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Behavioral and metabolic effects of S-adenosylmethionine and imipramine in the Flinders Sensitive Line rat model of depression
Behavioural Brain Research 364 (2019) 274–280
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Research report
Behavioral and metabolic effects of S-adenosylmethionine and imipramine in the Flinders Sensitive Line rat model of depression
T
Sandra Tillmanna, , Denise F. Happa, Per F. Mikkelsena, Juergen Geiselb, Gregers Wegenera, Rima Obeidb,c ⁎
a
Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, DK-8240 Risskov, Denmark Saarland University Hospital, Department of Clinical Chemistry and Laboratory Medicine, Building 57, D-66421 Homburg, Saar, Germany c Aarhus Institute of Advanced Studies, Aarhus University, DK-8000 Aarhus C, Denmark b
ARTICLE INFO
ABSTRACT
Keywords: Depression Flinders Sensitive Line rats Imipramine One-carbon metabolism S-Adenosylmethionine
Depression is associated with dysregulation of methyl group metabolism such as low S-adenosylmethionine (SAM). We previously reported that Flinders Sensitive Line (FSL) rats, an animal model of depression, had lower concentrations of liver SAM than the control rats, Flinders Resistant Line (FRL) rats. The present study investigated if SAM supplementation may correct liver SAM and behavioral abnormalities in this model. Moreover, we compared one-carbon (C1) metabolites, neurotransmitters, and gastrointestinal (GI) transit in SAM-treated versus imipramine (IMI)-treated animals. FSL rats received vehicle, IMI, SAM, or IMI + SAM (n = 9–10 per group) once daily through oral gavage for 4 weeks; FRL rats received vehicle. Behavior was assessed using standard tests for locomotion, cognition, and depressive-like behavior. Monoamine neurotransmitters and C1 metabolites were measured using UHPLC‐ECD and UPLC‐MS/MS, respectively. Compared to FRL rats, FSLs had lower liver SAM, higher plasma serotonin, lower hippocampal dopamine and serotonin turnover, and faster GI transit. Behaviorally, FSL rats showed impaired cognitive performance as well as increased depressive-like behavior compared to FRLs. Coadministration of IMI and SAM seemed to have adverse effects on spatial memory. SAM or IMI administration did not reverse C1 metabolites, neurotransmitters, or GI transit in FSLs. Despite low liver SAM in FSL rats, orally administered SAM did not show antidepressant effects in this specific animal model of depression.
1. Introduction The one-carbon (C1) cycle is the principal biochemical pathway regulating the transfer of C1 groups for methylation of DNA, RNA, neurotransmitters, and phospholipids [1]. S-adenosylmethionine (SAM) is a methyl group donor in over 100 methylation reactions in the body and is produced mainly in the liver from various dietary methyl donors such as methionine, folate, betaine, or choline [2]. Deficiency of methyl donors has been linked to psychiatric diseases such as depression, but it is unclear whether this association is causal [3]. For instance, depression caused by folate and vitamin B12 deficiency [4] may be partly mediated by low SAM levels in both conditions. Several clinical studies have investigated the antidepressant role of SAM supplementation alone [4–6] or in comparison to a classical antidepressant such as imipramine [7]. However, available studies are generally considered to be of poor quality [8] and SAM is currently not recommended as firstline monotherapy treatment for depression [9].
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In a previous study, we demonstrated that Flinders Sensitive Line (FSL) rats, a validated animal model of depression [10], had lower liver SAM concentrations than the control strain, Flinders Resistant Line (FRL) rats [11]. Moreover, FSL rats had higher plasma dopamine and norepinephrine concentrations than FRLs, which may be related to their lower concentrations of liver SAM, as SAM is needed to degrade monoamine neurotransmitters. Therefore, it can be hypothesized that providing SAM from external sources may correct neurotransmitter synthesis and depressive-like behavior in FSL rats. Given the role of C1 metabolism in the pathophysiology of depression [4], the therapeutic potential of SAM in depression warrants further investigation, and studying tissue concentration of C1-related markers in animals treated with SAM may provide novel insights into biochemical processes altered by this universal methyl donor. To our knowledge, low liver SAM has not been studied in other animal models of depression, making FSL rats an interesting model to investigate the effect of SAM on neurotransmitter synthesis or catabolism.
Corresponding author. E-mail address: [email protected] (S. Tillmann).
https://doi.org/10.1016/j.bbr.2019.02.011 Received 13 January 2019; Received in revised form 3 February 2019; Accepted 5 February 2019 Available online 06 February 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Experimental timeline. After an initial adaptation phase, FRL and FSL rats were randomly assigned to four intervention groups (VEH, IMI, SAM, IMI + SAM). The intervention was given from day 0 to day 28 once daily via oral gavage. Rats underwent behavioral tests (day21–23,26–27), the GITT (day 24), and euthanization (day 27–28). FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; FST, Forced Swim Test; GITT, Gastrointestinal transit time; IMI, Imipramine; NOR, Novel Object Recognition; OF, Open Field; SAM, Sadenosylmethionine; VEH, Vehicle.
Our overall aim was to investigate if chronic SAM supplementation may reverse the behavioral and neurotransmitter abnormalities in FSL rats, potentially by increasing liver SAM and affecting related plasma C1 metabolites (i.e. betaine, choline, dimethylglycine, homocysteine). Depression is associated with gastric motility symptoms such as constipation [12], but it is not known if gastric motility differs between FSL and FRLs and how these potential differences may be changed upon administration of SAM. We therefore assessed gastrointestinal (GI) transit time in this animal model as an exploratory outcome. Imipramine (IMI), a tricyclic antidepressant, has been found to speed the therapeutic onset of SAM (200 mg/day) in patients with moderate to severe depression [13], suggesting an interactive effect. We therefore performed all experiments with the following groups: I) vehicle (VEH); II) IMI; III) SAM; IV) IMI + SAM. Our study may provide novel physiological insights into the treatment with methyl donors and/or tricyclic antidepressants in FSL rats.
day) was purchased from Nature Made Nutritional Products (Mission Hills, CA, USA) in tablet form, crushed using a mortar and pestle, and dissolved in tap water. Animals in the combined IMI + SAM group received the same dose as the two individual groups (15 mg/kg IMI + 200 mg/kg SAM). The IMI dose was chosen based on studies showing antidepressant effects in FSL rats [14,15]; the SAM dose was based on its ability to raise SAM concentrations in the rat brain [16]. All drugs were freshly prepared each day just before administration and were given once daily towards the end of the rats’ dark phase (2 p.m. ± 1 h) for a duration of 4 weeks using oral gavage. The dosage was weightadjusted every other day; the administration volume was 4 mL/kg. After 3 weeks of intervention, we assessed mood-related behavior and gastrointestinal transit time (GITT). Rats were decapitated after 4 weeks of intervention. The last intervention was administered 24 h before euthanization. A graphical representation of the experimental design is provided in Fig. 1.
2. Materials and methods
2.3. Behavioral analysis
2.1. Animals
All behavioral tests were performed in specially-equipped rooms within the animal facility between 3 a.m. and 2 p.m. in the active phase of the animal under dim red light. The order of animals was determined randomly. To minimize stress, animals were habituated to the behavioral rooms 1 h before testing. Tests were ordered from the least to the most stressful to reduce carryover effects [17]; the order of the test descriptions corresponds to the chronological order. All arenas/tanks were thoroughly cleaned between each trial. All tests were scored by an observer blinded to strain and intervention groups.
Age-matched adult male Flinders Sensitive Line (FSL, n = 38) and Flinders Resistant Line (FRL, n = 10) rats were obtained from the breeding colony maintained at Aarhus University, Denmark. At baseline (day 0), rats were 10.4 ± 2.1 weeks old (mean ± SD) and weighed 318 ± 52.0 g. Animals were housed as previously reported [11]. Rats of the same strain and intervention were pair-housed in standard cages (Cage 1291H Eurostandard Type III H, 425 × 266 × 185 mm, Tecniplast, Italy) at 20 ± 2 °C and 60 ± 5 % relative humidity on a reversed 12 h light/dark cycle (lights on at 2 p.m.). The reversed cycle was introduced in 3-h increments right before a 2-week adaptation phase. After the adaptation phase, rats were weighed every other day. All rats received standard chow diet (#1324 Altromin, Brogaarden, Lynge, Denmark) and tap water ad libitum and had access to a tunnel shelter, nesting material, and a wooden stick. Cages were changed once a week by the same experimenter. All experiments were approved by the Danish Animal Experiments Inspectorate prior to initiation of the experiments (approval number: 2012-15-2934-00254) and were conducted in accordance with the European Community Council Directive.
2.3.1. Novel Object Recognition The Novel Object Recognition (NOR) test was performed to measure nonspatial memory as described previously [18]. To avoid neophobic responses stemming from an unfamiliar apparatus and to promote exploratory activity, all rats were habituated to the testing arena (an open field of 50 × 50 × 40 cm) 24 h before the testing day for a duration of 1 h. The testing day consisted of a brief re-habituation phase, a training phase, and a testing phase. During the re-habituation phase, rats were allowed to re-habituate to the arena for 1 min. They were then placed into holding cages while two identical objects were positioned in the center of the arena, with equal distances between the objects and the walls to allow passing. During the training phase, they were again placed in the center of the arena (facing the wall) and allowed to freely explore for 15 min before being returned to their home cages. After 45 min, the testing session commenced, where one of the two identical objects from the training session was replaced by a novel one. Rats were positioned facing the wall and were allowed to freely explore for 5 min. Objects (glass salt dispenser, tin can) were validated prior to use to exclude a baseline preference for one of them. The position of the novel object was randomly assigned and counterbalanced for each intervention group. All sessions were recorded by a camera mounted to the ceiling. Exploration time during the testing phase was analyzed manually with a stopwatch, and was defined as directing the nose towards an object at a close distance or touching it, but not sitting on it.
