Posted by jrbecker on November 17, 2004, at 11:00:37
http://www.sciencedaily.com/releases/2004/11/041116214900.htm
Source: Yale University
Date: 2004-11-17
Brain's Nicotine Receptors Also Target For Anti-depressantsNew Haven, Conn. -- The same receptors in the brain that are activated when a person smokes cigarettes also play a critical role in the effectiveness of antidepressants, according to a study by Yale researchers in the November issue of Biological Psychiatry.
What this means, particularly for patients who are suicidal, is that finding a way to activate these receptors will make anti-depressants work more quickly. Most anti-depressants now take up to three weeks to bring emotional relief.
"Just the ability to block the reuptake of serotonin isn't enough, otherwise it wouldn't take two to three weeks to be effective, " said Marina Picciotto, associate professor of psychiatry, pharmacology and neurobiology at Yale School of Medicine and senior author of the study. "This finding has implications for those patients who are depressed to the point of being suicidal, and for the 30 percent of people who are not responsive to anti-depressants that are now available."
The primary pharmacologic treatment for depression over the past several decades has been drugs that inhibit synaptic reuptake of monoamine neurotransmitters. Recent evidence indicates other neurotransmitter systems might play a role in the mechanism of action of antidepressants, Picciotto said.
In this study in mice, she and her colleagues tested the action of antidepressants with and without mecamylamine, a noncompetitive antagonist (blocker) in nicotinic acetylcholine receptors (nAChRs). In a separate study using knockout mice that lack these receptors, they found that the function of the nicotine receptor in the brain was an essential component of the therapeutic action of antidepressants.
Picciotto said the next step will be to study the role of nAChRs in regulating the behavioral and cellular responses to antidepressants. They will see if there is a direct effect mediated by nAChRs or an indirect effect of modulating neurotransmission in other cell types. "Use of more specific nAChRs antagonists, both alone and in combination with classic antidepressants, could lead to the development of novel and more effective treatments for individuals who suffer from depression," she said.
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Citation: Biological Psychiatry, Vol. 56 (9); pp 657-664.
Editor's Note: The original news release can be found here.
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This story has been adapted from a news release issued by Yale University.
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Biological Psychiatry
Volume 56, Issue 9 , 1 November 2004, Pages 657-664doi:10.1016/j.biopsych.2004.08.010
Copyright © 2004 Society of Biological Psychiatry Published by Elsevier Inc.
Original articlesHigh-affinity nicotinic acetylcholine receptors are required for antidepressant effects of amitriptyline on behavior and hippocampal cell proliferation
Barbara J. Caldaronea, Alexia Harrista, Muriel A. Clearya, Robert D. Beecha, Sarah L. Kinga and Marina R. Picciottoa, ,
aDepartment of Psychiatry, Yale University School of Medicine, New Haven, Connecticut
Received 26 April 2004; revised 12 August 2004; accepted 19 August 2004. Available online 30 October 2004.
Background
A wide variety of antidepressants act as noncompetitive antagonists of nicotinic acetylcholine receptors (nAChRs), but the relationship between this antagonism and the therapeutic effects of antidepressants is unknown.Methods
Antidepressant properties of the noncompetitive nAChR antagonist mecamylamine in the forced swim test were tested alone and in combination with the tricyclic antidepressant amitriptyline. Mice lacking high-affinity nAChRs were tested in three behavioral models to determine whether these receptors are required for behavioral effects of amitriptyline in common models of antidepressant action. Finally, the brains of wild-type and knockout animals treated with amitriptyline were examined to determine whether high-affinity nAChRs are required for antidepressant-induced increases in hippocampal cell proliferation.Results
Inhibition of nAChRs by mecamylamine had antidepressant-like effects in the forced swim test and potentiated the antidepressant activity of amitriptyline when the two drugs were used in combination. Mice lacking high-affinity nAChRs showed no behavioral response to amitriptyline. Finally, after chronic treatment with amitriptyline, nAChR knockout mice did not show the increase in hippocampal cell proliferation seen in wild-type mice.Conclusions
These data support the hypothesis that antagonism of nAChRs is an essential component of the therapeutic action of antidepressants.Key words: Nicotinic acetylcholine receptors; depression; learned helplessness; forced swim; mecamylamine; tail suspension
