of (R)-(+)-8-OH-DPAT to stimulate [35S]GTPγS binding in the dorsal raphe nucleus were also unchanged as a result of chronic sertraline administration. The present study confirms and extends our previous study in which we examined the effect of 21 day treatment with a lower dose of sertraline (7.5 mg/kg/day, s.c.) and measured 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal raphe nucleus using a single, maximal concentration of 8-OH-DPAT (1 μM) (Rossi et al. 2006). Taken together, our data suggest that the desensitization of somatodendritic 5-HT1A autoreceptors observed in the in vivo microdialysis experiments following chronic sertraline treatment is not due to a decrease in the capacity of 5-HT1A receptors to activate G proteins, but may occur distal to 5-HT1A receptor-G protein interaction.

In the dorsal raphe nucleus, 5-HT1A receptor activation opens potassium channels and inhibits cell firing (Innis and Aghajanian 1987; Penington et al. 1993). Activation of 5-HT1A receptors on dorsal raphe neurons also directly inhibits voltage-dependent calcium currents (Penington and Kelly 1990; Chen and Penington 1996). It is reasonable to speculate that desensitization of somatodendritic 5-HT1A autoreceptor function following chronic sertraline treatment occurs at the level of the effector (i.e. calcium or potassium channel).

Systemic injection of the 5-HT1A receptor agonist 8-OH-DPAT results in a dose-dependent decrease in extracellular 5-HT levels in the striatum as measured by in vivo microdialysis (Kreiss and Lucki 1994; Beyer et al. 2004; present study). This has been attributed to the activation of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus. It is prevented by administration of the 5-HT receptor antagonist (−)propranolol into the dorsal raphe nucleus (Kreiss and Lucki 1994), and by attenuation of Gαi-mediated receptor signaling as a result of over-expression of regulators of G protein signaling 4 (RGS4) mRNA in the dorsal raphe nucleus (Beyer et al. 2004). The activity of serotonergic neurons in the dorsal raphe nucleus, however, is also controlled by descending projections from the medial prefrontal cortex. Activation of post-synaptic 5-HT1A receptors on glutamatergic pyramidal cells, which project to the dorsal raphe nucleus, inhibit the firing of serotonergic neurons in the dorsal raphe nucleus (Hajós et al. 1998; Celeda et al. 2001) and decreases 5-HT release in the dorsal raphe nucleus and medial prefrontal cortex (Celeda et al. 2001). Activation of these receptors may result in a decrease in extracellular 5-HT levels in the striatum. The attenuation of the ability of systemic injection of 8-OH-DPAT to decrease 5-HT levels in striatum observed in the present study following chronic sertraline administration could therefore be because of desensitization of post-synaptic 5-HT1A receptors in the medial prefrontal cortex. Whether these receptors in medial prefrontal cortex are desensitized following chronic administration of sertraline, or other SSRIs, remains to be determined.

That chronic administration of sertraline did not decrease 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal raphe nucleus, is in marked contrast to what has been observed to follow chronic administration of the SSRIs fluoxetine (Hensler 2002; Pejchal et al. 2002; Shen et al. 2002; Castro et al. 2003) or escitalopram (D. V. Rossi and J. G. Hensler, unpublished observations). Thus, the desensitization of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus observed following chronic administration of fluoxetine or escitalopram (Kreiss and Lucki 1995; Le Poul et al. 1995; El Mansari et al. 2005), but not sertraline (present study), appears to be due to a decrease in the capacity of 5-HT1A receptors to activate G proteins. Our findings suggest that the SSRIs may not be a homogeneous class of drugs with regard to the regulation of somatodendritic 5-HT1A autoreceptor function.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by US PHS Grants MH 52369 (JGH). The authors wish to thank Drs David Morilak and William Morgan for helpful discussion. A preliminary account of these experiments was presented in abstract form (Rossi et al. 2007).

References

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
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