2.2. Experimental design and intervention FSL rats were randomized to receive VEH, IMI, SAM, or IMI + SAM (n = 10/group). FRL rats were used to ascertain the depressive-like phenotype of FSL rats and received VEH (n = 10). The number of animals in each group was based on previous experiments in these rats with similar outcomes [11]. Two FSL rats were excluded from analyses; one due to unforeseen death before treatment start and one due to noncompliance to oral gavage administration in week 1. The final FSL group numbers were as follows: VEH (n = 10), IMI (n = 10), SAM (n = 9), IMI + SAM (n = 9). VEH animals received tap water. Imipramine hydrochloride (15 mg/kg/day) was obtained from Sigma Aldrich (Taufkirchen, Germany) and dissolved in tap water. SAM (200 mg/kg/ 275
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assays, livers, hippocampi, and prefrontal cortices were homogenized in 1 N acetic acid (10 μL/mg tissue) and centrifuged at 10.000 × g for 10 min. Plasma samples for C1 metabolites (betaine, choline, dimethylglycine, homocysteine, cystathionine) were used without further processing. For monoamine measurements, 20 μL perchloric acid was added to 180 μL plasma followed by centrifugation. Brain tissue samples were sonicated in 0.2 N perchloric acid (5 μL/mg tissue), centrifuged at 10.000 × g for 10 min, and filtered using Costar Spin‐X (Corning, NY, USA). C1 metabolites were measured in plasma (betaine, choline, dimethylglycine, homocysteine, cystathionine) and liver extracts (SAH, SAM) using an Acquity Ultra Performance LC system coupled to a MicroMass Quattro Premier XE tandem quadrupole mass spectrometer (UPLC‐MS/MS) (Waters Corporation, Milford, MA, USA). Plasma concentrations of total homocysteine and cystathionine were measured using an in‐house gas‐chromatography mass spectrometry method. Concentrations of SAM and SAH in the acidified extracts of liver and brain were measured using established methods that depend on the use of labelled isotopes as internal standards. Concentrations of serotonin (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA), dopamine, 3,4‐dihydroxyphenylacetic acid (DOPAC), and norepinephrine were measured using ultra-high performance liquid chromatography with electrochemical detection (UHPLC‐ECD, Dionex Ultimate 3000 UHPLC, Thermo Scientific, Rockford, IL, USA) as described in our previous work [11].
The preference for a novel over a familiar object was calculated as exploration time of novel vs. all objects [%]. 2.3.2. Y-Maze The Y-Maze was performed to assess spatial working memory as described previously [18]. This task is based on the innate tendency of rodents to alternate between exploring different arms of a maze. Rats were placed into the center of the maze and were allowed to freely explore the three arms of the maze for 6 min. Each choice was recorded by a camera mounted to the ceiling and was analyzed manually. An alternation was defined as consecutive full entries (excluding the tail) into each of the three arms; and alternation percentage was calculated as the number of alternations vs. the total number of arm visits (minus 2). 2.3.3. Open Field Locomotion was measured in an Open Field (OF) arena (100 × 100 × 80 cm) immediately prior to the FST as described previously [18]. Briefly, animals were placed in the center of the square and allowed to move freely for 5 min. The total distance travelled (cm) was recorded by a camera mounted to the ceiling and scored with specialized software tracking the midpoint of the rats’ body contour (Noldus Ethovision XT, The Netherlands). 2.3.4. Forced Swim Test To measure depressive-like behavior, the FST was performed as described previously [18]. For the pre-FST, rats were placed into a perspex cylinder (height 60 cm, diameter 24 cm) filled with 24 ( ± 1)°C heated tap water. After 15 min of swimming, rats were removed from the water, dried with towels, placed under a heating lamp for 30 min, and returned to their home cages. On the testing day 24 h later, they were removed from the water after 5 min. Their behavior was recorded by a camera positioned in front of the four tanks. Three distinct parameters were scored manually with a time-sampling technique, whereby the predominant behavior over each 5-s period of the 300-s test was rated [23]. Behaviors distinguished were struggling, swimming, and immobility. Diving was classified as swimming behavior.
2.7. Data analysis Two separate one-way analysis of variance (ANOVA) tests were used to study differences in bodyweight, food/water intake, behavior, C1 metabolites, and neurotransmitters between rat strains (VEH-treated FSL/FRL rats) and interventions (VEH/IMI/SAM/IMI + SAM). In case of statistically significant intervention effects, the ANOVA was followed up by Bonferroni posthoc comparisons. GITT was analyzed using a Kaplan Meier log-rank survival analysis. Assumptions of normality and homogeneity of variances were tested by Shapiro-Wilk test and Levene’s test, respectively. Results in the graphs are expressed as means ± standard error of the mean (SEM). Alpha was set at 0.05, while a pvalue between 0.05 and 0.1 was interpreted as a trend. Statistical analyses were carried out using IBM SPSS 22.0 (IBM Corp., Armonk, NY, USA). Graphs were generated using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).
2.4. Gastrointestinal transit time The GITT test was performed as previously reported [19]. Rats were single-housed and fasted 1 h before and throughout the entire testing period. Carmine red (Sigma Aldrich, St. Louis, MO, USA) was prepared as a 6 % solution in 0.5 % methyl cellulose (Sigma Aldrich). Rats were gavaged with 0.3 mL of the solution between 2 and 3:30 p.m. and monitored every 5 min for the appearance of the first red fecal boli by two observers familiar with the rats. The maximum observation time was 8 h, after which rats were returned to their home cages. The latency from gavage to passage of the first red pellet was recorded as total GITT.
3. Results 3.1. Bodyweight and food/water intake VEH FSL rats weighed significantly less than VEH FRL rats at both baseline (day 0; mean ± SD: 314 ± 54.4 g vs. 366 ± 29.4 g; F (1,18) = 7.06, p = 0.016) and endpoint (decapitation day; 351 ± 34.5 g vs. 412 ± 34.9 g; F(1,18) = 15.43, p = 0.001), which is characteristic for their depressive-like phenotype [20]. There was no strain difference in weight gain from baseline to endpoint or food/water intake. The four intervention groups differed in endpoint bodyweight (F (3,33) = 2.91, p = 0.049), but multiple comparison analysis did not show any significant group differences. The IMI group consumed significantly more water than the IMI + SAM group (52.8 ± 14.2 mL/100 g bodyweight vs. 38.4 ± 6.73 mL/100 g bodyweight; main effect ANOVA: F(3,33) = 3.23, p = 0.035; post-hoc: p = 0.033). The groups did not differ in baseline bodyweight, weight gain, or food intake.
2.5. Tissue collection Rats were decapitated after 4 weeks of starting the intervention. Decapitation took place between 2 and 5 p.m. over 2 consecutive days and was conducted in a random order. To minimize stress, rats were housed in an adjacent room and were brought singly into the decapitation room. After decapitation, trunk blood was collected in EDTA‐coated tubes and immediately centrifuged at 3000 × g for 10 min. Plasma was aliquoted and stored at −80 °C. Liver tissue (from the left lateral lobe), hippocampi, and prefrontal cortices were quickly removed, snap‐frozen in pre‐cooled isopentane, and stored at −80 °C.
3.2. Behavioral analysis
2.6. Sample processing and biochemical measurements
3.2.1. Locomotion and cognition VEH FSLs moved a significantly greater total distance in the OF compared to VEH FRLs (3498 ± 585 cm vs. 2334 ± 496 cm; F
The samples were processed as described in our previous study [11]. Briefly, for SAM, S-adenosylhomocysteine (SAH), and other C1-related 276
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Fig. 2. Behavioral results after intervention with VEH, IMI, SAM, or IMI + SAM. A: FSL rats traveled a significantly greater distance in the OF than FRL rats (p < 0.001). B: FSL rats had a significantly lower preference for the novel over the familiar object in the NOR, indicating impaired recognition memory (p = 0.033). C: FSL rats in the IMI + SAM group made significantly fewer correct alternations in the Y-Maze than both VEH (p = 0.055) and IMI (p = 0.023). D: VEH FSLs struggled less in the pre-FST than VEH FRLs (p = 0.032). Moreover, IMI animals struggled more than both VEH (p = 0.008) and IMI + SAM (p = 0.042). E: IMI animals swam less in the pre-FST than VEH (p = 0.005) and SAM (p = 0.067). IMI + SAM animals swam less than VEH (p = 0.049). F: VEH FSLs were more immobile in the pre-FST than VEH FRLs (p = 0.024). Values are expressed as means ± SEM and were analyzed by two one-way ANOVAs (rat strain, intervention); n = 9–10/group. FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; FST, Forced Swim Test; IMI, Imipramine; NOR, Novel Object Recognition; OF, Open Field; SAM, S-adenosylmethionine; VEH, Vehicle. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, #: p < 0.07.