Article Outline
Methods and materials
Animals
Oral amitriptyline administration
Behavioral testing
Effects of mecamylamine in the forced swim test
Effects of amitriptyline in β2+/+ and β2-/- mice in the forced swim and learned helplessness tests
Learned helplessness
Effects of amitriptyline in β2+/+ and β2-/- mice in the tail suspension test
Serum levels of amitriptyline/nortriptyline and measurement of amitriptyline intake
Adult hippocampal cell proliferation
Statistical analysis
Results
The nAChR antagonist mecamylamine has antidepressant-like effects in the forced swim test
High-affinity nAChRs are required for the behavioral effects of amitriptyline in three behavioral models of antidepressant activity
Learned helplessness
Tail suspension and forced swim tests
Effects of amitriptyline on cell proliferation in the dentate gyrus require high-affinity (β2-subunit-containing) nAChRs
Discussion
Acknowledgements
References
The primary pharmacologic treatment for depression over the past several decades has been drugs that inhibit synaptic reuptake of monoamine neurotransmitters. Although the importance of monoamine neurotransmission in antidepressant efficacy cannot be discounted, recent evidence indicates other neurotransmitter systems might play a role in the mechanism of action of antidepressants (Shytle et al 2002a). Furthermore, the limitations of current antidepressant treatments, including slow onset of action and low efficacy, necessitate the development of novel compounds to treat depression.A growing body of evidence suggests that cholinergic neural systems might be potential targets for the development of novel antidepressant compounds. Cholinergic hyperactivity has been postulated to play a role in the pathophysiology of depression (Janowsky et al 1972). Stimulation of cholinergic systems with agents such as the cholinesterase inhibitor physostigmine can induce depressive-like symptoms in individuals with affective disorders, as well as in normal subjects (Janowsky et al 1972; Risch et al 1980). These findings are supported by animal studies demonstrating that the Flinders Sensitive line of rats, selectively bred for increased sensitivity to cholinergic agents, shows behavior that resembles symptoms of depression in humans (Overstreet 1993). Furthermore, inescapable footshock and swim stress, which are used to induce “depressive-like” states in rodents, can both induce a supersensitivity of the cholinergic system (Dilsaver and Alessi 1987; Dilsaver et al 1986). Taken together, these studies suggest that excessive activation of cholinergic systems might contribute to the pathophysiology of depression.
Whereas much of the research on the relationship between the cholinergic system and depression has focused on muscarinic neuropharmacology (Dilsaver 1986; Overstreet 1993), fewer studies have addressed the role that nicotinic acetylcholine receptors (nAChRs) play in depression and the action of antidepressants. Some evidence suggests that nAChRs are a common target of currently used antidepressant drugs. Studies at the cellular, physiologic, and behavioral levels have shown that a wide range of antidepressants, including tricyclic antidepressants, selective serotonin reuptake inhibitors, and atypical antidepressants, all act as noncompetitive antagonists of nAChRs (Fryer and Lukas 1999; Hennings et al 1997; Lopez-Valdes and Garcia-Colunga 2001; Schofield et al 1981; Slemmer et al 2000); however, to date there are no studies that have directly examined the role of specific nAChRs in an animal model of depression.
In the present study, we used three complementary approaches to examine the role of high-affinity nAChRs in animal models of antidepressant activity. First, we tested the antidepressant properties of the noncompetitive nAChR antagonist mecamylamine in the forced swim model of depression, both alone and in combination with the tricyclic antidepressant amitriptyline. Second, we tested knockout mice lacking high-affinity nAChRs in the forced swim, tail suspension, and learned helplessness models of antidepressant activity to determine whether these receptors are required for the behavioral effects of amitriptyline. Finally, we examined the brains of wild-type and nAChR knockout mice treated with amitriptyline to determine whether high-affinity nAChRs are required for antidepressant-induced increases in hippocampal cell proliferation. We found that direct antagonism of nAChRs by mecamylamine had antidepressant-like effects in the forced swim test and that mice lacking high-affinity nAChRs were resistant to amitriptyline across the three behavioral and cellular models of antidepressant efficacy. These data support the hypothesis that antagonism of high-affinity neuronal nAChRs is an essential component of the therapeutic mechanism of action of a classical antidepressant compound.
