Research ArticleArticle

Reduction of 5-Hydroxytryptamine (5-HT)1A-Mediated Temperature and Neuroendocrine Responses and 5-HT1A Binding Sites in 5-HT Transporter Knockout Mice

Qian Li, Christine Wichems, Armin Heils, Louis D. Van de Kar, Klaus-Peter Lesch and Dennis L. Murphy
Journal of Pharmacology and Experimental Therapeutics December 1999, 291 (3) 999-1007;

Abstract

The aim of the present study was to determine whether alterations in 5-hydroxytryptamine (5-HT)1A receptors would be found in knockout mice lacking the serotonin transporter (5-HTT). Hypothermic and neuroendocrine responses to the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) were used to examine the function of 5-HT1A receptors. Initial studies evaluated the dose-response and time course of 8-OH-DPAT-induced hypothermia and hormone secretion in normal CD-1 mice (the background strain of the 5-HTT knockout mice). 8-OH-DPAT dose-dependently produced hypothermic responses that peaked at 20 min postinjection. 8-OH-DPAT-induced hypothermia was blocked by the 5-HT1A antagonist WAY-100635. 8-OH-DPAT dose-dependently increased the concentrations of plasma oxytocin, corticotropin, and corticosterone. In the 5-HTT knockout (−/−) mice, the hypothermic response to 8-OH-DPAT (0.1 mg/kg s.c.) was completely abolished. Furthermore, 5-HTT−/− mice had significantly attenuated plasma oxytocin and corticosterone responses to 8-OH-DPAT. No significant changes in the hypothermic or hormonal responses to 8-OH-DPAT were observed in heterozygous (5-HTT+/−) mice. [3H]8-OH-DPAT- and [125I]MPPI [4-(2′-methoxyphenyl)-1-[2′-[N-(2"-pyridinyl)-iodobenzamido]ethyl]piperazine]-binding sites in the hypothalamus and [125I]MPPI-binding sites in the dorsal raphe were significantly decreased in 5-HTT−/− mice. The results indicate that lack of the 5-HTT is associated with a functional desensitization of 5-HT1A receptor responses to 8-OH-DPAT, which may be a consequence, at least in part, of the decrease in density of 5-HT1A receptors in the hypothalamus and dorsal raphe of 5-HTT−/− mice.

The 5-hydroxytryptamine (5-HT; serotonin) transporter (5-HTT) is an essential component in serotonergic neurotransmission. The 5-HTT regulates 5-HT concentrations in the synaptic cleft and adjacent brain extracellular fluid locations via recycling released serotonin, thus terminating the action of 5-HT. The 5-HTT is the target of serotonin reuptake inhibitors (SRIs), a group of widely used antidepressant and antianxiety agents. The exact mechanism of therapeutic action of the SRIs remains unknown because the onset of clinical improvement usually takes 2 to 4 weeks or longer after the initiation of SRI administration. Adaptive changes in the serotonergic system that are initiated by blockade of 5-HT reuptake have been implicated in these delayed therapeutic effects (Briley and Moret, 1993; Blier and De Montigny, 1998). A number of studies have demonstrated that chronic SRI administration leads to a desensitization of 5-HT1A receptors in the hypothalamus and dorsal raphe in rats (Le Poul et al., 1995; Li et al., 1996, 1997; Davidson and Stamford, 1998) and of responses to 5-HT1Aagonists in humans (Sargent et al., 1997; Berlin et al., 1998). Among the fourteen 5-HT receptors now identified, 5-HT1A receptors have been of special interest, because the combined administration of SRIs with 5-HT1A antagonists has led to earlier and enhanced therapeutic efficacy in some studies (Artigas et al., 1996;Blier et al., 1997). Thus, understanding the interaction between 5-HTT and 5-HT1A receptors may help in the development of better therapeutic approaches.

Although long-term treatment with SRIs has been used to study desensitization of 5-HT1A receptors induced by disruption of 5-HTT function, the limitations of these approaches are the relative short and variable duration of the treatment (2–12 weeks) and some other pharmacological effects of SRIs beside their effects on 5-HT transport (Romero et al., 1996). An alternative to pharmacological studies is to use molecularly manipulated mice with disrupted 5-HTT function to assess the hypothesized association between 5-HTT function and 5-HT1A receptor desensitization (Murphy et al., 1999). In the present study, we used mice in which 5-HTTs were genetically reduced (+/−, heterozygous) or deleted (−/−, homozygous) as described previously (Bengel et al., 1998). We hypothesized that 5-HT1A receptors would be desensitized in the 5-HTT−/− mice, with only slight or intermediate changes in the 5-HTT+/− mice. In addition, we expected greater 5-HT1A receptor system alterations in the 5-HTT−/− mice than in those treated with SRIs.

5-HT1A receptors function both as somatodendritic autoreceptors and as postsynaptic receptors (Hoyer et al., 1994). Somatodendritic 5-HT1A autoreceptors are principally located on 5-HT neurons and dendrites in the midbrain raphe nuclei. Activation of somatodendritic 5-HT1Aautoreceptors by 5-HT or 5-HT1A agonists decreases the firing rate of neurons and subsequently reduces the release of 5-HT from nerve terminals. Postsynaptic 5-HT1A receptors are widely distributed in brain regions that receive serotonergic input. Activation of postsynaptic 5-HT1A receptors by 5-HT produces physiological responses that depend on the function of the target cells.

