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. Author manuscript; available in PMC: 2012 Sep 13.
Published in final edited form as: Int J Neuropsychopharmacol. 2009 May 7;12(6):731–736. doi: 10.1017/S1461145709000418

Reduced effectiveness of escitalopram in the forced swimming test is associated with increased serotonin clearance rate in food restricted rats

CP France 1,2, J-X Li 1, WA Owens 3, W Koek 1,2, GM Toney 3, LC Daws 1,3
PMCID: PMC3440869  NIHMSID: NIHMS405214  PMID: 19419596

Abstract

Efficacy of antidepressant drugs is often limited. One of the limiting factors may be diet. This study shows that the effect of escitalopram in the forced swimming test is diminished in rats by food restriction that decreased body weight by 8%. The primary target for escitalopram is the serotonin (5-HT) transporter. Using high-speed chronoamperometry to measure 5-HT clearance in vivo in rats fed the same food restricted diet, the rate of 5-HT clearance from extracellular fluid in brain was dramatically increased. Increased 5-HT transporter function under conditions of dietary restriction might contribute to the decreased effect of escitalopram. These results suggest that diet plays an integral role in determining efficacy of antidepressant drugs, and might well generalize to other psychoactive drugs that impinge upon the 5-HT transporter.

Keywords: serotonin, antidepressant, SSRI, rat, serotonin transporter

INTRODUCTION

At least 50% of patients do not respond to antidepressant drugs (Baghai et al., 2002) and in those who show an initial response, symptoms often re-emerge (Tranter et al., 2002). The complex nature of depression undoubtedly contributes to variations in response among patients and includes factors such as age, gender, genetics, and diet (Khan et al., 2007). Dietary restriction is associated with decreased effectiveness of antidepressant drugs (Slaiman, 1989). Some of these drugs (e.g., the selective serotonin [5-HT] reuptake inhibitor [SSRI] fluoxetine) are not indicated in patients with eating disorders (e.g., anorexia nervosa [Barbarich et al., 2004]); they are often ineffective (Kaye et al., 1998) and they can further decrease food intake. Moreover, eating disorders are associated with long-term changes in 5-HT receptors and transporters (Bailer et al., 2005, 2007). These findings, along with a rapidly growing literature, support the view that nutritional factors alter 5-HT neurotransmission such that therapeutic effects of antidepressant and other psychoactive drugs are compromised. The significance of diet mediated regulation of 5-HT neurotransmission and therapeutic response is underlined, on the one hand, because loss of appetite and weight loss are common in depression and, on the other hand, because of the growing worldwide obesity epidemic and the fact that efforts to treat obesity (dieting) might alter the effectiveness of antidepressant drugs.

Food restriction can modify dopamine neurotransmission and the effects of drugs acting on dopamine systems (Sevak et al., 2008; Zhen et al., 2006), but much less is known about the effects of food restriction on 5-HT neurotransmission. 5-HT is important in the regulation of appetite, sleep and mood. After its release from neurons 5-HT can act on a variety of receptor subtypes, and its actions are terminated, in large part, by reuptake via the 5-HT transporter. SSRIs like escitalopram block reuptake of 5-HT and thereby prolong the actions of 5-HT. The current study examined how a diet that resulted in modest weight loss (8%) impacts the effects of the SSRI escitalopram in the forced swimming test, a behavioral test that predicts antidepressant activity in humans (Cryan et al., 2005; Porsolt et al., 1978; Sánchez et al., 2003). To gain insight into how food restriction might regulate the 5-HT transporter, in vivo electrochemistry was used to measure clearance of 5-HT from extracellular fluid in hippocampus and striatum.

METHOD

Animals

Male Sprague-Dawley rats (250-275 g upon arrival) were housed individually under a 12/12 hr light/dark cycle with free access to water. A total of 26 rats received 10 g/day of standard laboratory chow for 7 days; the remaining 28 rats had free access to standard laboratory chow. These studies were approved by the Institutional Animal Care and Use Committee, The University of Texas Health Science Center at San Antonio, and performed in accordance with the Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).

Forced swimming test

For the forced swimming test (Cryan et al., 2005; Detke et al., 1995; Porsolt et al., 1978; Sánchez et al., 2003) rats were placed in a transparent cylinder (20 cm diameter by 40 cm high) containing water (25° C, 30 cm deep). On the first day of the experiment, rats were placed in the cylinder for 15 min; 24 h later they were placed in the cylinder for a 5-min test. Vehicle or escitalopram oxalate (Shanco International, Inc., Hazlet, NJ, USA [dissolved in saline]) was administered s.c. 1 h before the 5-min test. During the 5-min test rats were videotaped for later scoring by two observers who were unaware of treatment condition. Every 5 s during a 5-min test the observers scored the rat as either immobile, swimming, or climbing according to methods of Lucki and colleagues (Cryan et al., 2005; Lucki and O'Leary, 2004). The inter-rater reliability was at least r=0.83 for observational measures.