Methods and materials
Animals
Knockout mice lacking the β2 subunit of the nAChR (β2-/-) were backcrossed at least 12 generations to C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) and were then generated from β2-/- matings. Control mice were age- and gender-matched C57BL/6J (β2+/+). The β2+/+ and β2-/- mice were group-housed in cages, with a maximum of five mice per cage. Mice were kept in a colony room maintained at 22°C on a 12-hour light/dark cycle, with lights on at 7:00 AM. Food and water were available at all times. All behavioral testing was carried out during the light portion of the light/dark cycle. All animal procedures were in strict accordance with the National Institutes of Health’s Care and Use of Laboratory Animals Guidelines and were approved by the Yale Animal Care and Use Committee.Oral amitriptyline administration
Amitriptyline hydrochloride (Sigma, St. Louis, Missouri) was administered in the drinking water as the sole source of fluid. The concentration of amitriptyline used was based on a previous study of learned helplessness in rats (Sherman et al 1979) and our previous work in mice (Caldarone et al 2003). Amitriptyline (200 μg/mL, free base) was dissolved in 2% saccharin to increase palatability; control mice received 2% saccharin alone. Mice were given amitriptyline or saccharin either chronically (21 days) (learned helplessness and forced swim test) or subchronically (tail suspension test) (4 days) before the start of behavioral testing, and solutions were changed twice per week. Mice continued to drink amitriptyline or saccharin throughout the duration of behavioral testing.Behavioral testing
Effects of mecamylamine in the forced swim test
The forced swim test was carried out in a glass cylinder (18.5-cm diameter, 25-cm height) filled with water to the height of 17 cm. Water was maintained at 23°C to 25°C. Mice were placed into the water, and immobility times were recorded by an observer for 15 min. A wide variety of clinically used antidepressants decrease immobility time in this test (Porsolt et al 1977), and it has been widely used as a screening test for new compounds with antidepressant activity. C57BL/6J male mice (2–3 months old) received a single intraperitoneal (IP) injection of mecamylamine hydrochloride (Sigma) (.50, .75, or 1.0 mg/kg) in an injection volume of 10 mL/kg or saline 30 min before forced swim testing. A separate group of C57BL/6J male mice (2–3 months old) received either subchronic treatment with amitriptyline (4 days amitriptyline [200 μg/mL] in the drinking water) in saccharin or saccharin alone. Mice were given an IP injection of .5 mg/kg mecamylamine or saline 30 min before forced swim testing.Effects of amitriptyline in β2+/+ and β2-/- mice in the forced swim and learned helplessness tests
β2+/+ and β2-/- mice were treated chronically (21 days) with amitriptyline in their drinking water in saccharin or with saccharin alone and were tested in the forced swim test (6–9 months old) as described above or in the learned helplessness paradigm (4–10 months old).Learned helplessness
Learned helplessness training was carried out in a shuttle box (Med Associates, St. Albans, Vermont) (43 × 17 × 25.5 cm) in which the front, back, and ceiling were clear Plexiglas, and the sides were aluminum. Scrambled shock was delivered by a shock source to a grid floor made of stainless steel bars spaced .50 cm apart. During training, a panel was inserted into the shock chamber, dividing it in half. Mice were placed on either side of the chamber so that two mice were administered shocks simultaneously. Escape testing was administered in the same chamber, except that a gate divided the chamber into 2 equal compartments. The gate was equipped with a door that opened manually into a 9 × 11.5-cm archway with a 1-cm hurdle.Learned helplessness procedures were based on those reported previously (Caldarone et al 2000, 2003; Shanks and Anisman 1988). Learned helplessness was induced by administering 120 inescapable, 4-sec footshocks (.3 mA) once every 26 sec over a 1-hour session. Training was given in two sessions that were spaced approximately 24 hours apart. A control group that did not receive shock during learned helplessness training was exposed to the apparatus for an equivalent period of time. Approximately 24 hours after the second learned helplessness training session, mice were tested on the shuttle escape task. Mice were given 30 shuttle escape trials with 30-sec intervals between the start of each trial. On the first five trials, the gate opened at the same time that the shock was turned on. For the remaining trials, the gate opened 2 sec after shock onset. Intensity of shock during the shuttle escape task was .3 mA, but duration was variable depending on when the mouse made an escape response. Each trial was terminated when the mouse crossed over the hurdle into the adjacent compartment. If an escape response was not made, the trial was terminated 24 sec after shock onset. If mice escaped the shock by jumping onto the hurdle on two consecutive trials, the hurdle was removed for the remainder of the escape test.
Effects of amitriptyline in β2+/+ and β2-/- mice in the tail suspension test
β2+/+ and β2-/- mice (4–5 months old) were treated subchronically (4 days) with amitriptyline and saccharin in drinking water or with saccharin alone and were tested in the tail suspension test. Approximately 1 cm from the end, each mouse’s tail was taped to a 36-cm piece of tubing. Mice were suspended by the tail approximately 120 cm above the floor, and the duration of immobility was recorded by an observer for 6 min.Serum levels of amitriptyline/nortriptyline and measurement of amitriptyline intake
Mice were sacrificed by rapid decapitation, and trunk blood was collected, centrifuged, and stored at −80°C. Serum levels of amitriptyline and its major metabolite nortriptyline (NOR) were measured by fluorescence polarization immunoassay (Abbott Laboratories, Abbott Park, Illinois). The assay is a competitive binding procedure that uses a fluorescein-labeled ligand. When the ligand is displaced from the antibody by the unlabeled drug in the sample, fluorescence polarization is reduced. The assay provides semiquantitative levels of amitriptyline plus NOR levels. The assay was carried out on an AxSYM analyzer (Abbott Laboratories).An independent group of mice was individually housed, and intake of amitriptyline was measured for 3 weeks. Mice were weighed each week, and intake of amitriptyline was calculated by averaging intake (milligrams amitriptyline per kilogram body) across days 1–7 (subchronic) and days 8–21 (chronic). Portions of the intake and serum measurement data that serve as a control for the current study have been reported previously (Caldarone et al 2003).