Two frequently used measurements of 5-HT1Afunction are the temperature and neuroendocrine responses to selective 5-HT1A agonists. For example, the hypothermia produced by the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) in mice has been generally attributed to activation of somatodendritic 5-HT1A autoreceptors (Goodwin et al., 1985; Bill et al., 1991; Martin et al., 1992). On the other hand, most evidence suggests that the increased secretion of several hormones, including corticotropin (ACTH), corticosterone, and oxytocin, that follows 8-OH-DPAT administration results from the stimulation of 5-HT1A receptors in the hypothalamic paraventricular nucleus (Bluet Pajot et al., 1995; Bagdy, 1996). Therefore, the magnitude of the hypothermic and hormone responses to 8-OH-DPAT can be used as indicators of the function of 5-HT1A receptor systems in the midbrain and hypothalamus.

In the present study, the hypothermic and hormone responses to 8-OH-DPAT were used to evaluate the function of 5-HT1A receptors in 5-HTT transgenic mice. To determine whether an alteration in the density of 5-HT1A receptors might contribute to a change in the function of 5-HT1A receptors, we also examined the density of 5-HT1A receptors in the hypothalamus. At the present, very little pharmacological data on hormone responses to 8-OH-DPAT are available in mice. Also, it has been reported that there is a large variation in behavioral and other physiological and pharmacological phenomena among mouse strains. Therefore, we investigated the pharmacological parameters of hypothermic and hormone responses to 8-OH-DPAT in normal mice.

Experimental Procedures

Animals

CD-1 mice used to establish time course and dose-responses to 8-OH-DPAT were purchased from Charles River Laboratories (Wilmington, MA). 5-HTT mutant mice with a CD-1 × 129 Sv/Ev background were created through homologous recombination as previously reported (Bengel et al., 1998). All of the purchased CD-1 mice used in the studies were 3 months old with a b.wt. of 25 to 30 g. The 5-HTT mutant mice were from the F3 generation and were 3 to 5 months of age, with b.wt. of 30 to 40 g. All mice were housed in groups of four or five per cage in a light- (12-h light/dark cycle, lights on at 6:00 AM), humidity-, and temperature-controlled room. Food and water were available ad libitum. All of the mice were handled every day for 1 week before the experiments. On the day before the experiments, the mice were separated into groups of two or three per cage. In all except one of the experiments, 7 to 10 male mice were included in each group, as specified in the figure and table legends. Experimental protocols adhered to National Institutes of Health guidelines and were approved by the National Institute of Mental Health Animal Care and Use Committee.

Materials

8-OH-DPAT was purchased from Research Biochemicals Inc. (Natick, MA). WAY-100635 [(N-[2-(4(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-2-pyridyl)cyclohexanecarboxamide] was a gift from Wyeth-Ayerst (UK). [3H]Corticosterone,125I-labeled oxytocin, [3H]8-OH-DPAT, and125I-labeled 4-(2′-methoxyphenyl)-1-[2′-[N-(2"-pyridinyl)-iodobenzamido]ethyl]piperazine (123I-MPPI) were purchased from NEN Life Science Products (Boston, MA). 125I-labeled ACTH was obtained from DIASORIN (Stillwater, MN). Corticosterone antiserum was purchased from ICN Biochemicals (Irvine, CA). ACTH antiserum (against the 5–18 segment of ACTH) was purchased from IgG Corp. (Nashville, TN). ACTH-(1–39) standard was purchased from Calbiochem (San Diego, CA).

Hypothermic Responses to 8-OH-DPAT

In all of the experiments, body temperature of the mice was measured with a digital thermometer with a temperature probe (Physitemp BAT-12; Physitemp Inst. Inc., Clifton, NJ) inserted 2 to 2.5 cm into the rectum, with mice slightly restrained by the tail. All temperatures were measured in a room with ambient temperature of 25°C.

Time Course and Dose-Response of 8-OH-DPAT-Induced Hypothermia.

Purchased CD-1 mice were injected with saline or 8-OH-DPAT in doses of 0.05, 0.1, 0.25, 0.5, or 1 mg/kg s.c. The body temperature of the mice was measured every 10 min from 20 min before to 60 min after 8-OH-DPAT injections. Mean values of the temperature from 20 and 10 min before 8-OH-DPAT injections were used for the zero temperature time point measures.

WAY-100635 Antagonism of 8-OH-DPAT-Induced Hypothermia.

5-HTT+/+ mice were injected with saline or WAY-100635 (0.1 mg/kg s.c.). Thirty minutes after the injection of WAY-100635, the mice received saline or 8-OH-DPAT (0.1 mg/kg s.c.). Their body temperature was measured 15 min before and after the injection of WAY-100635 and then every 10 min from 10 to 60 min after 8-OH-DPAT injections. The mean values of the temperatures at 15 min before and after the injection of WAY-100635 or saline were used for the zero temperature time point measures.

Hypothermic Responses to 8-OH-DPAT in Mutant Mice.