(1,18) = 23.01, p < 0.001; Fig. 2A), but there was no difference between intervention groups. VEH FSL rats had a lower preference for the novel over the familiar object in the NOR than VEH FRLs (60.2 ± 18.3 % vs. 77.2 ± 14.5 %; F(1,18) = 5.30, p = 0.033; Fig. 2B), but intervention groups did not differ. VEH FSL and FRL rats did not perform differently in the Y-Maze, but animals in the IMI + SAM group made fewer correct alternations (58.7 ± 10.9 %) than both VEH (69.5 ± 7.60 %; p = 0.055) and IMI (70.8 ± 7.93 %; p = 0.023; main effect ANOVA: F(3,34) = 4.0, p = 0.016; Fig. 2C).
Table 1 Gastrointestinal transit time assessed after intervention with VEH, IMI, SAM, or IMI + SAM. Intervention In FRL rats VEH In FSL rats VEH IMI SAM IMI + SAM Overall
3.2.2. Depressive-like behavior In the pre-FST, VEH FSLs struggled less than VEH FRLs (113 ± 33.9 s vs. 158 ± 51.0 s; F(1,18) = 5.40, p = 0.032; Fig. 2D) and were more immobile (98.5 ± 36.5 s vs. 62.0 ± 29.5 s; F (1,18) = 6.05, p = 0.024; Fig. 2F). IMI animals struggled more (172 ± 52.2 s) than both VEH (113 ± 33.9 s; p = 0.008) and IMI + SAM (122 ± 23.6 s; p = 0.042; main effect ANOVA: F(3,34) = 4.72, p = 0.007). IMI animals swam less (41.5 ± 33.6 s) than VEH (88.5 ± 26.9 s; p = 0.005) and SAM (76.7 ± 25.7 s; p = 0.067; main effect ANOVA: F(3,34) = 5.69, p = 0.003; Fig. 2E). Moreover, IMI + SAM animals swam less than VEH (51.7 ± 26.6 s vs. 88.5 ± 26.9 s; p = 0.049). There was no intervention difference in immobility. The aforementioned swimming effects in the pre-FST were also detected at the test session 24 h later; there were no other significant strain or intervention differences.
N of Events/ Total N
% Events
Mean Survival Estimate (min)
Mean Transit Time (min)
5/10
50.0
455 ± 12
430 ± 47
9/10 4/10 3/9 4/9 25/48
90.0 40.0 33.3 44.4 52.1
434 416 445 456 441
428 319 374 425 398
± ± ± ± ±
17 31 28 19 10
± ± ± ± ±
59 102 98 71 83
compared to FSLs (10 %). There was also a tendency for a significant difference between intervention groups (χ2(3) = 7.34, p = 0.062). To investigate any differences in GITT time among the animals that produced a red boli within the 8 h, we ran a one-way ANOVA with all uncensored cases. There was no difference in mean time between FRL and FSL rats or between the intervention groups among those producing a boli within 8 h (Table 1). 3.4. C1-related metabolites The concentrations of C1‐related metabolites in FRL and FSL rats are presented in Fig. 3 (plasma) and Fig. 4 (liver). In plasma, VEH FSL rats had higher dimethylglycine concentrations than VEH FRL rats (7.10 ± 0.96 μmol/L vs. 5.72 ± 0.77 μmol/L; F(1,18) = 12.62; p = 0.002; Fig. 3C), but the two VEH-treated strains did not differ in plasma betaine, choline, homocysteine, and cystathionine. The four intervention groups tended to differ in plasma cystathionine concentrations (F (3,33) = 2.73; p = 0.060; Fig. 3E), but Bonferroni analysis did not reach significance. There were no significant intervention differences in the aforementioned plasma markers. In liver, VEH FSL rats had lower SAM concentrations than VEH FRL rats (107 ± 11.5 nmol/g liver vs. 125 ± 16.2 nmol/g; F(1,18) = 8.16; p = 0.011; Fig. 4B), but SAH or the SAM/SAH ratio did not differ
3.3. Gastrointestinal transit time Table 1 represents the number of animals that produced a red boli within the 8-h observational period as well as the mean time of the first appearance of a red boli for each group. Within the observational period, 52.1 % of all animals produced a red boli (VEH FRL: 50 %, VEH FSL: 90 %, IMI: 40 %, SAM: 33.3 %, IMI + SAM: 44.4 %). A nonparametric log-rank test showed a significant difference in the survival distribution between VEH FRL and VEH FSL (χ2(1) = 4.06, p = 0.044) due to the higher proportion of censored cases in the FRL group (50 %) 277
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Fig. 3. Changes in plasma one-carbon metabolites after intervention with VEH, IMI, SAM, or IMI + SAM. There were no strain (VEH-treated FSL vs. FRL) or intervention differences in betaine (A) or choline concentrations (B). VEH FSL rats had significantly higher concentrations of dimethylglycine than VEH FRLs (p = 0.002) (C). The strain or intervention groups did not differ in homocysteine (D) or cystathionine (E). Values are expressed as means ± SEM and were analyzed by two one-way ANOVAs (rat strain, intervention); n = 9–10/group. FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; IMI, Imipramine; SAM, S-adenosylmethionine; VEH, Vehicle. **: p ≤ 0.01.
between the strains. There were no intervention differences in SAH, SAM, or the SAM/SAH ratio.
4. Discussion We investigated the effects of SAM and IMI administration on behavior, gut motility, C1 metabolites and neurotransmitters in an established animal model of depression that exhibits low liver SAM [11]. Compared to VEH, IMI + SAM reduced the percentage of correct alternations in the Y-Maze, indicating an impairment in spatial cognition. VEH-treated FSL rats showed preliminary evidence for faster intestinal transit than FRLs. Moreover, they had higher concentrations of plasma serotonin, but lower hippocampal dopamine and serotonin turnover than FRLs. There were no intervention effects on GITT, C1 metabolites, or neurotransmitters.
3.5. Monoamine neurotransmitters Table 2 shows the concentrations of monoamine neurotransmitters measured in plasma, hippocampus, and prefrontal cortex of FRL and FSL rats. In plasma, VEH FSL rats tended to have higher 5-HT concentrations than VEH FRLs (1625 ± 797 nmol/L vs. 1052 ± 495 nmol/L; F (1,18) = 3.73; p = 0.069). There were no strain differences in plasma DOPAC, dopamine, norepinephrine, or DOPAC/dopamine ratio. In the hippocampus, VEH FSL rats had lower DOPAC concentrations than VEH FRLs (0.01 ± 0.004 nmol/g tissue vs. 0.02 ± 0.01 nmol/g; F (1,18) = 5.47; p = 0.031). The ratio of DOPAC/DA, an indicator of dopamine turnover, was also lower in VEH FSL rats than in FRLs in the hippocampus (0.60 ± 0.31 vs. 1.37 ± 0.71; F(1,18) = 9.94; p = 0.006). Similarly, the ratio of 5-HIAA/5-HT, an indicator of serotonin turnover, was lower in VEH FSL rats than FRLs in the hippocampus (0.66 ± 0.27 vs. 1.04 ± 0.48; F(1,18) = 4.96; p = 0.039). There were no other significant strain differences in the hippocampus or in the prefrontal cortex. The intervention groups did not differ in their monoamine concentrations in plasma, hippocampus, or prefrontal cortex.
