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Specific hippocampal choline decrease in an animal model of depression

Published Online:https://doi.org/10.1259/bjr/25165760

Abstract

A decreased level of the hippocampal choline signal was found in patients with depression in previous proton magnetic resonance spectroscopy (1H-MRS) studies. The objective of this study is to compare choline levels before and after the forced swimming test (FST), an animal model of depression typically used for assessing antidepressant activity. 1H-MRS spectra were obtained from both the left and right hippocampus. After the FST, rats showed a significant decrease of the choline/creatine (Cho/Cr, p = 0.037) and choline/N-acetylaspartate (Cho/NAA, p = 0.048) ratios in the left hippocampus, but not in the right hippocampus. This finding was analogous to results from patients with depression. It suggests that decreased Cho/Cr and Cho/NAA ratios in the left hippocampal regions might be considered to be biomarkers in rats with depression.

Major depression is a severe, debilitating and life-threatening illness. Despite extensive pre-clinical and clinical investigations for several years, the specific neurobiological processes associated with depression and the mechanisms behind the therapeutic effects of antidepressants are not clearly understood [1].

The forced swimming test (FST) is one of the most widely used and reliable animal models of depression, allowing assessment of antidepressant activity and examination of the pre-clinical pathophysiology of depression [2]. This model is widely used in screening new antidepressants because of its ease of use, reproducibility across different laboratories and ability to detect a broad spectrum of antidepressant agents [3]. Immobility in the FST is assumed to reflect either a failure of persistence in escape-directed behaviour (i.e. behavioural despair) or the development of passive behaviour that disengages the animal from active forms of coping with stressful stimuli.

The hippocampus is a part of the limbic stress pathway that is substantially sensitive to the neurotoxic effects of elevated levels of glucocorticoids, as observed following recurrent stress episodes [4]. Impaired synaptic efficacy as a result of the FST procedure, which involves severe physical and emotional stress, has been reported in the rat hippocampus [5]. This synaptic efficacy shows significant improvement after treatment with repetitive transcranial magnetic stimulation (rTMS) [5]. N-methyl-D-aspartate (NMDA) receptors are a crucial component of the neuronal response to antidepressant drugs. In the FST, antidepressant-like effects were observed upon intrahippocampal administration of the NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 37489) and amino-7-phosphonoheptanoic acid (AP-7), suggesting that the hippocampus may be one of the major target sites with plentiful NMDA receptors [6, 7].

Localised proton magnetic resonance spectroscopy (1H-MRS) has been used as an efficient diagnostic methodology and non-invasive technique to elucidate the biochemical and molecular alterations that occur in the depressive disorder. A 1H-MRS study of patients with depression found a decreased concentration of choline-containing compounds (Cho) in the hippocampus of depressive patients when compared with non-depressed subjects [8]. This decreased level of Cho normalised under electroconvulsive shock (ECS) in parallel to the treatment response [8]. Using the learned helpless (LH) model, one of the valid depression models for rodents, ECS treatment produced a statistically greater increase in hippocampal choline/creatine (Cho/Cr) and choline/N-acetylaspartate (Cho/NAA) in LH animals than in not learned helpless (NLH) animals [9]. Considering all of these results together, it was believed that a decreased hippocampal Cho/Cr ratio might reflect metabolic dysfunction in the pathology of major depression and in animal models of depression, leading to a depression-like symptomatology.

We have recently reported a significantly increased Cho/Cr ratio in the dorsolateral pre-frontal cortex (DLPFC) of rats exposed to the FST as compared with animals not subjected to the FST [10], a finding similar to that obtained in investigations of patients with depression [11]. To extend our previous findings and to characterise variation of the hippocampal Cho/Cr ratio in an animal model of depression, we systematically examined the Cho/Cr ratio in the hippocampus of rats before and after the FST. Using a relatively small volume of interest (VOI), we were able to focus specifically on the homogeneous hippocampus tissue, whereas our previous study was performed with relatively large VOI and thus an increased possibility of contamination by heterogeneous DLPFC tissue [10]. This previous 1H-MRS study in the hippocampus of patients with depression, gave rise to our hypothesis that the Cho/Cr ratio in the hippocampus of rats would be lower after exposure to the FST than before exposure to the FST.