5-HTT+/+, 5-HTT+/−, and 5-HTT−/− mice were injected with saline or 8-OH-DPAT (0.1 mg/kg s.c.). Body temperatures of the mice were measured every 10 min from 20 min before to 60 min after 8-OH-DPAT injections. The mean values of the temperatures from 20 and 10 min before 8-OH-DPAT injections were used for the zero temperature time point measures.

Hormone Responses to 8-OH-DPAT

Time Course of ACTH and Corticosterone Responses to 8-OH-DPAT.

Purchased male CD-1 mice were injected with saline or 8-OH-DPAT (0.1 mg/kg s.c.) and decapitated at 15, 30, and 45 min after the injections. Mice used for zero time point were injected with saline and decapitated 15 min after the saline injection. Trunk blood was collected in 1.8-ml Eppendorf vials containing 50 μl of 3 M EDTA for plasma hormone assays.

Dose-Response of Oxytocin, ACTH, and Corticosterone Responses to 8-OH-DPAT.

Purchased male CD-1 mice were injected with saline or 8-OH-DPAT (0.05, 0.1, 0.2, or 0.5 mg/kg s.c.) and decapitated 15 min after the injections. Trunk blood was collected for the plasma hormone assays.

Hormone Responses to 8-OH-DPAT in 5-HTT Mutant Mice.

Male 5-HTT+/+, 5-HTT+/−, and 5-HTT−/− mice were injected with 8-OH-DPAT (0.1 mg/kg s.c.) or saline and decapitated 15 min later. To examine the basal concentrations of plasma ACTH and corticosterone, other groups of transgenic mice were decapitated without saline injections or injection-related handling.

Radioimmunoassays for Plasma Hormone Concentrations

Plasma oxytocin, ACTH, and corticosterone were measured by radioimmunoassays as described previously (Li et al., 1993, 1994). Briefly, 20-μl plasma samples or ACTH(1–39) standards (0.1–20 pg) were incubated with ACTH antiserum (final dilution, 1:30,000) at 4°C overnight in 0.01 M PBS, pH 7.6, containing 1% BSA, 0.025 M EDTA, 0.5% normal rabbit serum, and 25 kallikrein-inactivating units (KIU)/ml aprotinin. 125I-ACTH (2000 cpm/0.1 ml) was added and incubated for 24 h at 4°C in a total volume of 0.3 ml. After incubation with the second antibody (1:50 final dilution, goat anti-rabbit γ-globulin; Calbiochem) at 4°C for 24 h, 1.5 ml of cold PBS was added to the tubes, and they were then centrifuged at 15,000g and 4°C for 40 min. The radioactivity of the pellet was counted with a Micromedic 4/600 gamma counter. The sensitivity of the assay was 0.2 pg/tube, and the intra-assay and interassay variations were 4.2 and 14.6%, respectively.

In the plasma corticosterone assay, 3 μl of plasma or corticosterone standards (0.01–5 ng) were added into 0.5 ml of total volume assay buffer (0.1 M PBS, 0.1% gelatin, and 0.1% sodium azide) and incubated in 75°C for 20 min to denature corticosterone-binding proteins. After the tubes were cooled to room temperature, corticosterone antiserum (final dilution, 1:11,200) and [3H]corticosterone (at 7000 cpm) were added and incubated at 4°C overnight. The free and bound [3H]corticosterone were separated by incubating 0.2 ml of Carcoal suspension (62.5 mg% Destran-T70 and 0.625% Norit A in assay buffer) for 20 min at 4°C, followed with centrifugation at 15,000g and 4°C for 20 min. The supernatant was decanted into scintillation vials containing 5 ml of scintillation fluid (Scint 30, Fisher) and counted in a Beckman scintillation counter. The sensitivity was 0.02 ng/tube, and the intra-assay and interassay variations were 4.5 and 11.9%, respectively.

Oxytocin in 0.25 ml of mouse plasma was extracted into 0.5 ml of ice-cold acetone, followed by 1.25 ml of cold petroleum ether, and then dried. The dried extract was dissolved in 0.5 ml of assay buffer (0.05 M phosphate buffer, pH 7.4, containing 0.125% BSA, 0.01% sodium azide, and 0.001 M EDTA). The plasma extract (100 μl) and oxytocin standards (0–1 ng) were used for the oxytocin radioimmunoassay (Li et al., 1997). The concentration of plasma oxytocin was calculated using a correction factor based on the extraction recovery. The sensitivity limit of this assay was 1 pg/tube, and the intra-assay and interassay variations were 8.1 and 8.6%, respectively.

Receptor Binding Assays

Tissue Preparation.

The hypothalamus from each individual mouse was homogenized in 1 ml of 50 mM Tris (pH 7.5) using a Tissuetaker at the highest speed for 8 s. The homogenates were centrifuged at 4°C at 35,000g for 30 min. The pellets were resuspended in 50 mM Tris (pH 7.5) to yield concentrations of 30 mg tissue/ml. The protein concentrations of the tissue homogenates were measured according to the method of Lowry et al. (1951).

[125I]MPPI Binding.