4.1. Effects on behavior Monotherapy of both IMI and SAM has generally shown procognitive effects in animal models and patients. IMI treatment significantly improved cognitive function in a mouse model of traumatic brain injury [21]. In humans, IMI improved cognitive function in depressed patients [22], whereas the effect in Alzheimer’s patients was minimal [23]. A meta-analysis on the effect of SAM on Y-Maze performance in mice showed that SAM was associated with improvements in cognitive
Fig. 4. Changes in liver one-carbon metabolites after intervention with VEH, IMI, SAM, or IMI + SAM. There were no strain (VEH-treated FSL vs. FRL) or intervention differences in SAH concentrations (A). VEH FSL rats had significantly lower concentrations of SAM than VEH FRLs (p = 0.011) (B). The SAM/SAH ratio was similar between all groups (C). Values are expressed as means ± SEM and were analyzed by two one-way ANOVAs (rat strain, intervention); n = 9–10/group. FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; IMI, Imipramine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; VEH, Vehicle. *: p ≤ 0.05. 278
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Table 2 Plasma and tissue concentrations of monoamine neurotransmitters after intervention with VEH, IMI, SAM, or IMI + SAM. Intervention Strain Number
VEH 10
FRL
EDTA plasma (nmol/L) Norepinephrine 40.3 ± 7 Dopamine 8.38 ± 2.07 DOPAC 3.08 ± 1.03 DOPAC/dopamine 0.40 ± 0.20 5-HT 1052 ± 4951 Hippocampal extract (nmol/g tissue) Norepinephrine 0.60 ± 0.09 Dopamine 0.01 ± 0.004 DOPAC 0.02 ± 0.012 DOPAC/dopamine 1.37 ± 0.713 5-HT 0.08 ± 0.07 5-HIAA 0.08 ± 0.05 5-HIAA/5-HT 1.04 ± 0.484 Prefrontal cortex extract (nmol/g tissue) Norepinephrine 0.56 ± 0.11 Dopamine 0.21 ± 0.06 DOPAC 0.12 ± 0.04 DOPAC/dopamine 0.57 ± 0.26 5-HT 0.55 ± 0.27 5-HIAA 0.35 ± 0.12 5-HIAA/5-HT 0.72 ± 0.23
IMI 10
FSL
9
FSL
SAM 9
FSL
IMI + SAM 9
FSL
39.5 ± 12 8.15 ± 3.17 3.02 ± 1.66 0.42 ± 0.24 1625 ± 797
44.8 ± 11 7.02 ± 2.51 5.23 ± 3.65 0.71 ± 0.32 1285 ± 502
37.0 ± 11 7.58 ± 1.79 3.39 ± 1.80 0.46 ± 0.27 1310 ± 501
49.6 ± 14 6.85 ± 1.34 2.94 ± 1.52 0.42 ± 0.19 1105 ± 462
0.60 ± 0.12 0.02 ± 0.01 0.01 ± 0.004 0.60 ± 0.31 0.07 ± 0.06 0.06 ± 0.06 0.66 ± 0.27
0.56 ± 0.10 0.02 ± 0.01 0.01 ± 0.01 0.57 ± 0.42 0.10 ± 0.05 0.09 ± 0.06 0.77 ± 0.33
0.62 ± 0.04 0.02 ± 0.01 0.01 ± 0.01 0.58 ± 0.34 0.10 ± 0.06 0.11 ± 0.11 0.91 ± 0.50
0.61 ± 0.09 0.03 ± 0.02 0.01 ± 0.01 0.45 ± 0.28 0.11 ± 0.06 0.10 ± 0.08 0.85 ± 0.36
0.60 ± 0.12 0.22 ± 0.05 0.11 ± 0.03 0.54 ± 0.15 0.62 ± 0.24 0.39 ± 0.07 0.68 ± 0.17
0.60 ± 0.17 0.21 ± 0.05 0.09 ± 0.04 0.46 ± 0.15 0.66 ± 0.24 0.41 ± 0.13 0.68 ± 0.26
0.68 ± 0.17 0.23 ± 0.04 0.10 ± 0.02 0.47 ± 0.15 0.67 ± 0.29 0.44 ± 0.13 0.72 ± 0.27
0.70 ± 0.16 0.23 ± 0.05 0.11 ± 0.03 0.50 ± 0.16 0.73 ± 0.32 0.42 ± 0.11 0.65 ± 0.21
Data are presented as mean ± SD. Rats received VEH (tap water), IMI (15 mg/kg), SAM (200 mg/kg) or a combination of IMI + SAM once daily for 4 weeks through oral gavage. 1 Tendency to be significantly different from VEH FSL, p = 0.069 (one-way ANOVA) 2 Significantly different from VEH FSL, p = 0.031 (one-way ANOVA) 3 Significantly different from VEH FSL, p = 0.006 (one-way ANOVA) 4 Significantly different from VEH FSL, p = 0.039 (one-way ANOVA) 5-HIAA, 5‐hydroxyindoleacetic acid; 5‐HT, 5‐hydroxytryptamine (serotonin); DOPAC, 3,4‐dihydroxyphenylacetic acid; FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; IMI, Imipramine; SAM, S-adenosylmethionine; VEH, Vehicle.
ability [24], although it should be noted that all mice received a folatedeficient diet along with SAM. Adjunctive SAM has also shown beneficial cognitive effects in non-responding depressed patients [25]. In our study, monotherapy with IMI or SAM had no effects on cognition, whereas coadministration of IMI and SAM decreased the percentage of correct alternations in the Y-Maze compared to VEH and IMI. As both substances act on the serotonergic system (SAM e.g. by serotonin synthesis [26] and IMI by inhibition of serotonin reuptake [27]), we speculated whether their coadministration may have caused excess release of serotonin. Serotonin syndrome is a potentially life-threatening adverse drug reaction that has been associated with cognitive malfunction [28], which may explain our findings. However, our measurements of serotonin levels in plasma, hippocampus, and prefrontal cortex did not differ between the intervention groups. Chronic IMI treatment has also been associated with increased methionine in the cerebral cortex [29], which may lead to subsequent increases in SAM and homocysteine. Homocysteine concentrations may also be increased by SAM, and the combined administration of IMI + SAM may have increased homocysteine concentrations to abnormal levels. Hyperhomocysteinemia is known as an independent risk factor for cognitive dysfunction, and has also been linked directly to poor spatial cognition skills [30]. However, our measurements of homocysteine did not support this hypothesis. VEH FSL rats performed worse in the NOR than FRL rats, which has been reported previously [31]. Previous studies suggested that SAM could speed the action of IMI [13], potentially by preventing 5-HT1A receptor upregulation in the frontal cortex induced by IMI [32]. In our study, IMI significantly increased struggling behavior in the FST, SAM had no effect, and IMI + SAM decreased swimming behavior. Our data do not support a potential beneficial role of SAM in the FSL model of depression, which may be in line with other studies casting doubt on the efficacy of SAM in depressed patients [8]. The characteristic depressive-like phenotype of FSL rats was confirmed in the FST.
4.2. Effects on gastrointestinal transit Depression has been associated with constipation [12], but it is unknown if FSL rats have slower GI transit than FRL rats, and whether this potential difference may be mitigated by SAM or IMI treatment. Within our chosen observation period (8 h), approximately half of the animals expelled a red fecal pellet, indicative for successful transit. Since a large number of animals therefore did not complete the transit within the given time frame, the results should be interpreted with caution, as it is unknown if they would have produced a boli within the next 5 min or 5 h. With this caveat in mind, VEH-treated FSL rats might exhibit faster intestinal transit than FRLs, as 90 % produced a boli within 8 h compared to 50 % in FRLs. Importantly, no animals showed any signs of diarrhea, so a faster transit time cannot be attributed to a higher occurrence of diarrhea in FSL rats. The potential difference between the two strains may also be related to a higher sensitivity to fasting and social isolation conditions during the observational period, especially since our group has previously shown differences in metabolism [11] and stress reactivity [18] between VEH-treated FSL and FRL rats. However, given the high number of rats not completing the transit within the 8 h, these results should be considered preliminary and replication is necessary before any potential mechanisms can be discussed. 4.3. Effects on C1 metabolites and neurotransmitters Administration of oral SAM did not affect SAM or related C1 metabolites. Because the liver is the main site of SAM production, the administered dose in our study could be insufficient to raise liver SAM, especially when given orally due to the intestinal absorption barrier. Given the relatively short half-life of SAM (about 100 min in humans [33]), a more frequent administration may be necessary to obtain 279
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metabolic effects. Moreover, potential changes in C1 metabolites may have been short term and could have been restored within the 24-h gap between the last dose and euthanization. Alternatively, changes in plasma and liver tissue may require longer intervention periods than 4 weeks. We previously reported that VEH-treated FSL rats have decreased liver SAM compared to FRL rats [11], which was confirmed in the present independent experiment, suggesting that low liver SAM may be a metabolic phenotype of FSL rats. We replicated our previous finding that FSL rats had higher concentrations of plasma serotonin than FRLs [11]. FSL rats had lower dopamine and serotonin turnover in the hippocampus, as measured by DOPAC/dopamine and 5-HIAA/5-HT ratios, respectively. The intervention induced no changes in neurotransmitter concentrations, which is in line with the lack of effect on SAM that is necessary for neurotransmitters synthesis and degradation.
[7]
[8] [9]
[10] [11] [12] [13]
4.4. Limitations and conclusions
[14]
Several limitations deserve mentioning. First, our results were gathered from a relatively small sample size (n = 9–10/group) typically used for such experiments. However, several metabolic phenotypes (C1 metabolites and neurotransmitters [11]) were replicated in the present study, suggesting that the lack of effect of SAM supplements was not due to a low sample size. Other reasons for the lack of effect could be the route of SAM or IMI administration, the frequency of the dose, or the specific model of depression (FSL rats). It can, however, be argued that low liver SAM in FSL rats makes these animals an interesting model to test the hypothesis that SAM has an antidepressant effect. It may also be possible that low liver SAM in FSLs is a result of elevated neurotransmitters due to high metabolic demand, especially since our previous experiments showed that only liver SAM but not brain SAM was lowered in those animals. Finally, a further limitation of the present study is that SAM was not measured in brain tissues. Overall, our study has confirmed the previously reported low liver SAM concentrations in FSL compared to FRL rats, but SAM supplementation did not reverse imbalances in C1 metabolism. Neither SAM nor IMI had major effects on behavior, intestinal transit, neurotransmitters, or intestinal transit. There was some evidence for adverse behavioral effects of coadministration of IMI and SAM, but the underlying molecular mechanisms need to be determined by further studies. While FSL rats may still be an appropriate model to study SAM deficiency in the context of depression, restoring their metabolic and behavioral abnormalities may require other approaches.
[15]
[16] [17]
[18] [19] [20] [21]
[22] [23] [24] [25]
Declaration of interest
[26]
GW reported having received lecture/consultancy fees from H. Lundbeck A/S, Servier SA, AstraZeneca AB, Eli Lilly A/S, Sun Pharma Pty Ltd., Pfizer, Inc., Shire A/S, HB Pharma A/S, Arla Foods Amba., and Mundipharma International Ltd. No other conflicts of interests are reported.