Methods and materials

Animals

12 experimentally naive male Sprague–Dawley rats (Charles River, Yokohama, Japan) weighting 160–180 g were used as subjects. The rats were acclimatised for 1 week in specific pathogen-free (SPF) environmental conditions in a room kept at a constant temperature (23±2°C) on a 12-h light/12-h dark cycle. Food and water were available during the entire study. To investigate the pure effect of the FST, all rats underwent 1H-MRS examinations twice but the control group of animals did not undergo the FST. The treatment group (i.e. depression group) were exposed to the FST within 2–3 days of the first 1H-MRS examination. A second 1H-MRS examination was performed 10–15 min after the FST to allow observation of rapid changes in neurotransmitters, and to dry and relax the rats exposed to the FST. All animal experiments were performed in accordance with institutional animal care guidelines for the care and use of laboratory animals (The Catholic University of Korea, Seoul, South Korea) and the ethics committee of our university approved all protocols in this study. The minimum number of animals required to analyse statistical differences was employed.

Forced swimming test

Each rat was placed into a vertical glass cylinder (height 40 cm, diameter 18 cm) containing 25 cm of water (maintained at 25°C) and left there for 15 min. After 15 min in the cylinder, the rat was removed and allowed to dry before being returned to its cage. 24 h later, the rat was again plunged into the cylinder for 5 min. Water was replaced between each individual test. According to an original version of the FST [2], the FST was performed on each rat just once.

Localised proton magnetic resonance spectroscopy

Magnetic resonance (MR) experiments were conducted using a 4.7T BIOSPEC scanner (Bruker Medical GmbH, Ettlingen, Germany) with a 400-mm-bore magnet and 150 mT m–1 actively shielded gradient coils. To execute the 1H-MRS measurements, rats were initially anaesthetised by inhalation of isoflurane at a 4–6% concentration in a mixture of N2O and O2 gas (50%/50%). Anaesthesia was maintained by inhalation of a 1.5–2% concentration of isoflurane in a mixture of N2O and O2 gas (50%/50%). Anaesthetised rats were placed in the prone position with the head firmly fixed on a palate holder equipped with an adjustable nose cone. A scout image was initially obtained to verify the position of the subject and the image quality. The positions of the rectangular VOIs (1.5 × 2.5 × 2.5 mm3) were carefully selected in the left and right dorsal hippocampus of the rat brain from multislice axial T2 weighted MR images obtained using rapid acquisition with a relaxation enhancement (RARE) sequence (repetition time (TR) 5000 ms, echo time (TE) 22 ms, slice thickness 1.0 mm, number of excitations (NEX) 2, matrix size 256 × 192) (Figure 1). 1H-MR spectra were obtained with use of a point resolved spectroscopy (PRESS) localisation sequence performed according to the following parameters: TR 2500 ms, TE 144 ms, 512 average, 2048 complex data points, voxel dimensions 1.5 × 2.5 × 2.5 mm3, acquisition time 25 min. Adjustment of all first- and second-order shim terms was accomplished with the fast automatic shimming technique by mapping along the projections (FASTMAP). Water suppression was accomplished by variable power RF pulses with optimised relaxation delay (VAPOR) by controlling the transmit gain to maximise water suppression. The rectal temperature was maintained at 30–35°C by a heating blanket positioned around the body of the animals and was verified by a thermo sensor. In addition, the respiratory cycle and heart rate were continuously observed.

Spectral quantification

Raw data were processed using the TOPSPIN data analysis program (Bruker) by an unbiased analyser who was experienced in 1H-MRS data processing. All spectra were processed with an exponential filter corresponding to a line broadening of 5 Hz. Time-domain data were converted to frequency domain by Fourier transformation. Zero/first-order phase correction and baseline correction were applied to the frequency domain spectra. 1H-MRS spectra with extreme baseline distortion, which make it difficult to recognise the NAA, Cho and Cr resonances, were disregarded from the analysis. Proton resonances in the spectra obtained from brain tissue were assigned on the basis of prior assignments. Resonance peak assignments of the major 1H-MRS observable neurometabolites were the CH3 of NAA, 2.0 ppm; the N-CH3 of Cr, 3.0 ppm; and the N-(CH3)3 of Cho, 3.2 ppm. Quantitative expression of results was evaluated as values relative to the Cr resonance at 3.0 ppm, thereby minimising the errors caused by variations in the magnetic field homogeneity and tissue volume.

Peak ratio was calculated using the area of each peak. In addition, to exclude possible alterations in the Cho/Cr resonance ratio that reflect changes in Cr resonance , the ratio of Cho/NAA resonance was also determined.