Binding sites for the 5-HT1A antagonist [125I]MPPI in the hypothalamus were measured as described (Kung et al., 1995). Briefly, 25 μl of the hypothalamic homogenates (40–50 μg protein) were incubated with 50 mM Tris (pH 7.5), 2 mM MgCl2, and 0.2 nM [125I]MPPI (2200 Ci/mmol) in a total volume of 0.25 ml at 37°C for 20 min. Nonspecific binding was defined using 10−5 M 5-HT. After the incubations, the bound and free [125I]MPPI were separated by filtering the solution through 1% polyethylenimine-pretreated Whatman GF/C filter papers (Brandel Inc., Gaithersburg, MD), which were then washed three times with 5 ml of 50 mM Tris buffer. The filter papers were counted in a gamma counter (Tilertek Instruments Inc., Huntsville, AL).

[3H]8-OH-DPAT Binding.

[3H]8-OH-DPAT-binding sites in the hypothalamus were determined as described previously (Li et al., 1994). Briefly, the hypothalamic membrane suspensions (40 μl, corresponding to 80 μg protein) were incubated with 50 mM Tris (pH 7.5), 10 mM MgSO4, 0.5 mM EDTA, and 2 nM [3H]8-OH-DPAT (127 Ci/mmol) in a final volume of 0.5 ml at room temperature for 60 min. Nonspecific binding was defined using 10−5 M 5-HT. After the incubations, the solutions were filtered using GF/C filter papers (Brandel Inc.), and the filter papers were washed three times with 5 ml of 50 mM Tris buffer (pH 7.5). The filter papers were added to 5 ml of scintillation fluid (Scintisafe 30%; Fisher Scientific) and quantified in a scintillation counter (Beckman).

Autoradiography of [125I]MPPI Binding.

[125I]MPPI-binding sites in the dorsal raphe were determined by an autoradiographic assay as described previously (Kung et al., 1995). The assay was performed in 15-μm midbrain sections of 5-HTT+/+, 5-HTT+/−, and 5-HTT−/− mice. The 5-HTT+/+, 5-HTT+/−, and 5-HTT−/− mice were decapitated, and brains were sectioned into 15-μm slices. The sections were thaw-mounted onto chromalum/gelatin-coated glass slides and stored in −80°C for less than 1 month before the assays. The midbrain sections were used for examination of [125I]MPPI-binding sites in the dorsal raphe. The brain sections were preincubated for 30 min at room temperature in assay buffer (50 mM Tris, pH 7.4, containing 2 mM MgCl2). The slides were incubated with [125I]MPPI (0.14 nM in assay buffer) for 2 h at room temperature. The concentration of [125I]MPPI was below the Kd value of MPPI for 5-HT1A receptors. Nonspecific binding was defined using 10−5 M 5-HT. The slides were then washed twice with assay buffer at 4°C and rinsed with cold ddH2O. After air blow-drying, the slides were exposed to 3H Hyperfilm (Amersham, Arlington Heights, IL) for 1 to 3 days. 125I microscales (Amersham) were exposed with each slide film to calibrate the absorbance in the fmol/mg tissue equivalent. Brain images were captured and analyzed with the use of National Institutes of Health Image software.

Statistical Analysis

The data are presented as mean ± S.E. and compared with the use of Student's t test or two-way ANOVA or one-way ANOVA followed by Student-Newman-Keuls post hoc tests with SuperANOVA software programs (Abacus Concepts Inc., Berkeley, CA).

Results

Hypothermic Responses to 8-OH-DPAT.

8-OH-DPAT dose-dependently decreased the body temperature of purchased CD-1 mice (Fig.1). The minimum dose of 8-OH-DPAT to produce statistically significant hypothermia was 0.1 mg/kg, and the hypothermic response was maximal in extent and duration at 0.5 mg/kg 8-OH-DPAT. The time of peak 8-OH-DPAT-induced hypothermia was 20 min. Although body temperature returned to normal 60 min after the injection of the lowest dose of 8-OH-DPAT (0.1 mg/kg s.c., P= N.S.), body temperature did not return to baseline within 60 min after all higher doses of 8-OH-DPAT, with statistically significant differences at all 10- to 60-min time points.

Figure 1
Figure 1

Time course and dose-response of 8-OH-DPAT-induced hypothermia. Data represent mean ± S.E. of 10 CD-1 mice per group. Two-way ANOVA: main effect of 8-OH-DPAT,F5,175 = 116.7, P< .0001; main effect of time, F6,175 = 36.4, P < .0001; and interaction between 8-OH-DPAT and time, F30,175 = 7.26,P < .0001. For all time points and all doses of 8-OH-DPAT of 0.1 mg/kg or higher (except for the 60-min time point for the 0.1-mg/kg dose), temperature responses were significantly different from those of the saline-treated mice.

The 5-HT1A antagonist WAY-100635 (0.1 mg/kg s.c.) completely blocked 8-OH-DPAT-induced hypothermia in 5-HTT+/+ mice (Fig. 2). WAY-100635 alone did not alter body temperature in these mice (Fig. 2).

Figure 2
Figure 2

Antagonism by WAY-100635 of 8-OH-DPAT-induced hypothermia. Data represent mean ± S.E. of 10 5-HTT+/+ mice per group. Two-way ANOVA: main effect of time,F8,341 = 21.64, P< .0001; main effect of drugs, F3,341= 137, P < .0001; and interaction between time and drugs, F24,341 = 13.1,P < .0001. ∗, significant difference from those in the zero time points of the same treatment group,P < .05 (Student-Newman-Keuls post hoc test).