Adult hippocampal cell proliferation
β2+/+ and β2-/- mice (6 months old) received either chronic (at least 1 month) amitriptyline (200 μg/mL) in saccharin, or saccharin alone in their drinking water. Bromodeoxyuridine (BrdU) labeling of proliferating cells was performed essentially as described by Eisch et al (2000). Mice received a single IP injection of BrdU (75 mg/kg) (Roche Diagnostics, Indianapolis, Indiana) to measure the number of cells in S phase. Two hours after BrdU injection, mice were deeply anesthetized with chloral hydrate and perfused transcardially with 4% paraformaldehyde. After perfusion, brains were postfixed overnight in 4% paraformaldehyde at 4°C and stored in 30% sucrose. Serial 30-μm coronal sections of the brain were cut on a freezing microtome through the entire hippocampus and stored in .1% sodium adize/.1 mol/L phosphate-buffered saline (PBS) at 4°C. Every ninth section of the hippocampus was mounted, and slides were coded to ensure objectivity. Bromodeoxyuridine immunohistochemistry was performed as previously described (Eisch et al 2000). Deoxyribonucleic acid denaturation was conducted by incubating slides in .01 mol/L citric acid (pH 6.0) at 100°C for 13 min. Membrane permeabilization and acidification was then carried out by incubating slides for 10 min in .1% trypsin/.1 mol/L Tris/.1% calcium chloride and for 30 min in 2 N HCl at room temperature. Slides were blocked in 3% normal goat serum (NGS) (Vector Laboratories, Burlingame, California)/.5% Triton X/.1 mol/L PBS for 1 hour, incubated in 1:100 mouse anti-BrdU (Becton Dickinson, Franklin Lakes, New Jersey) in 3% NGS/.5% Tween 20/.1 mol/L PBS for 20 hours and subsequently incubated with biotinylated goat-antimouse immunoglobulin G (1:400; Sigma)/1.5% NGS for 1 hour. Endogenous peroxidases were quenched by incubation in .3% hydrogen peroxide/.1 mol/L PBS for 30 min, and slides were incubated with an avidin-biotin complex (Vector Laboratories) for 1 hour. Visualization of BrdU-positive cells was achieved by incubation with diaminobenzidine (Vector Laboratories) for 10 min. This was followed by counterstaining for 1.5 min with Fast Red (Vector Laboratories) to label cell nuclei.Bromodeoxyuridine-positive cells in the dentate gyrus were counted under a light microscope (Olympus BX-60; Tokyo, Japan) at 400× magnification in every ninth section of the dentate gyrus from each brain (approximately Bregma −.82 mm to 4.16 mm) (Franklin and Paxinos 1997). Cells were counted throughout both the granule cell layer and hilus, and any positive cells touching the granule cell layer were considered part of that layer. Cells counted were multiplied by 9 to obtain total cell counts for each animal. Portions of the control data have been reported previously (Harrist et al, in press).
Statistical analysis
The effects of mecamylamine in the forced swim test were analyzed by t tests with a Bonferroni correction for multiple comparisons. Behavioral data from the knockout studies were analyzed with repeated-measures analysis of variance (ANOVA) with drug (saccharin, amitriptyline) and genotype (β2+/+, β2-/-) as the between-subjects variables and block (learned helplessness, 1–6; tail suspension, 1–6; forced swim 1–3) as the within-subjects variables. Intake and cellular proliferation data were analyzed by ANOVA with drug (saccharin, amitriptyline) and genotype (β2+/+, β2-/-) as the between-subjects variables. Significant effects were followed up with the least significant difference post hoc test (α = .05).Results
The nAChR antagonist mecamylamine has antidepressant-like effects in the forced swim test
The antidepressant properties of the high-affinity, noncompetitive nAChR antagonist mecamylamine were evaluated in the forced swim model of antidepressant activity. Mecamylamine dose-dependently reduced immobility in the forced swim test, and this decrease reached significance at the 1.0 mg/kg dose (Figure 1A) [t(19) = 2.7, p = .013, saline vs. 1.0 mg/kg mecamylamine]. Thus, in this test, acute mecamylamine administration has effects similar to those seen with currently used antidepressants.