Statistical analysis

Data are presented as the mean±standard deviation. Statistical analysis was performed with commercial SPSS software (SPSS 15.0 for Windows, SPSS, Chicago, IL). The paired samples t-test was used to test for an FST effect, that is, to compare data from before and after each rat was exposed to FST. Data from two rats were excluded from further statistical analysis because of inappropriate MR spectral quality. The left and right hippocampi were not averaged for each animal but were treated as individual hippocampi (n = 40). Statistical significance was set at p<0.05.

Results

Before performing the FST, we investigated interhemispheric differences (i.e. differences between the left and right hippocampi) in the metabolite ratios and no significant difference was found (Cho/Cr and NAA/Cr; p>0.05). As shown in Figure 2, the proton MR spectra obtained from the left hippocampus after exposing the rats to the FST exhibited a marked reduction in the relative intensity of the Cho signal as compared with the spectra obtained before the FST. The paired samples t-test comparing metabolite signals measured before and after the FST determined that the NAA/Cr ratio remained stable (1.60±0.29 vs 1.57±0.33; df = 9, t = 0.236, p = 0.819), that the Cho/Cr ratio was significantly decreased (0.88 ± 0.28 vs 0.65±0.13; df = 9, t = 2.443, p = 0.037), and that the Cho/NAA ratio was also significantly decreased (0.55±0.16 vs 0.42±0.11; df = 9, t = 2.287, p = 0.048) in the left hippocampus following FST (Figure 3a).

However, no significant differences in the Cho/Cr ratio (0.79±0.33 vs 0.84±0.31; df = 9, t = −0.323, p = 0.754), Cho/NAA ratio (0.47±0.12 vs 0.48±0.21; df = 9, t = −0.082, p = 0.936) or NAA/Cr ratio (1.65±0.32 vs 1.83±0.53; df = 9, t = −0.987, p = 0.349) were observed before and after the FST in the right hippocampal region (Figure 3b).

Discussion

To the best of our knowledge, this is the first study to demonstrate a decreased hippocampal Cho/Cr ratio in rats undergoing FST by the use of 1H-MRS as a general method for exploring the pathophysiology of depression. This important finding provides new evidence of a localised biochemical abnormality in the hippocampus in rats after exposure to the FST. This result is consistent with early findings from 1H-MRS studies demonstrating decreased hippocampal levels of Cho in tree shrews exposed to psychosocial stress that led to a depression-like symptomatology [12, 13] and in patients with depression [8]. It implies that decreased Cho/Cr and Cho/NAA ratios in the hippocampal regions might represent neurobiological markers of both rats with depressive characteristics induced by the FST and patients with depression.

The decreased hippocampal Cho/Cr ratio in the rats after exposure to the FST may be associated with the hippocampal atrophy observed in a psychosocial stress model [13] and in patients with major depression [14]. Dwivedi et al [15] reported reduced cytosolic phospholipase C (PLC) activity and protein expression within the hippocampus in a stress-induced learned helplessness model. In a previous study from the same research group, a decreased level of PLC in platelets was observed in bipolar but not in unipolar patients [16]. These results suggest that a differential application of the PLC isoenzyme may be associated with the pathophysiology of depression, and that PLC downregulation induced by the FST could be responsible for the decreased Cho/Cr ratio seen in the hippocampus in this study.

The present result showing a stable NAA/Cr ratio is in good agreement with a previous clinical study [8] assuming that no neuronal and axonal losses occurred as a consequence of the FST. These findings were controversial, however, because earlier investigations found high levels of corticosterone both after the FST [17] and in other stress models that induce depression, such as aversive tailshock [18] and massed water maze trainings [19], resulting in neuronal loss in the hippocampus [20]. One possible explanation is to consider the physiological role of the glia in the aetiology of depression: a previous study reported the contribution of glial cell loss to hippocampal damage in depression patients [21]. Changes in glial cells may have an effect on regulating several neurotransmitters, such as γ-aminobutyric acid (GABA), glutamate and acetylcholine, and on modulating synaptic activity [22, 23], resulting in altered Cho/Cr ratio without a change in NAA/Cr ratio.

The previous studies revealed a simultaneous decrease of NAA, Cr and Cho-containing compounds in a psychosocial stress model of depression [13] and a significantly increased Cr/NAA ratio in a LH model after ECS treatment [9], precluding a comprehensive analysis to adequately discriminate obvious neurochemical profiles in rats before and after the FST. Future 1H-MRS studies should involve absolute quantification to investigate specific alterations of NAA, Cr and Cho-containing compound concentrations in rats exposed to the FST.