The hypothermic response to 8-OH-DPAT was absent in 5-HTT−/− mice (Fig. 3). Although there was a slight attenuation of the hypothermia in 5-HTT+/− mice relative to 5-HTT+/+ mice, the hypothermic responses to 8-OH-DPAT were significantly greater in 5-HT+/+ mice than in 5-HT−/− mice. The body temperature was not different between genotypes in mice injected with saline (data not presented).

Figure 3
Figure 3

Absence of 8-OH-DPAT (0.1 mg/kg)-induced hypothermia in 5-HTT−/− mice. Data represent mean ± S.E. of 10 mice per group. Two-way ANOVA: main effect of genotype,F2,147 = 31.07, P< .0001; main effect of time, F6,147 = 8.77, P < .0001; and interaction between genotype and time, F12,147 = 1.49,P = .132. *Significant difference from those in the zero time points of the same genotype of 5-HTT, P< .05 (Student-Newman-Keuls post hoc test). #, significant difference from the 5-HTT+/+ mice (at same time point), P < .05 (Student-Newman-Keuls post hoc test).

Hormonal Responses to 8-OH-DPAT.

The time course of the 8-OH-DPAT-induced increases in plasma ACTH and corticosterone in the purchased CD-1 mice is presented in Fig.4. Relative to saline injections, 8-OH-DPAT (0.1 mg/kg s.c.) was associated with an approximate 2-fold increase in ACTH (peak concentration at 15 min) and with an approximate 2.5-fold increase in corticosterone (peak concentration at 30 min; Fig.4). Because corticosterone elevations were not significantly different among the 15-, 30-, and 45-min time points, the 15-min time point was chosen for subsequent 8-OH-DPAT studies of ACTH and corticosterone responses. Time course responses for oxytocin could not be determined because of the insufficient plasma available.

Figure 4
Figure 4

Time course of ACTH and corticosterone responses to 8-OH-DPAT. The mice received saline (0 time point) or 8-OH-DPAT (0.1 mg/kg s.c.). Data represent mean ± S.E. of eight CD-1 mice per group. One-way ANOVA: for ACTH, F3,26 = 2.47, P = .084; and for corticosterone,F3,27 = 5.47, P < .01. ∗, significant difference from saline group (the zero time points), P < .05 (Student-Newman-Keuls post hoc test).

At the 15-min time point, 8-OH-DPAT significantly increased the plasma concentrations of oxytocin, ACTH, and corticosterone in a dose-related manner in the CD-1 purchased mice (Fig.5). Plasma oxytocin concentrations were significantly increased at the 0.1-mg/kg dose of 8-OH-DPAT and were further increased at the 0.5-mg/kg dose of 8-OH-DPAT (Fig. 5A). Plasma ACTH concentrations were significantly increased after injections of 0.1 mg/kg 8-OH-DPAT and did not increase further after the 0.5 mg/kg 8-OH-DPAT dose (Fig. 5B). Corticosterone levels were significantly increased by the 0.05-mg/kg dose of 8-OH-DPAT, and the corticosterone response reached maximum values after the 0.5-mg/kg dose of 8-OH-DPAT (Fig. 5C).

Figure 5
Figure 5

Dose-response curves of 8-OH-DPAT-induced increases of plasma oxytocin (A), ACTH (B), and corticosterone (C). The mice received saline (0 dose of 8-OH-DPAT) or 8-OH-DPAT (0.1 mg/kg s.c.) 15 min before sacrifice. Data represent mean ± S.E. of eight CD-1 mice per group. One-way ANOVA for oxytocin,F3,29 = 12.22, P < .001; for ACTH, F3,29 = 3.32,P < .05; and for corticosterone,F3,27 = 17.5, P < .001. ∗, significant difference from saline group (0 dose of 8-OH-DPAT), P < .05 (Student-Newman-Keuls post hoc test).

In the 5-HTT+/+ mice, 8-OH-DPAT significantly increased plasma oxytocin concentrations approximately 2.5-fold relative to saline-treated controls (Fig. 6). The oxytocin response to 8-OH-DPAT was diminished and not statistically different from saline-treated mice in 5-HTT−/− and 5-HTT+/− mice. There was no significant genotype-related difference in oxytocin levels after saline injections, although the somewhat greater increases in 5-HTT−/− > 5-HTT+/− > 5-HTT+/+ mice might have partially contributed to the different responses to 8-OH-DPAT across genotypes.

Figure 6
Figure 6

The oxytocin response to 8-OH-DPAT in 5-HTT knockout mice. The mice received saline (0 dose of 8-OH-DPAT) or 8-OH-DPAT (s.c.) and were decapitated 15 min after the injections. Data represent mean ± S.E. of eight mice per group. Two-way ANOVA was performed on log-transformed data to maintain homogeneity of variance: main effect of genotype, F2,33 = 0.29,P = .75; main effect of 8-OH-DPAT,F1,33 = 18.97, P < .001; and interaction between genotype and 8-OH-DPAT,F2,33 = 7.01, P = .029. ∗, significant difference from saline group of the same 5-HTT genotype mice, P < .05 (Student-Newman-Keuls post hoc test). #, significant difference from the 5-HTT+/+ mice with the same treatment, P < .05 (Student-Newman-Keuls post hoc test).