[27] [28] [29]
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Research report
Behavioral and metabolic effects of S-adenosylmethionine and imipramine in the Flinders Sensitive Line rat model of depression
T
Sandra Tillmanna, , Denise F. Happa, Per F. Mikkelsena, Juergen Geiselb, Gregers Wegenera, Rima Obeidb,c ⁎
a
Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, DK-8240 Risskov, Denmark Saarland University Hospital, Department of Clinical Chemistry and Laboratory Medicine, Building 57, D-66421 Homburg, Saar, Germany c Aarhus Institute of Advanced Studies, Aarhus University, DK-8000 Aarhus C, Denmark b
ARTICLE INFO
ABSTRACT
Keywords: Depression Flinders Sensitive Line rats Imipramine One-carbon metabolism S-Adenosylmethionine
Depression is associated with dysregulation of methyl group metabolism such as low S-adenosylmethionine (SAM). We previously reported that Flinders Sensitive Line (FSL) rats, an animal model of depression, had lower concentrations of liver SAM than the control rats, Flinders Resistant Line (FRL) rats. The present study investigated if SAM supplementation may correct liver SAM and behavioral abnormalities in this model. Moreover, we compared one-carbon (C1) metabolites, neurotransmitters, and gastrointestinal (GI) transit in SAM-treated versus imipramine (IMI)-treated animals. FSL rats received vehicle, IMI, SAM, or IMI + SAM (n = 9–10 per group) once daily through oral gavage for 4 weeks; FRL rats received vehicle. Behavior was assessed using standard tests for locomotion, cognition, and depressive-like behavior. Monoamine neurotransmitters and C1 metabolites were measured using UHPLC‐ECD and UPLC‐MS/MS, respectively. Compared to FRL rats, FSLs had lower liver SAM, higher plasma serotonin, lower hippocampal dopamine and serotonin turnover, and faster GI transit. Behaviorally, FSL rats showed impaired cognitive performance as well as increased depressive-like behavior compared to FRLs. Coadministration of IMI and SAM seemed to have adverse effects on spatial memory. SAM or IMI administration did not reverse C1 metabolites, neurotransmitters, or GI transit in FSLs. Despite low liver SAM in FSL rats, orally administered SAM did not show antidepressant effects in this specific animal model of depression.
1. Introduction The one-carbon (C1) cycle is the principal biochemical pathway regulating the transfer of C1 groups for methylation of DNA, RNA, neurotransmitters, and phospholipids [1]. S-adenosylmethionine (SAM) is a methyl group donor in over 100 methylation reactions in the body and is produced mainly in the liver from various dietary methyl donors such as methionine, folate, betaine, or choline [2]. Deficiency of methyl donors has been linked to psychiatric diseases such as depression, but it is unclear whether this association is causal [3]. For instance, depression caused by folate and vitamin B12 deficiency [4] may be partly mediated by low SAM levels in both conditions. Several clinical studies have investigated the antidepressant role of SAM supplementation alone [4–6] or in comparison to a classical antidepressant such as imipramine [7]. However, available studies are generally considered to be of poor quality [8] and SAM is currently not recommended as firstline monotherapy treatment for depression [9].
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In a previous study, we demonstrated that Flinders Sensitive Line (FSL) rats, a validated animal model of depression [10], had lower liver SAM concentrations than the control strain, Flinders Resistant Line (FRL) rats [11]. Moreover, FSL rats had higher plasma dopamine and norepinephrine concentrations than FRLs, which may be related to their lower concentrations of liver SAM, as SAM is needed to degrade monoamine neurotransmitters. Therefore, it can be hypothesized that providing SAM from external sources may correct neurotransmitter synthesis and depressive-like behavior in FSL rats. Given the role of C1 metabolism in the pathophysiology of depression [4], the therapeutic potential of SAM in depression warrants further investigation, and studying tissue concentration of C1-related markers in animals treated with SAM may provide novel insights into biochemical processes altered by this universal methyl donor. To our knowledge, low liver SAM has not been studied in other animal models of depression, making FSL rats an interesting model to investigate the effect of SAM on neurotransmitter synthesis or catabolism.
Corresponding author. E-mail address: [email protected] (S. Tillmann).
https://doi.org/10.1016/j.bbr.2019.02.011 Received 13 January 2019; Received in revised form 3 February 2019; Accepted 5 February 2019 Available online 06 February 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Experimental timeline. After an initial adaptation phase, FRL and FSL rats were randomly assigned to four intervention groups (VEH, IMI, SAM, IMI + SAM). The intervention was given from day 0 to day 28 once daily via oral gavage. Rats underwent behavioral tests (day21–23,26–27), the GITT (day 24), and euthanization (day 27–28). FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; FST, Forced Swim Test; GITT, Gastrointestinal transit time; IMI, Imipramine; NOR, Novel Object Recognition; OF, Open Field; SAM, Sadenosylmethionine; VEH, Vehicle.
Our overall aim was to investigate if chronic SAM supplementation may reverse the behavioral and neurotransmitter abnormalities in FSL rats, potentially by increasing liver SAM and affecting related plasma C1 metabolites (i.e. betaine, choline, dimethylglycine, homocysteine). Depression is associated with gastric motility symptoms such as constipation [12], but it is not known if gastric motility differs between FSL and FRLs and how these potential differences may be changed upon administration of SAM. We therefore assessed gastrointestinal (GI) transit time in this animal model as an exploratory outcome. Imipramine (IMI), a tricyclic antidepressant, has been found to speed the therapeutic onset of SAM (200 mg/day) in patients with moderate to severe depression [13], suggesting an interactive effect. We therefore performed all experiments with the following groups: I) vehicle (VEH); II) IMI; III) SAM; IV) IMI + SAM. Our study may provide novel physiological insights into the treatment with methyl donors and/or tricyclic antidepressants in FSL rats.
day) was purchased from Nature Made Nutritional Products (Mission Hills, CA, USA) in tablet form, crushed using a mortar and pestle, and dissolved in tap water. Animals in the combined IMI + SAM group received the same dose as the two individual groups (15 mg/kg IMI + 200 mg/kg SAM). The IMI dose was chosen based on studies showing antidepressant effects in FSL rats [14,15]; the SAM dose was based on its ability to raise SAM concentrations in the rat brain [16]. All drugs were freshly prepared each day just before administration and were given once daily towards the end of the rats’ dark phase (2 p.m. ± 1 h) for a duration of 4 weeks using oral gavage. The dosage was weightadjusted every other day; the administration volume was 4 mL/kg. After 3 weeks of intervention, we assessed mood-related behavior and gastrointestinal transit time (GITT). Rats were decapitated after 4 weeks of intervention. The last intervention was administered 24 h before euthanization. A graphical representation of the experimental design is provided in Fig. 1.
2. Materials and methods
2.3. Behavioral analysis
2.1. Animals
All behavioral tests were performed in specially-equipped rooms within the animal facility between 3 a.m. and 2 p.m. in the active phase of the animal under dim red light. The order of animals was determined randomly. To minimize stress, animals were habituated to the behavioral rooms 1 h before testing. Tests were ordered from the least to the most stressful to reduce carryover effects [17]; the order of the test descriptions corresponds to the chronological order. All arenas/tanks were thoroughly cleaned between each trial. All tests were scored by an observer blinded to strain and intervention groups.
Age-matched adult male Flinders Sensitive Line (FSL, n = 38) and Flinders Resistant Line (FRL, n = 10) rats were obtained from the breeding colony maintained at Aarhus University, Denmark. At baseline (day 0), rats were 10.4 ± 2.1 weeks old (mean ± SD) and weighed 318 ± 52.0 g. Animals were housed as previously reported [11]. Rats of the same strain and intervention were pair-housed in standard cages (Cage 1291H Eurostandard Type III H, 425 × 266 × 185 mm, Tecniplast, Italy) at 20 ± 2 °C and 60 ± 5 % relative humidity on a reversed 12 h light/dark cycle (lights on at 2 p.m.). The reversed cycle was introduced in 3-h increments right before a 2-week adaptation phase. After the adaptation phase, rats were weighed every other day. All rats received standard chow diet (#1324 Altromin, Brogaarden, Lynge, Denmark) and tap water ad libitum and had access to a tunnel shelter, nesting material, and a wooden stick. Cages were changed once a week by the same experimenter. All experiments were approved by the Danish Animal Experiments Inspectorate prior to initiation of the experiments (approval number: 2012-15-2934-00254) and were conducted in accordance with the European Community Council Directive.
2.3.1. Novel Object Recognition The Novel Object Recognition (NOR) test was performed to measure nonspatial memory as described previously [18]. To avoid neophobic responses stemming from an unfamiliar apparatus and to promote exploratory activity, all rats were habituated to the testing arena (an open field of 50 × 50 × 40 cm) 24 h before the testing day for a duration of 1 h. The testing day consisted of a brief re-habituation phase, a training phase, and a testing phase. During the re-habituation phase, rats were allowed to re-habituate to the arena for 1 min. They were then placed into holding cages while two identical objects were positioned in the center of the arena, with equal distances between the objects and the walls to allow passing. During the training phase, they were again placed in the center of the arena (facing the wall) and allowed to freely explore for 15 min before being returned to their home cages. After 45 min, the testing session commenced, where one of the two identical objects from the training session was replaced by a novel one. Rats were positioned facing the wall and were allowed to freely explore for 5 min. Objects (glass salt dispenser, tin can) were validated prior to use to exclude a baseline preference for one of them. The position of the novel object was randomly assigned and counterbalanced for each intervention group. All sessions were recorded by a camera mounted to the ceiling. Exploration time during the testing phase was analyzed manually with a stopwatch, and was defined as directing the nose towards an object at a close distance or touching it, but not sitting on it.