(42K)Figure 1. Mecamylamine has antidepressant-like effects in the forced swim test. (A) C57BL/6J male mice were administered .50 (n = 8), .75 (n = 8), or 1.0 mg/kg (n = 12) of mecamylamine or saline (n = 9) 30 min before forced swim testing. Mecamylamine dose-dependently reduced immobility, but the reduction reached significance only at the 1.0 mg/kg dose (*p < .05 vs. saline). (B) Mice were treated subchronically (4 days) with either saccharin or amitriptyline (200 μg/mL) and given an IP injection of either .5 mg/kg mecamylamine or saline 30 min before forced swim testing (saccharin-saline, n = 12; saccharin-mecamylamine, n = 13; amitriptyline-saline, n = 11; amitriptyline-mecamylamine, n = 13). Neither amitriptyline nor mecamylamine delivered in this regimen affected immobility time on its own, but the combination of amitriptyline and mecamylamine produced a significant reduction in immobility (*p < .05 vs. saccharin-saline). In both panels, data are presented as mean time immobile (±SE) averaged over the 15-min test.
Next, we examined the effects of combining suboptimal treatments of mecamylamine and the classic tricyclic antidepressant amitriptyline in the forced swim test. Mice were given subchronic amitriptyline in the drinking water, which was previously shown to be ineffective in the forced swim test (Caldarone et al 2003). Then, a subeffective .5-mg/kg IP injection of mecamylamine was administered 30 min before forced swim testing. Whereas subthreshold treatment with either amitriptyline or mecamylamine alone had no effect, the combination of these treatments caused a significant reduction in immobility (Figure 1B) [t(23) = 3.8, p = .001, saccharin-saline vs. amitriptyline-mecamylamine].
High-affinity nAChRs are required for the behavioral effects of amitriptyline in three behavioral models of antidepressant activity
The results described above demonstrate that the high-affinity nAChR antagonist mecamylamine has antidepressant-like properties in the forced swim test. We next tested whether nAChRs are required for the behavioral effects of amitriptyline, using β2 subunit nAChR knockout mice, which lack high-affinity nAChRs in the brain (Picciotto et al 1995). β2+/+ and β2-/- mice were treated with amitriptyline at doses that have previously been shown to be effective in C57BL/6 mice (Caldarone et al 2003) in the learned helplessness (Maier 1984), tail suspension (Steru et al 1985), and forced swim (Porsolt et al 1977) models of antidepressant efficacy.Learned helplessness
In the learned helplessness test, both β2+/+ and β2-/- mice became helpless, as shown by increased escape latencies after exposure to inescapable shock compared with nontrained controls (Figure 2A and B). Amitriptyline-treated β2+/+ mice showed reduced escape latencies after training compared with mice treated with saccharin, demonstrating the expected antidepressant effect. In contrast, β2-/- mice treated with amitriptyline showed no reduction in escape latency after training compared with β2-/- saccharin-treated control mice (Figure 2A). Analysis of variance showed a significant drug × genotype interaction [F(1,76) = 4.2, p < .05], with post hoc tests confirming that β2+/+ mice that received saccharin had longer escape latencies than β2+/+ mice treated with amitriptyline. There were no significant differences between β2-/- saccharin-treated and β2-/- amitriptyline-treated mice. In control animals that did not receive training, amitriptyline had no effect on escape latencies in either β2+/+ or β2-/- mice, although there was an increased baseline escape latency in β2-/- mice (Figure 2B) [ANOVA significant main effect of genotype: F(1,48) = 6.4, p < .05]. Therefore, the effects of amitriptyline are specific for blocking learned helplessness rather than nonspecific effects on general motor activity.
(60K)Figure 2. High-affinity nicotinic acetylcholine receptors are required for the effects of amitriptyline in the learned helplessness, tail suspension, forced swim, and tests. (A) β2+/+ and β2-/- mice treated chronically with amitriptyline or saccharin were given 2 days of learned helplessness training (β2+/+-saccharin, n = 21; β2+/+-amitriptyline, n = 19; β2-/--saccharin, n = 21; β2-/--amitriptyline, n = 19). β2+/+ mice treated with amitriptyline showed lower escape latencies than β2+/+ mice treated with saccharin alone. In contrast, β2-/- mice treated with amitriptyline mice showed no difference in escape latency compared with β2-/- mice treated with saccharin (*p < .05, β2+/+-saccharin vs. β2+/+-amitriptyline). (B) Mice that did not receive learned helplessness training showed no effect of amitriptyline, regardless of genotype, although β2-/- mice overall had longer escape latencies than β2+/+ mice (β2+/+-saccharin, n = 13; β2+/+-amitriptyline, n = 13; β2-/--saccharin, n = 14; β2-/--amitriptyline, n = 12). In A and B, data are shown as mean escape latencies (±SE) averaged in six blocks of five trials. (C) In the tail suspension test, mice (n = 10 per group) received either saccharin or amitriptyline for 4 days before testing. β2+/+ mice treated with amitriptyline showed a reduction in immobility, whereas β2-/- mice showed no effect of amitriptyline (*p < .05, β2+/+-saccharin vs. β2+/+-amitriptyline). Data are shown as mean immobility time (±SE) across six 1-min blocks. (D) In the forced swim test, mice were treated chronically (21 days) with either saccharin or amitriptyline before testing (β2+/+-saccharin, n = 13; β2+/+-amitriptyline, n = 13; β2-/--saccharin, n = 14; β2-/--amitriptyline, n = 12). β2+/+ mice treated with amitriptyline showed a reduction in immobility, whereas β2-/- mice showed no effect of amitriptyline. (*p < .05, β2+/+-saccharin vs. β2+/+-amitriptyline). Data are shown as mean time immobile (±SE) across three 5-min blocks.