8-OH-DPAT significantly increased plasma corticosterone concentrations in 5-HTT+/+ and 5-HTT+/− mice relative to saline-treated mice (Fig.7). However, the corticosterone response to 8-OH-DPAT was absent in the 5-HTT knockout mice. There were no significant genotype-related changes in corticosterone concentrations after saline injections.

Figure 7
Figure 7

The corticosterone response to 8-OH-DPAT in 5-HTT knockout mice. The mice received saline (0 dose of 8-OH-DPAT) or 8-OH-DPAT (s.c.) and were decapitated 15 min after the injections. Data represent mean ± S.E. of eight mice per group. Two-way ANOVA: main effect of genotype, F2,41 = 7.98,P < .01; main effect of 8-OH-DPAT,F1,41 = 10.8, P < .05; and interaction between genotype and 8-OH-DPAT,F2,41 = 3.46, P < .05. ∗, significant difference from saline group of same 5-HTT genotype mice, P < .05 (Student-Newman-Keuls post hoc test). #, significant difference from the 5-HTT+/+ mice with the same treatment, P < .05 (Student-Newman-Keuls post hoc test).

8-OH-DPAT relative to saline significantly increased plasma ACTH concentrations in the 5-HTT+/+ mice (Fig.8). However, unlike corticosterone, plasma ACTH concentrations were increased in saline-injected 5-HTT+/− and 5-HTT−/− mice. ACTH concentrations after injection of 8-OH-DPAT in the 5-HTT+/− and 5-HTT−/− mice were of similar magnitude as those in 5-HTT+/+ mice. Thus, the lack of difference from saline-injected mice could probably be accounted for by the significantly increased ACTH responses to saline injection-related handling relative to differences in plasma ACTH concentrations in mice decapitated without saline injections (Table 1).

Figure 8
Figure 8

The ACTH response to 8-OH-DPAT in 5-HTT knockout mice. The mice received saline (0 dose of 8-OH-DPAT) or 8-OH-DPAT (s.c.) and were decapitated 15 min after the injections. Data represent mean ± S.E. of eight mice per group. Two-way ANOVA: main effect of genotype, F2,40 = 1.76,P = .185; main effect of 8-OH-DPAT,F1, 40 = 1.9, P = .175; and interaction between genotype and 8-OH-DPAT,F2,40 = 6.39, P = .01. ∗, significant difference from saline group of same 5-HTT genotype mice, P < .05 (Student-Newman-Keuls post hoc test). #, significant difference from the 5-HTT+/+ mice with the same treatment, P < .05 (Student-Newman-Keuls post hoc test).

View this table:
Table 1

Basal plasma ACTH and corticosterone concentrations in the 5-HTT transgenic mice

Density of 5-HT1A Receptors in Hypothalamus and Dorsal Raphe.

Both [125I]MPPI- and [3H]8-OH-DPAT-binding sites were significantly reduced approximately 30% in the hypothalamus of 5-HTT−/− mice compared with 5-HTT+/+ mice (Fig. 9). Neither [125I]MPPI- nor [3H]8-OH-DPAT binding -sites in the hypothalamus were significantly different between 5-HTT+/+ and 5-HTT+/− mice. [125I] MPPI-binding sites in the dorsal raphe were significantly reduced in the 5-HTT−/− mice (Fig. 10). The reduction in [125I]MPPI-binding sites in the dorsal raphe was genotype related. Because all of these binding assays were conducted using a single ligand concentration, the changes could be due to either a decrease in the Bmax or an increase in theKd.

Figure 9
Figure 9

[125I]MPPI- (top) and [3H]8-OH-DPAT- (bottom) binding sites in the hypothalamus of 5-HTT mutant mice. Data represent mean ± S.E. of eight mice per group. One-way ANOVA: for [125I]MPPI-binding sites,F2,18 = 11.4, P < .01; and for [3H]8-OH-DPAT-binding sites,F2,18 = 7.51, P < .01. ∗, significant difference from the 5-HTT+/+ mice,P < .05 (Student-Newman-Keuls post hoc test).

Figure 10
Figure 10

[125I]MPPI-binding sites in the dorsal raphe of 5-HTT mutant mice. Data represent mean ± S.E. of five to seven mice per group. One-way ANOVA:F2,15 = 7.66, P < .01. ∗, significant difference from the 5-HTT+/+ mice,P < .05 (Student-Newman-Keuls post hoc test).

Discussion

The present results suggest a desensitization of 5-HT1A receptors in both the raphe nuclei and the hypothalamus of mice lacking 5-HTTs. Electrophysiologically measured 5-HT1A receptors desensitization has previously been reported to follow repeated SRI administration (Blier and De Montigny, 1998). In the present study, we are inferring desensitization on the basis of a functional response to 5-HT1Aagonists, together with directly measured decreases in the number of 5-HT1A receptors in these two brain regions (Davidson and Stamford, 1998). The present results suggest that 5-HTT knockout-related desensitization of 5-HT1Areceptors may, at least in part, result from observed reductions in the density of 5-HT1A-binding sites in the hypothalamus and the dorsal raphe, although other mechanisms, such as in postreceptor signaling mechanisms, also are likely involved.