2.2. Experimental design and intervention FSL rats were randomized to receive VEH, IMI, SAM, or IMI + SAM (n = 10/group). FRL rats were used to ascertain the depressive-like phenotype of FSL rats and received VEH (n = 10). The number of animals in each group was based on previous experiments in these rats with similar outcomes [11]. Two FSL rats were excluded from analyses; one due to unforeseen death before treatment start and one due to noncompliance to oral gavage administration in week 1. The final FSL group numbers were as follows: VEH (n = 10), IMI (n = 10), SAM (n = 9), IMI + SAM (n = 9). VEH animals received tap water. Imipramine hydrochloride (15 mg/kg/day) was obtained from Sigma Aldrich (Taufkirchen, Germany) and dissolved in tap water. SAM (200 mg/kg/ 275
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assays, livers, hippocampi, and prefrontal cortices were homogenized in 1 N acetic acid (10 μL/mg tissue) and centrifuged at 10.000 × g for 10 min. Plasma samples for C1 metabolites (betaine, choline, dimethylglycine, homocysteine, cystathionine) were used without further processing. For monoamine measurements, 20 μL perchloric acid was added to 180 μL plasma followed by centrifugation. Brain tissue samples were sonicated in 0.2 N perchloric acid (5 μL/mg tissue), centrifuged at 10.000 × g for 10 min, and filtered using Costar Spin‐X (Corning, NY, USA). C1 metabolites were measured in plasma (betaine, choline, dimethylglycine, homocysteine, cystathionine) and liver extracts (SAH, SAM) using an Acquity Ultra Performance LC system coupled to a MicroMass Quattro Premier XE tandem quadrupole mass spectrometer (UPLC‐MS/MS) (Waters Corporation, Milford, MA, USA). Plasma concentrations of total homocysteine and cystathionine were measured using an in‐house gas‐chromatography mass spectrometry method. Concentrations of SAM and SAH in the acidified extracts of liver and brain were measured using established methods that depend on the use of labelled isotopes as internal standards. Concentrations of serotonin (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA), dopamine, 3,4‐dihydroxyphenylacetic acid (DOPAC), and norepinephrine were measured using ultra-high performance liquid chromatography with electrochemical detection (UHPLC‐ECD, Dionex Ultimate 3000 UHPLC, Thermo Scientific, Rockford, IL, USA) as described in our previous work [11].
The preference for a novel over a familiar object was calculated as exploration time of novel vs. all objects [%]. 2.3.2. Y-Maze The Y-Maze was performed to assess spatial working memory as described previously [18]. This task is based on the innate tendency of rodents to alternate between exploring different arms of a maze. Rats were placed into the center of the maze and were allowed to freely explore the three arms of the maze for 6 min. Each choice was recorded by a camera mounted to the ceiling and was analyzed manually. An alternation was defined as consecutive full entries (excluding the tail) into each of the three arms; and alternation percentage was calculated as the number of alternations vs. the total number of arm visits (minus 2). 2.3.3. Open Field Locomotion was measured in an Open Field (OF) arena (100 × 100 × 80 cm) immediately prior to the FST as described previously [18]. Briefly, animals were placed in the center of the square and allowed to move freely for 5 min. The total distance travelled (cm) was recorded by a camera mounted to the ceiling and scored with specialized software tracking the midpoint of the rats’ body contour (Noldus Ethovision XT, The Netherlands). 2.3.4. Forced Swim Test To measure depressive-like behavior, the FST was performed as described previously [18]. For the pre-FST, rats were placed into a perspex cylinder (height 60 cm, diameter 24 cm) filled with 24 ( ± 1)°C heated tap water. After 15 min of swimming, rats were removed from the water, dried with towels, placed under a heating lamp for 30 min, and returned to their home cages. On the testing day 24 h later, they were removed from the water after 5 min. Their behavior was recorded by a camera positioned in front of the four tanks. Three distinct parameters were scored manually with a time-sampling technique, whereby the predominant behavior over each 5-s period of the 300-s test was rated [23]. Behaviors distinguished were struggling, swimming, and immobility. Diving was classified as swimming behavior.
2.7. Data analysis Two separate one-way analysis of variance (ANOVA) tests were used to study differences in bodyweight, food/water intake, behavior, C1 metabolites, and neurotransmitters between rat strains (VEH-treated FSL/FRL rats) and interventions (VEH/IMI/SAM/IMI + SAM). In case of statistically significant intervention effects, the ANOVA was followed up by Bonferroni posthoc comparisons. GITT was analyzed using a Kaplan Meier log-rank survival analysis. Assumptions of normality and homogeneity of variances were tested by Shapiro-Wilk test and Levene’s test, respectively. Results in the graphs are expressed as means ± standard error of the mean (SEM). Alpha was set at 0.05, while a pvalue between 0.05 and 0.1 was interpreted as a trend. Statistical analyses were carried out using IBM SPSS 22.0 (IBM Corp., Armonk, NY, USA). Graphs were generated using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).
2.4. Gastrointestinal transit time The GITT test was performed as previously reported [19]. Rats were single-housed and fasted 1 h before and throughout the entire testing period. Carmine red (Sigma Aldrich, St. Louis, MO, USA) was prepared as a 6 % solution in 0.5 % methyl cellulose (Sigma Aldrich). Rats were gavaged with 0.3 mL of the solution between 2 and 3:30 p.m. and monitored every 5 min for the appearance of the first red fecal boli by two observers familiar with the rats. The maximum observation time was 8 h, after which rats were returned to their home cages. The latency from gavage to passage of the first red pellet was recorded as total GITT.
3. Results 3.1. Bodyweight and food/water intake VEH FSL rats weighed significantly less than VEH FRL rats at both baseline (day 0; mean ± SD: 314 ± 54.4 g vs. 366 ± 29.4 g; F (1,18) = 7.06, p = 0.016) and endpoint (decapitation day; 351 ± 34.5 g vs. 412 ± 34.9 g; F(1,18) = 15.43, p = 0.001), which is characteristic for their depressive-like phenotype [20]. There was no strain difference in weight gain from baseline to endpoint or food/water intake. The four intervention groups differed in endpoint bodyweight (F (3,33) = 2.91, p = 0.049), but multiple comparison analysis did not show any significant group differences. The IMI group consumed significantly more water than the IMI + SAM group (52.8 ± 14.2 mL/100 g bodyweight vs. 38.4 ± 6.73 mL/100 g bodyweight; main effect ANOVA: F(3,33) = 3.23, p = 0.035; post-hoc: p = 0.033). The groups did not differ in baseline bodyweight, weight gain, or food intake.
2.5. Tissue collection Rats were decapitated after 4 weeks of starting the intervention. Decapitation took place between 2 and 5 p.m. over 2 consecutive days and was conducted in a random order. To minimize stress, rats were housed in an adjacent room and were brought singly into the decapitation room. After decapitation, trunk blood was collected in EDTA‐coated tubes and immediately centrifuged at 3000 × g for 10 min. Plasma was aliquoted and stored at −80 °C. Liver tissue (from the left lateral lobe), hippocampi, and prefrontal cortices were quickly removed, snap‐frozen in pre‐cooled isopentane, and stored at −80 °C.
3.2. Behavioral analysis
2.6. Sample processing and biochemical measurements
3.2.1. Locomotion and cognition VEH FSLs moved a significantly greater total distance in the OF compared to VEH FRLs (3498 ± 585 cm vs. 2334 ± 496 cm; F
The samples were processed as described in our previous study [11]. Briefly, for SAM, S-adenosylhomocysteine (SAH), and other C1-related 276
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Fig. 2. Behavioral results after intervention with VEH, IMI, SAM, or IMI + SAM. A: FSL rats traveled a significantly greater distance in the OF than FRL rats (p < 0.001). B: FSL rats had a significantly lower preference for the novel over the familiar object in the NOR, indicating impaired recognition memory (p = 0.033). C: FSL rats in the IMI + SAM group made significantly fewer correct alternations in the Y-Maze than both VEH (p = 0.055) and IMI (p = 0.023). D: VEH FSLs struggled less in the pre-FST than VEH FRLs (p = 0.032). Moreover, IMI animals struggled more than both VEH (p = 0.008) and IMI + SAM (p = 0.042). E: IMI animals swam less in the pre-FST than VEH (p = 0.005) and SAM (p = 0.067). IMI + SAM animals swam less than VEH (p = 0.049). F: VEH FSLs were more immobile in the pre-FST than VEH FRLs (p = 0.024). Values are expressed as means ± SEM and were analyzed by two one-way ANOVAs (rat strain, intervention); n = 9–10/group. FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; FST, Forced Swim Test; IMI, Imipramine; NOR, Novel Object Recognition; OF, Open Field; SAM, S-adenosylmethionine; VEH, Vehicle. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, #: p < 0.07.