High-speed chronoamperometry. High speed chronoamperometry is an electrochemical recording technique, which affords the kinetics of 5-HT clearance to be measured in vivo

Detailed methods are published elsewhere (Daws and Toney, 2007; Daws et al., 2005). Rats were anesthetized by i.p. injection of chloralose (85 mg/kg) and urethane (850 mg/kg) followed by tracheal intubation and placement into a stereotaxic frame. Body temperature was maintained at 37°C and blood oxygen levels monitored (MouseOximeter, StarrLifeSciences.com) and maintained above 90%. A Nafion-coated carbon fiber electrode was attached to a glass micropipette containing 5-HT. The assembly was lowered into the brain region of interest (stereotaxic coordinates in mm: CA3 region of hippocampus AP-4.1, ML+/-3.3, DV-2.8; striatum AP+1.2, ML+/-2.2, DV-3.5 to -5.5) and 5-HT pressure ejected to achieve concentrations at the recording electrode ranging from approximately 0.2 to 4.0 μM. When possible, chronoamperometric recordings were made in both brain regions in the same rat. High-speed chronoamperometric recordings were made using the FAST-12 and FAST-16 systems (Quanteon, Nicholasville, KY). Oxidation potentials consisted of 100 msec pulses of +0.55 volts. Each pulse was separated by a 900 msec interval during which the electrode potential was maintained at 0.0 V. Voltage at the active electrode was applied with respect to a Ag/AgCl reference electrode positioned in the extracellular fluid of the ipsilateral superficial cortex. Electrode placement was confirmed by making an electrolytic lesion at the recording site at the conclusion of the experiment

Brain mass and hydration

For examining possible effects of food restriction on brain mass and hydration, rats were anesthetized with isoflurane (3% in O2) and decapitated. The spinal cord was transected at the C1 level and brains (including olfactory cortex) were removed and weighed on a Mettler-Toledo analytical scale (model AB54, 10-4 gram resolution). Brains were placed in sealed desiccators containing Drierite® (H.A. Hammond Co., LTD., Zenia, OH, USA) and weighed daily for 7 days (i.e., until a nadir was reached and maintained for 2 days). Hematocrit was determined from duplicate capillary tubes measured with a Lancer microhematocrit tube reader (St. Louis, MO, USA). Blood samples (0.4 ml) were centrifuged (8,400 g, 3 min) and plasma osmolality was measured using a freezing point depression osmometer (Advanced Instruments, Inc., model 3320, Norwood, MA, USA). Plasma protein concentration was determined by refractometry (VWR International, Inc., Buffalo Grove, IL, USA).

RESULTS

Food restriction decreases body weight

Body weights of free-feeding and food-restricted rats were not significantly different prior to the start of food restriction; restricting rats (n=26) to 10 g/day of standard laboratory chow for 7 days resulted in significant (paired-t test, t=7.15, df=25, p<0.001) body weight loss (from 358.6±7.5 to 329.3±7.3 g [mean±SEM]; 8.2±1.1% [mean±SEM]). During the same 7-day period the body weight of rats (n=28) with free access to standard laboratory chow increased significantly (t=11.59, df=27, p<0.001; from 350.3±5.8 to 369.4±4.4 g; 5.5±0.5%).

Food restriction decreases the effect of escitalopram in the forced swimming test

There was no difference in immobility, swimming, or climbing between food-restricted and free-feeding rats that received vehicle (compare open bars, panels a-f, Fig. 1). In free-feeding rats escitalopram significantly (two-way ANOVA with Bonferroni post hoc test; p<0.05) decreased immobility and increased swimming without affecting climbing (panels a-c, Fig. 1). Escitalopram had no significant effect on immobility, swimming or climbing in food-restricted rats. The effects of escitalopram (10 mg/kg) on immobility and swimming also were significantly (p<0.05) different between free-feeding and food-restricted rats.

Fig. 1.

Fig. 1

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Effects of escitalopram on immobility (a,d), swimming (b,e), and climbing (c,f) in the forced swimming test (n=4 per dose in each condition [total n=24]). Counts in a 5-min observation period (±SEM) are plotted as a function of dose (mg/kg) of escitalopram (*=significantly different from vehicle control, “0”).

Food restriction increases 5-HT clearance from brain extracellular fluid

Food restriction resulted in a marked and significant increase in 5-HT clearance rate (i.e., transporter activity) in both regions (hippocampus, upper panels, Fig. 2; striatum, lower panels, Fig. 2). Panels a and c (Fig. 2) show representative oxidation currents (converted to μM values using a pre-determined calibration factor) generated by local application of equal amounts of 5-HT into hippocampus and striatum, respectively. While the resulting maximum signal amplitude was unaffected, food restriction markedly increased the clearance rate of 5-HT as reflected by the leftward shift in the clearance function in food-restricted rats (dark tracing) compared with free-feeding rats (grey tracing). Panels b and d (Fig. 2) summarize data over a range of 5-HT concentrations and show significantly increased clearance of 5-HT in food-restricted rats compared with free-feeding rats, both in hippocampus and in striatum.

Fig. 2.