Although desensitization of 5-HT1A receptors was observed in 5-HTT knockout mice and has been reported after chronic SRI treatment, the mechanism of the desensitization of 5-HT1A receptors may be different in these two models. Most studies have shown that repeated administration of SRIs did not reduce the density of 5-HT1A-binding sites. It has been hypothesized that the effects of SRIs on 5-HT1A receptors may be mediated by changes in G protein coupling and second messenger systems (Broekkamp et al., 1995). In contrast, 5-HTT knockout mice have a decrease in both 5-HT1A antagonist [125I-MPPI]- and 5-HT1Aagonist ([3H]8-OH-DPAT)-binding sites, indicating that there are reductions in total receptor numbers as well as active 5-HT1A receptor numbers. This difference in the density of 5-HT1A-binding sites between the 5-HTT knockout mice and rodents treated with chronic SRIs could be due to the extent and/or duration of the reduction in 5-HTT capacity, which is likely to be more complete and is present since gestation in 5-HTT−/− mice, compared with rodents chronically administered SRIs for 2 to 12 weeks.

Hypothermic responses to 5-HT1A agonists have been used as a marker to evaluate the function of 5-HT1A receptors. However, the location of 5-HT1A receptors that mediate the 5-HT1A agonist-induced hypothermia may be different between rats and mice. Most studies reported that hypothermic responses to 8-OH-DPAT in mice are mediated by 5-HT1A autoreceptors in the raphe nuclei (Goodwin et al., 1985; Bill et al., 1991; Martin et al., 1992), whereas 8-OH-DPAT-induced hypothermia in rats is mediated by postsynaptic 5-HT1A receptors (Bill et al., 1991; Millan et al., 1993). These conclusions are based on evidence that 5,7-dihydroxytryptamine lesions attenuated 8-OH-DPAT-induced hypothermia in mice but not in rats (Goodwin et al., 1985; Bill et al., 1991; Martin et al., 1992). However, the effect ofp-chlorophenylalanine on 8-OH-DPAT-induced hypothermia in mice remains controversial across different studies (Goodwin et al., 1985; Matsuda et al., 1990; Meller et al., 1992). These differences may be due to the different doses of p-chlorophenylalanine used in these studies. Also, variations between mice strains may contribute to these different results. In addition, the effects of antagonists on 8-OH-DPAT-induced hypothermia are different between rats and mice (Moser, 1991; Meller et al., 1992). For example, hypothermic responses to 8-OH-DPAT were blunted by pretreatment with the 5-HT1A antagonist and β-receptor antagonist pindolol in rats but not in mice. All together, it is likely that 8-OH-DPAT-induced hypothermia is mediated by 5-HT1A autoreceptors in the dorsal raphe. However, other mechanisms still cannot be completely ruled out.

Considering the variation between mice strains and the inconsistent effects of 5-HT1A antagonists on 8-OH-DPAT-induced hypothermia, we determined the pharmacological profile of 8-OH-DPAT-induced hypothermia in CD-1 mice and transgenic 5-HTT+/+ normal littermates. We found that 8-OH-DPAT dose-dependently induced hypothermic responses with a maximum dose of 0.5- mg/kg 8-OH-DPAT. The peak time for the hypothermic response to 8-OH-DPAT was 20 min. These results are consistent with the observations from other investigators using other mouse strains (Goodwin et al., 1985; Bill et al., 1991). In addition, our data demonstrate that 8-OH-DPAT-induced hypothermia was eliminated by the selective 5-HT1A antagonist WAY-100635, confirming that the hypothermic response to 8-OH-DPAT is mediated by 5-HT1A receptors. The present results showed that 8-OH-DPAT-induced hypothermia was eliminated in 5-HTT−/− mice, demonstrating an essentially complete desensitization of 5-HT1A receptor function in 5-HTT−/− mice, consonant with predictions based on data obtained from rodents chronically administered SRIs. Using electrophysiological or microdialysis techniques, several investigators reported that repeated administration of SRIs induces desensitization of 5-HT1A autoreceptors in the raphe nuclei (Chaput et al., 1991; Le Poul et al., 1995; Romero et al., 1996; Davidson and Stamford, 1998). The SRI-induced desensitization of raphe 5-HT1A receptors has been hypothesized to contribute to the therapeutic effects of SRIs (Blier and De Montigny, 1998).

The hypothalamus is involved in the regulation of the secretion of several hormones as well as eating behaviors, sexual behaviors, and mood changes (Kandel et al., 1995; Hoffman et al., 1998). 5-HT1A receptors in the hypothalamus may be important for these functions. Although hormonal responses to 5-HT1A agonists have been extensively used to evaluate the functional status of hypothalamic 5-HT1A receptors in both humans and rats, few studies have reported hormonal responses to 5-HT1A agonists in mice. In the present studies, we first examined the dose-response and (for ACTH and corticosterone only) the time course of the oxytocin, ACTH, and corticosterone responses to 8-OH-DPAT in CD-1 and, to a partial extent, in transgenic 5-HTT+/+ mice. In general, the CD-1 and 5-HTT+/+ mice had similar dose-related responses to 8-OH-DPAT (e.g., the ED50 value for the corticosterone response to 8-OH-DPAT in CD-1 mice was 0.065 mg/kg, and that in transgenic 5-HTT+/+ mice was 0.07 mg/kg). The time course of 8-OH-DPAT-induced increases in ACTH and corticosterone in mice is similar to that in rats (Di Sciullo et al., 1990). However, the magnitude of 8-OH-DPAT-induced elevations of ACTH is much lower in mice than in rats, an observation consistent with previous investigations (Matsuda et al., 1991; Li et al., 1993).