1H-MRS studies of major depression patients reported that metabolite alterations were more common in the left hemisphere than in the right hemisphere [24, 25], which is consistent with previous studies indicating that left hemispheric lesions are more linked to depression, whereas right hemispheric lesions are more general in mania [24]. In line with these findings, transcranial magnetic stimulation (TMS) has been used to reveal significantly lower excitability in the left hemisphere in patients with refractory major depressive disorder (MDD) who were off medication, with no difference in controls [26]. The present results of metabolite differences in the left but not right hippocampal region may suggest a similar pathophysiology in major depression and FST-induced depression.

The strengths of this study include the selection of homogeneous hippocampal tissue and the use of a reliable method that accurately resolves the levels of NAA, Cr and Cho. Nevertheless, the findings of the present study should be interpreted cautiously because of several limitations. We did not include other small coupled-spin metabolites, such as glutamate (Glu), glutamine (Gln) and GABA, because of irreproducible resolved peaks and unreliable quantification in the post-processing step. Previous authors have reported reduced GABA levels in the nucleus accumbens, cortex and brainstem after a session of the FST [27], and decreased Glx (Glu+Glx) concentrations in the left DLPFC of depressed patients [28]. Another possible confounder is the relatively small sample size, which reduced the statistical power of our analysis. However, we obtained MR spectra twice from the same rats, before exposure to the FST and after exposure to the FST, which were processed by an unbiased analyser, thereby adding statistical strength to the present results. Finally, the 1H-MRS parameters chosen for this study may account for the apparent differences between the resonance of Cho-containing compounds in this study and TR and TE values previously measured in rat hippocampus at 4.0 T [29]. Changes in the relaxation time of metabolites in the FST model could contribute to metabolite variability and could potentially confound the metabolite ratio measurements.

Conclusions

The present study demonstrates that the FST causes a significantly decreased Cho/Cr ratio in the left dorsal hippocampal regions, which is in good agreement with the results of a previous human study. Our results indicate that biochemical perturbation, i.e. a decreased Cho/Cr ratio, may be an important factor in the pathophysiology of depression, and also suggests a decelerated turnover of the membrane in the hippocampus of rats with depression. Finally, a larger number of subjects and the use of antidepressant drug responses need to be investigated to confirm this finding, and to allow us to understand more clearly the implications of the decreased hippocampal Cho/Cr ratio in rats exposed to the FST.

Figure 1.
Figure 1.

Axial magnetic resonance imaging with the superimposed location of the volume of interest (1.5 × 2.5 × 2.5 mm3) in the left and right hippocampal regions.

Figure 2.
Figure 2.

Proton magnetic resonance spectroscopy showing the effect of the forced swimming test (FST). An arrow indicates a statistically significant reduction in the relative intensity of the Cho signal after FST.

Figure 3.
Figure 3.

Cho/Cr and Cho/NAA metabolite ratios in (a) the left hippocampus and (b) the right hippocampus in rats before exposure to the forced swimming test (FST) (white) and after exposure to the FST (grey). The error bar indicates the 95% confidence interval. *p<0.05 as compared with the rats before exposure to the FST.

This study was supported by grants from the Seoul R&BD Program (10550), the Korea Health 21 R&D Project, the Ministry of Health & Welfare, Republic of Korea (02-PJ3-PG6-EV07-0002) (A081057) and the Purpose Basic Research Grant of the KOSEF (R01-2007-000-20782-0).

References

  • 1 Manji HK, Drevets WC, Charney DS. The cellular neurobiology of depression. Nat Med 2001;7:541–7. Crossref Medline ISIGoogle Scholar

  • 2 Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature 1977;266:730–2. Crossref Medline ISIGoogle Scholar

  • 3 Bourin M, Fiocco AJ, Clenet F. How valuable are animal models in defining antidepressant activity? Hum Psychopharmacol Clin Exp 2001;16:9–21. Crossref Medline ISIGoogle Scholar

  • 4 McEwen BS. Effects of adverse experiences for brain structure and function. Biol Psychiatry 2000;48:721–31. Crossref Medline ISIGoogle Scholar

  • 5 Kim EJ, Kim WR, Chi SE, Lee KH, Park EH, Chae JH, et al. Repetitive transcranial magnetic stimulation protects hippocampal plasticity in an animal model of depression. Neurosci Lett 2006;405:79–83. Crossref Medline ISIGoogle Scholar