Tail suspension and forced swim tests
β2+/+ and β2-/- mice were treated with amitriptyline or saccharin and tested in two models of antidepressant activity that do not rely on footshock: the tail suspension and forced swim tests. In both tail suspension (Figure 2C) and forced swim (Figure 2D) tests, β2+/+ mice treated with amitriptyline showed a robust decrease in immobility time compared with saccharin-treated controls, demonstrating the expected antidepressant effect. In contrast, β2-/- mice treated with amitriptyline showed no change in immobility time compared with β2-/- mice treated with saccharin. Analysis of variance revealed a significant drug × genotype interaction for both the tail suspension [F(1,36) = 7.6, p < .01] and forced swim tests [F(1,63) = 6.8, p < .05]. In both the tail suspension and forced swim tests, post hoc tests confirmed that β2+/+ saccharin-treated mice were more immobile than β2+/+ mice that received amitriptyline. There were no significant differences between β2-/- saccharin-treated and β2-/- amitriptyline-treated mice. Analysis of variance also showed a significant main effect of genotype in both the tail suspension test [F(1,36) = 19.8, p < .0001] and the forced swim test [F(1,63) = 17.7, p < .0001], indicating that β2-/- mice overall were less immobile than β2+/+ mice in these tests.The intake and serum levels of amitriptyline (Table 1) and locomotor activity (data not shown) did not differ between β2+/+ and β2-/- mice. These data suggest that the lack of antidepressant response in β2-/- mice was not due to nonspecific effects on drug intake, metabolism, or locomotor response.
Table 1.
Amitriptyline Intake and Blood Levels β2+/+ β2−/−
Subchronic Intake 16.7 ± 1.4 15.2 ± 1.9
Chronic Intake 20.7 ± .94 21.3 ± .81
AMI/NOR Levels 189.3 ± 44.1 186.3 ± 32.6
Mean (±SE) intake (mg/kg/day) of amitriptyline after subchronic and chronic treatment and mean (±SE) serum levels (ng/mL) of amitriptyline and nortriptyline (AMI/NOR) in β2+/+ (intake, n = 9; serum, n = 16) and β2−/− (intake, n = 7; serum, n = 16) mice. There were no differences between β2+/+ and β2−/− mice in either intake or serum levels of amitriptyline.
Effects of amitriptyline on cell proliferation in the dentate gyrus require high-affinity (β2-subunit-containing) nAChRs
Previous studies have shown that chronic treatment with antidepressants increases neurogenesis in the hippocampus (Malberg et al 2000; Santarelli et al 2003). Therefore, we examined antidepressant-induced increases in hippocampal cell proliferation in β2+/+ and β2-/- mice to determine whether a cellular marker of antidepressant responsiveness might be correlated with the behavioral response to amitriptyline. As expected, amitriptyline-treated β2+/+ mice showed an increase in the number of proliferating cells in the hippocampus compared with β2+/+ mice given saccharin (Figure 3); however, amitriptyline-treated β2-/- mice showed no increase in the number of BrdU-labeled cells compared with β2-/- mice treated with saccharin. A baseline decrease in the number of proliferating cells was observed in β2-/- mice, which might suggest a role for endogenous acetylcholine in supporting adult hippocampal cellular proliferation (Harrist et al, in press). These observations were confirmed with ANOVA that showed both a main effect of drug [F(1,17) = 7.1, p < .05] and genotype [F(1,17) = 26.9, p < .01]. Post hoc tests showed that β2+/+ mice treated with amitriptyline had more BrdU-labeled cells than β2+/+ mice treated with saccharin and that β2-/- mice treated with saccharin or amitriptyline were not different from each other.