Oxytocin is released directly from hypothalamic magnocellular neurons. ACTH and corticosterone are released from the pituitary and adrenal glands, respectively, although their secretions are controlled by the hypothalamic hormone corticortropin-releasing hormone. Therefore, the magnitude of 8-OH-DPAT-induced increases of oxytocin provides potentially more direct indicator of the function of hypothalamic 5-HT1A receptors than those of ACTH and corticosterone. The present results demonstrate the absence of an oxytocin response to 8-OH-DPAT in 5-HTT−/− mice, indicating an essentially complete desensitization of 5-HT1Areceptors in the hypothalamus.

An unexpected observation was that ACTH levels in the saline-injected 5-HTT+/− and 5-HTT−/− mice were significantly higher than those in 5-HTT+/+ mice. This could be due to a stress effect induced by the handling and saline injection procedure, because the basal ACTH levels (i.e., those measured immediately after decapitation without saline injections or attendant handling) were not increased in either 5-HTT−/− or 5-HTT+/− mice (Table 1). These data suggest that 5-HTT−/− and 5-HTT+/− mice are more reactive to stressful conditions than 5-HTT+/+ mice. Indeed, a similar observation has been reported in transgenic mice expressing an antisense oligonucleotide that blocks glucocorticoid receptors (Karanth et al., 1997; Dijkstra et al., 1998). It is possible that the 5-HTT knockout induces a change in the hypothalamic-pituitary-adrenal axis because serotonin is an important regulator in the development of the central nervous system (Azmitia and Whitaker-Azmitia, 1997; Hoffman et al., 1998; Persico et al., 1998;Vitalis et al., 1998).

Although ACTH concentrations were increased in the saline-injected 5-HTT+/− and 5-HTT−/− mice relative to mice decapitated without saline injection and the subsequent 15-min period before decapitation, corticosterone levels in these mice were not increased. At present, there is no good explanation for the differential responses observed in plasma ACTH and corticosterone responses. One hypothesis might be that the sensitivity of the adrenal gland to ACTH is altered by a deficiency or reduction in 5-HTT function, because 5-HTT-binding sites are known to be present in the adrenal gland (Blakely et al., 1991; Lefebvre et al., 1998). 5-HT in the adrenal gland stimulates the secretion of corticosterone through 5-HT4 receptors in frogs and humans, although not in rats; similar studies in mice have apparently not been reported (Hegde and Eglen, 1996; Lefebvre et al., 1998). 5-HT4 receptors share the same second messenger with ACTH receptors. It is possible that 5-HTT knockout-induced changes in adrenal 5-HT concentrations trigger changes in the second messenger system of 5-HT4receptors, which influence the efficiency of ACTH receptors. Future studies, however, are needed to evaluate this hypothesis.

In conclusion, the present results suggest a desensitization of 5-HT1A receptors in 5-HTT−/− mice. These results are consistent with the observations that chronic administration of SRIs desensitizes 5-HT1Areceptors in the hypothalamus and the dorsal raphe nuclei. However, the components of the 5-HT1A receptor system that are involved in the desensitization of 5-HT1Areceptors might be different in 5-HTT knockout mice and rodents treated with SRIs. Desensitization of 5-HT1A receptors in mice lacking the 5-HTT may be mediated, at least in part, by the observed down-regulation of the number of 5-HT1Areceptor sites. Further studies are necessary to determine the basis of differences between these two ways in which 5-HTT function is altered.

Acknowledgments

We thank Francisca Garcia, Teresa Tolliver, and Su-Jan Huang for their excellent technical assistance with the experiments.

Footnotes

  • Send reprint requests to: Qian Li, Ph.D., Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health Clinical Center, Room 3D41, 10 Center Dr., MSC-1264, Bethesda, MD 20892-1264. E-mail:qianli{at}codon.nih.gov

  • ↵1 K.-P.L. is supported by the Hermann and Lilly Schilling Foundation.

  • Abbreviations:
    5-HT
    5-hydroxytryptamine (serotonin)
    5-HTT
    5-hydroxytryptamine transporter
    8-OH-DPAT
    8-hydroxy-2-(di-n-propylamino)tetraline
    MPPI
    4-(2′-methoxyphenyl)-1-[2′-[N-(2"-pyridinyl)-iodobenzamido]ethyl]piperazine
    WAY-100635
    (N-[2-(4(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-2-pyridyl)cyclohexanecarboxamide
    ACTH
    corticotropin
    SRI
    serotonin reuptake inhibitor
    • Received March 26, 1999.
    • Accepted August 7, 1999.

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Reduction of 5-Hydroxytryptamine (5-HT)1A-Mediated Temperature and Neuroendocrine Responses and 5-HT1A Binding Sites in 5-HT Transporter Knockout Mice

Qian Li, Christine Wichems, Armin Heils, Louis D. Van de Kar, Klaus-Peter Lesch and Dennis L. Murphy
Journal of Pharmacology and Experimental Therapeutics December 1, 1999, 291 (3) 999-1007;
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