(1,18) = 23.01, p < 0.001; Fig. 2A), but there was no difference between intervention groups. VEH FSL rats had a lower preference for the novel over the familiar object in the NOR than VEH FRLs (60.2 ± 18.3 % vs. 77.2 ± 14.5 %; F(1,18) = 5.30, p = 0.033; Fig. 2B), but intervention groups did not differ. VEH FSL and FRL rats did not perform differently in the Y-Maze, but animals in the IMI + SAM group made fewer correct alternations (58.7 ± 10.9 %) than both VEH (69.5 ± 7.60 %; p = 0.055) and IMI (70.8 ± 7.93 %; p = 0.023; main effect ANOVA: F(3,34) = 4.0, p = 0.016; Fig. 2C).
Table 1 Gastrointestinal transit time assessed after intervention with VEH, IMI, SAM, or IMI + SAM. Intervention In FRL rats VEH In FSL rats VEH IMI SAM IMI + SAM Overall
3.2.2. Depressive-like behavior In the pre-FST, VEH FSLs struggled less than VEH FRLs (113 ± 33.9 s vs. 158 ± 51.0 s; F(1,18) = 5.40, p = 0.032; Fig. 2D) and were more immobile (98.5 ± 36.5 s vs. 62.0 ± 29.5 s; F (1,18) = 6.05, p = 0.024; Fig. 2F). IMI animals struggled more (172 ± 52.2 s) than both VEH (113 ± 33.9 s; p = 0.008) and IMI + SAM (122 ± 23.6 s; p = 0.042; main effect ANOVA: F(3,34) = 4.72, p = 0.007). IMI animals swam less (41.5 ± 33.6 s) than VEH (88.5 ± 26.9 s; p = 0.005) and SAM (76.7 ± 25.7 s; p = 0.067; main effect ANOVA: F(3,34) = 5.69, p = 0.003; Fig. 2E). Moreover, IMI + SAM animals swam less than VEH (51.7 ± 26.6 s vs. 88.5 ± 26.9 s; p = 0.049). There was no intervention difference in immobility. The aforementioned swimming effects in the pre-FST were also detected at the test session 24 h later; there were no other significant strain or intervention differences.
N of Events/ Total N
% Events
Mean Survival Estimate (min)
Mean Transit Time (min)
5/10
50.0
455 ± 12
430 ± 47
9/10 4/10 3/9 4/9 25/48
90.0 40.0 33.3 44.4 52.1
434 416 445 456 441
428 319 374 425 398
± ± ± ± ±
17 31 28 19 10
± ± ± ± ±
59 102 98 71 83
compared to FSLs (10 %). There was also a tendency for a significant difference between intervention groups (χ2(3) = 7.34, p = 0.062). To investigate any differences in GITT time among the animals that produced a red boli within the 8 h, we ran a one-way ANOVA with all uncensored cases. There was no difference in mean time between FRL and FSL rats or between the intervention groups among those producing a boli within 8 h (Table 1). 3.4. C1-related metabolites The concentrations of C1‐related metabolites in FRL and FSL rats are presented in Fig. 3 (plasma) and Fig. 4 (liver). In plasma, VEH FSL rats had higher dimethylglycine concentrations than VEH FRL rats (7.10 ± 0.96 μmol/L vs. 5.72 ± 0.77 μmol/L; F(1,18) = 12.62; p = 0.002; Fig. 3C), but the two VEH-treated strains did not differ in plasma betaine, choline, homocysteine, and cystathionine. The four intervention groups tended to differ in plasma cystathionine concentrations (F (3,33) = 2.73; p = 0.060; Fig. 3E), but Bonferroni analysis did not reach significance. There were no significant intervention differences in the aforementioned plasma markers. In liver, VEH FSL rats had lower SAM concentrations than VEH FRL rats (107 ± 11.5 nmol/g liver vs. 125 ± 16.2 nmol/g; F(1,18) = 8.16; p = 0.011; Fig. 4B), but SAH or the SAM/SAH ratio did not differ
3.3. Gastrointestinal transit time Table 1 represents the number of animals that produced a red boli within the 8-h observational period as well as the mean time of the first appearance of a red boli for each group. Within the observational period, 52.1 % of all animals produced a red boli (VEH FRL: 50 %, VEH FSL: 90 %, IMI: 40 %, SAM: 33.3 %, IMI + SAM: 44.4 %). A nonparametric log-rank test showed a significant difference in the survival distribution between VEH FRL and VEH FSL (χ2(1) = 4.06, p = 0.044) due to the higher proportion of censored cases in the FRL group (50 %) 277
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Fig. 3. Changes in plasma one-carbon metabolites after intervention with VEH, IMI, SAM, or IMI + SAM. There were no strain (VEH-treated FSL vs. FRL) or intervention differences in betaine (A) or choline concentrations (B). VEH FSL rats had significantly higher concentrations of dimethylglycine than VEH FRLs (p = 0.002) (C). The strain or intervention groups did not differ in homocysteine (D) or cystathionine (E). Values are expressed as means ± SEM and were analyzed by two one-way ANOVAs (rat strain, intervention); n = 9–10/group. FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; IMI, Imipramine; SAM, S-adenosylmethionine; VEH, Vehicle. **: p ≤ 0.01.
between the strains. There were no intervention differences in SAH, SAM, or the SAM/SAH ratio.
4. Discussion We investigated the effects of SAM and IMI administration on behavior, gut motility, C1 metabolites and neurotransmitters in an established animal model of depression that exhibits low liver SAM [11]. Compared to VEH, IMI + SAM reduced the percentage of correct alternations in the Y-Maze, indicating an impairment in spatial cognition. VEH-treated FSL rats showed preliminary evidence for faster intestinal transit than FRLs. Moreover, they had higher concentrations of plasma serotonin, but lower hippocampal dopamine and serotonin turnover than FRLs. There were no intervention effects on GITT, C1 metabolites, or neurotransmitters.
3.5. Monoamine neurotransmitters Table 2 shows the concentrations of monoamine neurotransmitters measured in plasma, hippocampus, and prefrontal cortex of FRL and FSL rats. In plasma, VEH FSL rats tended to have higher 5-HT concentrations than VEH FRLs (1625 ± 797 nmol/L vs. 1052 ± 495 nmol/L; F (1,18) = 3.73; p = 0.069). There were no strain differences in plasma DOPAC, dopamine, norepinephrine, or DOPAC/dopamine ratio. In the hippocampus, VEH FSL rats had lower DOPAC concentrations than VEH FRLs (0.01 ± 0.004 nmol/g tissue vs. 0.02 ± 0.01 nmol/g; F (1,18) = 5.47; p = 0.031). The ratio of DOPAC/DA, an indicator of dopamine turnover, was also lower in VEH FSL rats than in FRLs in the hippocampus (0.60 ± 0.31 vs. 1.37 ± 0.71; F(1,18) = 9.94; p = 0.006). Similarly, the ratio of 5-HIAA/5-HT, an indicator of serotonin turnover, was lower in VEH FSL rats than FRLs in the hippocampus (0.66 ± 0.27 vs. 1.04 ± 0.48; F(1,18) = 4.96; p = 0.039). There were no other significant strain differences in the hippocampus or in the prefrontal cortex. The intervention groups did not differ in their monoamine concentrations in plasma, hippocampus, or prefrontal cortex.