(100K)Figure 3. β2-containing nicotinic acetylcholine receptors are essential for amitriptyline-induced increases in cell proliferation in the hippocampus. (A) β2+/+ and β2-/- mice given chronic treatment with either saccharin or amitriptyline (β2+/+-saccharin, n = 5; β2+/+-amitriptyline, n = 5; β2-/--saccharin, n = 6; β2-/--amitriptyline, n = 5) received an injection of bromodeoxyuridine (BrdU) 2 hours before perfusion. The number of BrdU-positive cells was increased after chronic amitriptyline treatment in β2+/+ mice. This increase was absent in β2-/- mice (*p < .05, β2+/+-saccharin vs. β2+/+-amitriptyline and β2-/--saccharin). Data are presented as mean number of BrdU-positive cells (±SE) in the hippocampus. (B) Representative sections of BrdU immunoreactivity in β2+/+ and β2-/- mice that received chronic treatment with either saccharin or amitriptyline. Arrows indicate BrdU-labeled cells. Insets show representative clusters of BrdU-labeled cells.
Discussion
The nAChR antagonist mecamylamine had antidepressant-like effects when used alone in the forced swim test and potentiated the effects of a subchronic regimen of amitriptyline when the two drugs were given in combination. These data suggest that acute inhibition of nAChR activity might accelerate the response to a typical antidepressant, which typically takes several weeks. Furthermore, combination therapy with mecamylamine and a classic antidepressant might be beneficial in treatment-resistant individuals. These findings are consistent with the clinical observation that mecamylamine decreased symptoms of depression and anxiety in patients with Tourette’s syndrome (Shytle et al 2002b) and animal studies showing that the nAChR antagonists mecamylamine and dihydro-β-erythroidine potentiated antidepressant effects of imipramine and citalopram in the tail suspension test (Popik et al 2003). Other studies have found that nicotine itself has antidepressant-like effects in both animals (Djuric et al 1999; Ferguson et al 2000; Semba et al 1998; Tizabi et al 1999) and humans (Salin-Pascual et al 1996). The idea that both nicotine and nicotinic receptor antagonists can produce antidepressant-like effects seems paradoxical; however, chronic nicotine administration can result in desensitization and inactivation of nAChRs, resulting in a functional antagonism (Gentry and Lukas 2002). Thus, the antidepressant-like effects of chronic nicotine might be related to its action as a functional antagonist of nAChRs rather than its agonist properties at those receptors.The antidepressant activity of amitriptyline is generally considered to be due to the inhibition of monoamine reuptake. Although the mechanism by which mecamylamine enhances the effects of amitriptyline in the forced swim test is not known, one possibility is that the combination of mecamylamine and amitriptyline might increase synaptic levels of 5-hydroxytryptamine (5-HT), which would result in decreased immobility time in the forced swim test. Evidence for this idea comes from studies in which mecamylamine induced 5-HT release in both rat hippocampal slices (Kenny et al 2000) and dorsal raphe neurons (Mihailescu 1998). Alternatively, mecamylamine might act through a mechanism that is independent of monoamine neurotransmission.
A wide range of classic antidepressants can act as noncompetitive nAChR antagonists (Shytle et al 2002a). It has been suggested that this common action might be related to antidepressant efficacy, although to date no studies have directly tested this hypothesis in an animal model. Therefore, we compared β2+/+ and β2-/- in the tail suspension, forced swim, and learned helplessness tests to determine whether nAChRs are essential for amitriptyline action. These three models were chosen because they all show a robust response to antidepressant treatment in mice (Caldarone et al 2003; Porsolt et al 1977; Shanks and Anisman 1989; Steru et al 1985) and because the use of several behavioral models can aid in the interpretation of complex behaviors in transgenic and knockout mice (Crawley et al 1997; Picciotto and Wickman 1998). We also examined whether chronic amitriptyline would increase hippocampal cell proliferation in β2+/+ and β2-/- mice. Our results demonstrate that β2-/- mice are resistant to the effects of amitriptyline across the three behavioral depression models as well as the cellular proliferation model of antidepressant responsiveness. These findings, which demonstrate that the antidepressant effects of amitriptyline require high-affinity nAChRs, provide compelling evidence that the therapeutic effects of antidepressants might be mediated, in part, through actions at nAChRs.
Baseline differences between β2+/+ and β2-/- were noted in the tail suspension and forced swim tests, in untrained mice in the learned helplessness model, as well as hippocampal cell proliferation. In the forced swim and tail suspension test, control β2-/- mice showed less immobility than control β2+/+ mice, which would suggest a less “antidepressive-like” phenotype. In contrast, β2-/- mice that were not given learned helplessness training showed greater escape latencies in the active escape response than untrained β2+/+ mice. Furthermore, untreated β2-/- mice exhibited less hippocampal cell proliferation than β2+/+ mice. Both the reduced hippocampal cell proliferation and the poor escape performance in β2-/- could be interpreted as a more “depressive-like” phenotype. Alternatively, the poor escape performance could reflect a learning or performance impairment in the active escape task. Thus, depending on the measure used, β2-/- mice can exhibit either “depressive” or “antidepressant”-like phenotypes. These differences preclude a definitive conclusion on the role of high-affinity nAChRs in baseline “depressive-like” behavior.