Fig. 2

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Effects of food restriction on 5-HT clearance in hippocampus (a,b) and striatum (c,d). Panels a and c show the oxidation current (converted to μM concentration using a calibration factor determined in vitro) produced by pressure ejection of 40 pmol of 5-HT into the CA3 region and striatum, respectively, of anesthetized free-feeding (grey trace) and food-restricted rats (black trace). Panels b and d show corresponding summary data where the rates of 5-HT clearance (nM/sec) are plotted as a function of increasing concentrations of 5-HT. Data represent the mean ±SEM for 6 food restricted and 5 free-feeding rats (hippocampus; two-way ANOVA, p<0.05) and for 6 food restricted and 9 free-feeding rats (striatum; two-way ANOVA,p<0.01). Panel e and f show brain mass and brain water content, respectively.

Brain water content and mass

Brain mass and hydration level were unchanged by food restriction (panels e and f, Fig. 2), supporting the view that the increased clearance rate of 5-HT in food restricted rats was due primarily to increased 5-HT transporter activity. Other indices of body fluid regulation did not differ between free-feeding and food-restricted rats. For example, in free-feeding and food restricted rats, respectively, measures of body fluid regulation were as follows: 298.2 ± 1.3 and 295.0 ± 0.7 mOsm/kg for plasma osmolality; 5.86 ± 0.05 and 5.88 ± 0.11 g/100 ml for plasma protein; and 46.8 ± 0.7 and 49.4 ± 0.9 percent cell volume for hematocrit. Thus, multiple indices of body fluid balance show that food-restricted rats maintained normal body fluid homeostasis. In addition, because the wet and dry weights of brains were not different between free-feeding and food-restricted rats, food restriction also had no effect on protein/lipid contents of brain. These data show that increased 5-HT clearance rate and loss of antidepressant-like effects of escitalopram are not due to disruption of body fluid homeostasis or brain metabolic balance in food-restricted rats.

DISCUSSION

Results of this study show that diet, in particular dietary restriction, might be a significant contributor to lack of antidepressant efficacy. In the forced swimming test, which predicts antidepressant drug activity in humans, escitalopram decreased immobility and increased swimming in free-feeding rats, consistent with previous findings (Sánchez et al., 2003), but was ineffective in food-restricted rats. These results support an earlier observation (Soubrié et al., 1989) that food restriction can attenuate the behavioral effects of antidepressant drugs.

The SSRI escitalopram decreases clearance of 5-HT by selectively blocking its reuptake at 5-HT transporters (Sánchez et al., 2003), thereby increasing extracellular concentrations of 5-HT that act at pre- and postsynaptic receptors. Increasing the uptake of 5-HT decreases extracellular concentrations of 5-HT which, in turn, might diminish the effectiveness of drugs that act by blocking 5-HT reuptake. Consistent with this view, food restriction significantly increased the rate of 5-HT clearance from extracellular fluid in hippocampus, a brain region important in mediating mood as well as actions of antidepressant drugs (Campell and Macqueen, 2004), and striatum, a structure important in motor activity and reward (Wise, 2004). Food restriction can also decrease water consumption (Fitzsimons and Le Magnen, 1969); moreover, volume fraction and tortuosity are important determinants of the rate at which molecules diffuse through the extracellular matrix. Multiple indices of fluid balance were not different between free-feeding and food-restricted rats, suggesting that changes in 5-HT clearance and in the effects of escitalopram are not likely due to reduced water intake or consequent changes in fluid balance in food-restricted rats. Food restriction can also decrease sensitivity to drugs acting directly at 5-HT receptors (Li and France, 2008) and can reduce the availability of the 5-HT precursor tryptophan. However, under the conditions used in our study brain tryptophan levels, and therefore 5-HT levels, were unlikely affected (Haleem, 2009); that not-with-standing, in our clearance experiments, 5-HT was exogenously applied in equivalent amounts (see panels a and c, Fig. 2) in free-feeding and food-restricted rats, thus, averting potential confounds of baseline differences in endogenous 5-HT.

Collectively, these data show that one week of food restriction with modest (8%) weight reduction can profoundly affect behavior and brain neurochemistry in a neurotransmitter system that is the target of many psychotherapeutic drugs. Given the importance of 5-HT systems in the therapeutic effects of many drugs used in psychiatry, along with the fact that most drugs are developed and evaluated in relatively homogeneous populations comprising individuals with relatively normal weight, age and physiological disposition, it is important to understand how common differences and fluctuations in physiological state (e.g., dieting) impact this and other neurotransmitter systems. These findings might help to better understand why drugs, such as SSRIs, used to treat depression are effective in less than half of patients (Baghai et al., 2006; Tranter et al., 2002). Such understanding might help to identify drugs with antidepressant activity that is not affected by dietary factors, which would improve the treatment of depression.

Acknowledgements

Supported by USPHS grant MH64489 (LCD), HL71645 (GMT), and a Senior Scientist Award to CPF (DA17918) as well as NARSAD Independent Investigator Awards to WK and LCD.

Footnotes

Statement of Interest

None

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