4.1. Effects on behavior Monotherapy of both IMI and SAM has generally shown procognitive effects in animal models and patients. IMI treatment significantly improved cognitive function in a mouse model of traumatic brain injury [21]. In humans, IMI improved cognitive function in depressed patients [22], whereas the effect in Alzheimer’s patients was minimal [23]. A meta-analysis on the effect of SAM on Y-Maze performance in mice showed that SAM was associated with improvements in cognitive
Fig. 4. Changes in liver one-carbon metabolites after intervention with VEH, IMI, SAM, or IMI + SAM. There were no strain (VEH-treated FSL vs. FRL) or intervention differences in SAH concentrations (A). VEH FSL rats had significantly lower concentrations of SAM than VEH FRLs (p = 0.011) (B). The SAM/SAH ratio was similar between all groups (C). Values are expressed as means ± SEM and were analyzed by two one-way ANOVAs (rat strain, intervention); n = 9–10/group. FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; IMI, Imipramine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; VEH, Vehicle. *: p ≤ 0.05. 278
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Table 2 Plasma and tissue concentrations of monoamine neurotransmitters after intervention with VEH, IMI, SAM, or IMI + SAM. Intervention Strain Number
VEH 10
FRL
EDTA plasma (nmol/L) Norepinephrine 40.3 ± 7 Dopamine 8.38 ± 2.07 DOPAC 3.08 ± 1.03 DOPAC/dopamine 0.40 ± 0.20 5-HT 1052 ± 4951 Hippocampal extract (nmol/g tissue) Norepinephrine 0.60 ± 0.09 Dopamine 0.01 ± 0.004 DOPAC 0.02 ± 0.012 DOPAC/dopamine 1.37 ± 0.713 5-HT 0.08 ± 0.07 5-HIAA 0.08 ± 0.05 5-HIAA/5-HT 1.04 ± 0.484 Prefrontal cortex extract (nmol/g tissue) Norepinephrine 0.56 ± 0.11 Dopamine 0.21 ± 0.06 DOPAC 0.12 ± 0.04 DOPAC/dopamine 0.57 ± 0.26 5-HT 0.55 ± 0.27 5-HIAA 0.35 ± 0.12 5-HIAA/5-HT 0.72 ± 0.23
IMI 10
FSL
9
FSL
SAM 9
FSL
IMI + SAM 9
FSL
39.5 ± 12 8.15 ± 3.17 3.02 ± 1.66 0.42 ± 0.24 1625 ± 797
44.8 ± 11 7.02 ± 2.51 5.23 ± 3.65 0.71 ± 0.32 1285 ± 502
37.0 ± 11 7.58 ± 1.79 3.39 ± 1.80 0.46 ± 0.27 1310 ± 501
49.6 ± 14 6.85 ± 1.34 2.94 ± 1.52 0.42 ± 0.19 1105 ± 462
0.60 ± 0.12 0.02 ± 0.01 0.01 ± 0.004 0.60 ± 0.31 0.07 ± 0.06 0.06 ± 0.06 0.66 ± 0.27
0.56 ± 0.10 0.02 ± 0.01 0.01 ± 0.01 0.57 ± 0.42 0.10 ± 0.05 0.09 ± 0.06 0.77 ± 0.33
0.62 ± 0.04 0.02 ± 0.01 0.01 ± 0.01 0.58 ± 0.34 0.10 ± 0.06 0.11 ± 0.11 0.91 ± 0.50
0.61 ± 0.09 0.03 ± 0.02 0.01 ± 0.01 0.45 ± 0.28 0.11 ± 0.06 0.10 ± 0.08 0.85 ± 0.36
0.60 ± 0.12 0.22 ± 0.05 0.11 ± 0.03 0.54 ± 0.15 0.62 ± 0.24 0.39 ± 0.07 0.68 ± 0.17
0.60 ± 0.17 0.21 ± 0.05 0.09 ± 0.04 0.46 ± 0.15 0.66 ± 0.24 0.41 ± 0.13 0.68 ± 0.26
0.68 ± 0.17 0.23 ± 0.04 0.10 ± 0.02 0.47 ± 0.15 0.67 ± 0.29 0.44 ± 0.13 0.72 ± 0.27
0.70 ± 0.16 0.23 ± 0.05 0.11 ± 0.03 0.50 ± 0.16 0.73 ± 0.32 0.42 ± 0.11 0.65 ± 0.21
Data are presented as mean ± SD. Rats received VEH (tap water), IMI (15 mg/kg), SAM (200 mg/kg) or a combination of IMI + SAM once daily for 4 weeks through oral gavage. 1 Tendency to be significantly different from VEH FSL, p = 0.069 (one-way ANOVA) 2 Significantly different from VEH FSL, p = 0.031 (one-way ANOVA) 3 Significantly different from VEH FSL, p = 0.006 (one-way ANOVA) 4 Significantly different from VEH FSL, p = 0.039 (one-way ANOVA) 5-HIAA, 5‐hydroxyindoleacetic acid; 5‐HT, 5‐hydroxytryptamine (serotonin); DOPAC, 3,4‐dihydroxyphenylacetic acid; FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; IMI, Imipramine; SAM, S-adenosylmethionine; VEH, Vehicle.
ability [24], although it should be noted that all mice received a folatedeficient diet along with SAM. Adjunctive SAM has also shown beneficial cognitive effects in non-responding depressed patients [25]. In our study, monotherapy with IMI or SAM had no effects on cognition, whereas coadministration of IMI and SAM decreased the percentage of correct alternations in the Y-Maze compared to VEH and IMI. As both substances act on the serotonergic system (SAM e.g. by serotonin synthesis [26] and IMI by inhibition of serotonin reuptake [27]), we speculated whether their coadministration may have caused excess release of serotonin. Serotonin syndrome is a potentially life-threatening adverse drug reaction that has been associated with cognitive malfunction [28], which may explain our findings. However, our measurements of serotonin levels in plasma, hippocampus, and prefrontal cortex did not differ between the intervention groups. Chronic IMI treatment has also been associated with increased methionine in the cerebral cortex [29], which may lead to subsequent increases in SAM and homocysteine. Homocysteine concentrations may also be increased by SAM, and the combined administration of IMI + SAM may have increased homocysteine concentrations to abnormal levels. Hyperhomocysteinemia is known as an independent risk factor for cognitive dysfunction, and has also been linked directly to poor spatial cognition skills [30]. However, our measurements of homocysteine did not support this hypothesis. VEH FSL rats performed worse in the NOR than FRL rats, which has been reported previously [31]. Previous studies suggested that SAM could speed the action of IMI [13], potentially by preventing 5-HT1A receptor upregulation in the frontal cortex induced by IMI [32]. In our study, IMI significantly increased struggling behavior in the FST, SAM had no effect, and IMI + SAM decreased swimming behavior. Our data do not support a potential beneficial role of SAM in the FSL model of depression, which may be in line with other studies casting doubt on the efficacy of SAM in depressed patients [8]. The characteristic depressive-like phenotype of FSL rats was confirmed in the FST.
4.2. Effects on gastrointestinal transit Depression has been associated with constipation [12], but it is unknown if FSL rats have slower GI transit than FRL rats, and whether this potential difference may be mitigated by SAM or IMI treatment. Within our chosen observation period (8 h), approximately half of the animals expelled a red fecal pellet, indicative for successful transit. Since a large number of animals therefore did not complete the transit within the given time frame, the results should be interpreted with caution, as it is unknown if they would have produced a boli within the next 5 min or 5 h. With this caveat in mind, VEH-treated FSL rats might exhibit faster intestinal transit than FRLs, as 90 % produced a boli within 8 h compared to 50 % in FRLs. Importantly, no animals showed any signs of diarrhea, so a faster transit time cannot be attributed to a higher occurrence of diarrhea in FSL rats. The potential difference between the two strains may also be related to a higher sensitivity to fasting and social isolation conditions during the observational period, especially since our group has previously shown differences in metabolism [11] and stress reactivity [18] between VEH-treated FSL and FRL rats. However, given the high number of rats not completing the transit within the 8 h, these results should be considered preliminary and replication is necessary before any potential mechanisms can be discussed. 4.3. Effects on C1 metabolites and neurotransmitters Administration of oral SAM did not affect SAM or related C1 metabolites. Because the liver is the main site of SAM production, the administered dose in our study could be insufficient to raise liver SAM, especially when given orally due to the intestinal absorption barrier. Given the relatively short half-life of SAM (about 100 min in humans [33]), a more frequent administration may be necessary to obtain 279
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metabolic effects. Moreover, potential changes in C1 metabolites may have been short term and could have been restored within the 24-h gap between the last dose and euthanization. Alternatively, changes in plasma and liver tissue may require longer intervention periods than 4 weeks. We previously reported that VEH-treated FSL rats have decreased liver SAM compared to FRL rats [11], which was confirmed in the present independent experiment, suggesting that low liver SAM may be a metabolic phenotype of FSL rats. We replicated our previous finding that FSL rats had higher concentrations of plasma serotonin than FRLs [11]. FSL rats had lower dopamine and serotonin turnover in the hippocampus, as measured by DOPAC/dopamine and 5-HIAA/5-HT ratios, respectively. The intervention induced no changes in neurotransmitter concentrations, which is in line with the lack of effect on SAM that is necessary for neurotransmitters synthesis and degradation.
[7]
[8] [9]
[10] [11] [12] [13]
4.4. Limitations and conclusions
[14]
Several limitations deserve mentioning. First, our results were gathered from a relatively small sample size (n = 9–10/group) typically used for such experiments. However, several metabolic phenotypes (C1 metabolites and neurotransmitters [11]) were replicated in the present study, suggesting that the lack of effect of SAM supplements was not due to a low sample size. Other reasons for the lack of effect could be the route of SAM or IMI administration, the frequency of the dose, or the specific model of depression (FSL rats). It can, however, be argued that low liver SAM in FSL rats makes these animals an interesting model to test the hypothesis that SAM has an antidepressant effect. It may also be possible that low liver SAM in FSLs is a result of elevated neurotransmitters due to high metabolic demand, especially since our previous experiments showed that only liver SAM but not brain SAM was lowered in those animals. Finally, a further limitation of the present study is that SAM was not measured in brain tissues. Overall, our study has confirmed the previously reported low liver SAM concentrations in FSL compared to FRL rats, but SAM supplementation did not reverse imbalances in C1 metabolism. Neither SAM nor IMI had major effects on behavior, intestinal transit, neurotransmitters, or intestinal transit. There was some evidence for adverse behavioral effects of coadministration of IMI and SAM, but the underlying molecular mechanisms need to be determined by further studies. While FSL rats may still be an appropriate model to study SAM deficiency in the context of depression, restoring their metabolic and behavioral abnormalities may require other approaches.
[15]
[16] [17]
[18] [19] [20] [21]
[22] [23] [24] [25]
Declaration of interest
[26]
GW reported having received lecture/consultancy fees from H. Lundbeck A/S, Servier SA, AstraZeneca AB, Eli Lilly A/S, Sun Pharma Pty Ltd., Pfizer, Inc., Shire A/S, HB Pharma A/S, Arla Foods Amba., and Mundipharma International Ltd. No other conflicts of interests are reported.
[27] [28] [29]
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