Despite the baseline differences between β2+/+ and β2-/- mice, the resistance of β2-/- mice to the antidepressant effects of amitriptyline is consistent across all depression models tested. Although saccharin-treated β2-/- mice showed less immobility than β2+/+ mice in both the forced swim and tail suspension tests, the lack of response to amitriptyline in the β2-/- mice was unlikely to be the result of a floor effect. First, immobility time increased in β2-/- mice over the course of the tests to levels at which β2+/+ mice showed an antidepressant-like response. Second, β2-/- mice show a reduction in immobility in response to amphetamine in the tail suspension test (data not shown). Finally, even in measures in which the β2-/- mice tended to show a greater “depressive-like” phenotype (learned helplessness and hippocampal cell proliferation), amitriptyline was still ineffective. Therefore, the baseline differences in both behavior and hippocampal cell proliferation between β2+/+ and β2-/- mice do not preclude interpretation of the antidepressant responses in these tests.
An emerging hypothesis proposes that the mechanisms underlying the therapeutic actions of antidepressants might involve neuronal plasticity, including adult hippocampal neurogenesis, rather than simple changes in the synaptic level of monoamine neurotransmitters (Duman et al 1997). Previous studies have shown that chronic administration of a wide variety of antidepressants can increase neurogenesis in the hippocampus (Malberg et al 2000), and exposure to stress reduces hippocampal cell proliferation, an effect that is reversed by antidepressant treatment (Alonso et al 2003; Malberg and Duman 2003). In addition, X-irradiation of the hippocampus blocks both the proliferation-inducing and behavioral effects of antidepressants in the novelty-suppressed feeding test (Santarelli et al 2003), leading to the suggestion that increasing neurogenesis might be required for the behavioral effects of antidepressants. Consistent with previous studies of other antidepressants (Malberg and Duman 2003; Malberg et al 2000; Santarelli et al 2003), we observed an increase in hippocampal cell proliferation in β2+/+ mice treated chronically with amitriptyline; however, β2-/- mice treated with amitriptyline showed no increase in proliferation. Thus, β2-subunit-containing nAChRs are required for the proliferation-inducing effects of amitriptyline.
Interestingly, β2-/- mice had fewer BrdU-labeled cells at baseline compared with β2+/+ mice, suggesting that high-affinity nAChRs regulate cell proliferation in the hippocampus (Harrist et al, in press). In support of these findings, β2-subunit-containing nAChRs have been shown to be essential for the neuroprotective effects of nicotine (Laudenbach et al 2002; Stevens et al 2003). In addition, activation of nAChRs with nicotine can increase levels of neurotrophic factors, such as fibroblast growth factor (Belluardo et al 1998), in the hippocampus, providing one possible mechanism by which nAChRs can support hippocampal cell proliferation. Recent data have shown no differences between β2+/+ and β2-/- BrdU-labeled cells in the hippocampus (Mechawar et al 2004); however, 2–4-month old mice were used in that study, whereas we used mice more than 6 months of age. We did not see any difference in baseline hippocampal cell proliferation in 3-month-old mice in our studies (Harrist et al, in press), suggesting that the difference in hippocampal proliferation between β2+/+ and β2-/- mice is likely to be age-dependent.
In summary, our results demonstrate that nAChRs are required for both behavioral and hippocampal proliferation-inducing effects of chronic amitriptyline. These results suggest that antagonism of high-affinity nAChRs might be both necessary and sufficient for antidepressant activity in a rodent model. Further studies will be required to determine whether the role of nAChRs in regulating the behavioral and cellular responses to antidepressants is a direct effect mediated by nAChRs or an indirect effect of modulating neurotransmission in other cell types. Further investigation into more specific nAChRs antagonists, both alone and in combination with classic antidepressants, could lead to the development of novel and more effective treatments for individuals who suffer from depression.
This work was supported by grants DA00436, DA10455, DA13334, and DA00167 from the National Institutes of Health. We thank Dr. Peter Jatlow for serum measurements of amitriptyline and Drs. Ronald Duman and Tony George for critical reading of the manuscript.
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Biological Psychiatry
Volume 56, Issue 9 , 1 November 2004, Pages 657-664
poster:jrbecker
thread:417063
URL: http://www.dr-bob.org/babble/20041113/msgs/417063.html