  • 6 Przegalinski E, Tatarczynska E, Deren-Wesolek A, Chojnacka-Wojcik E. Antidepressant-like effects of a partial agonist at strychnine-insensitive glycine receptors and a competitive NMDA receptor antagonist. Neuropharmacology 1997;36:31–7. Crossref Medline ISIGoogle Scholar

  • 7 Padovan CM, Guimaraes FS. Antidepressant-like effects of NMDA-receptor antagonist injected into the dorsal hippocampus of rats. Pharmacol Biochem Behav 2004;77:15–9. Crossref Medline ISIGoogle Scholar

  • 8 Ende G, Braus DF, Walter S, Weber-Fahr W, Henn FA. The hippocampus in patients treated with electroconvulsive therapy: a proton magnetic resonance spectroscopic imaging study. Arch Gen Psychiatry 2000;57:937–43. Crossref MedlineGoogle Scholar

  • 9 Sartorius A, Vollmayr B, Neumann-Haefelin C, Ende G, Hoehn M, Henn FA. Specific creatine rise in learned helplessness induced by electroconvulsive shock treatment. NeuroReport 2003;14:2199–201. Crossref Medline ISIGoogle Scholar

  • 10 Hong ST, Choi CB, Park CS, Hong KS, Cheong CJ, Jeon YW, Choe BY. Variation of the choline signal intensity in the dorsolateral prefrontal cortex of rats exposed to the forced swimming test as detected by in vivo 1H MR spectroscopy. J Neurosci Methods 2007;165:89–94. Crossref Medline ISIGoogle Scholar

  • 11 Kumar A, Thomas A, Lavretsky H, Yue K, Huda A, Curran J, et al. Frontal white matter biochemical abnormalities in late-life major depression detected with proton magnetic resonance spectroscopy. Am J Psychiatry 2002;159:630–6. Crossref Medline ISIGoogle Scholar

  • 12 Fuchs E, Kramer M, Hermes B, Netter P, Hiemke C. Psychosocial stress in tree shrews: clomipramine counteracts behavioral and endocrine changes. Pharmacol Biochem Behav 1996;54:219–28. Crossref Medline ISIGoogle Scholar

  • 13 Czeh B, Michaelis T, Watanabe T, Frahm J, de Biurrun G, van Kampen M, et al. Stress-induced changes in cerebral metabolites, hippocampal volume, and cell proliferation are prevented by antidepressant treatment with tianeptine. Proc Natl Acad Sci USA 2001;98:12796–801. Crossref Medline ISIGoogle Scholar

  • 14 Bremner JD, Narayan M, Anderson ER, Staib LH, Miller HL, Charney DS. Hippocampal volume reduction in major depression. Am J Psychiatry 2000;157:115–7. Crossref Medline ISIGoogle Scholar

  • 15 Dwivedi Y, Mondal AC, Rizavi HS, Shukla PK, Pandey GN. Single and repeated stress-induced modulation of phospholipase C catalytic activity and expression: role in LH behavior. Neuropsychopharmacology 2005;30:473–83. Crossref Medline ISIGoogle Scholar

  • 16 Pandey GN, Dwivedi Y, SridharaRao J, Ren X, Janicak PG, Sharma R. Protein kinase C and phospholipase C activity and expression of their specific isozymes is decreased and expression of MARCKS is increased in platelets of bipolar but not in unipolar patients. Neuropsychopharmacology 2002;26:216–28. Crossref Medline ISIGoogle Scholar

  • 17 Abel EL. Ontogeny of immobility and response to alarm substance in the forced swim test. Physiol Behav 1993;54:713–6. Crossref Medline ISIGoogle Scholar

  • 18 Yang CH, Huang CC, Hsu KS. Behavioral stress modifies hippocampal synaptic plasticity through corticosterone-induced sustained extracellular signal-regulated kinase/mitogen-activated protein kinase activation. J Neurosci 2004;24:11029–34. Crossref Medline ISIGoogle Scholar

  • 19 Kim JJ, Lee HJ, Han JS, Packard MG. Amygdala is critical for stress-induced modulation of hippocampal long-term potentiation and learning. J Neurosci 2001;21:5222–8. Crossref Medline ISIGoogle Scholar

  • 20 Watanabe Y, Gould E, McEwen BS. Stress induces atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. Brain Res 1992;588:341–5. Crossref Medline ISIGoogle Scholar

  • 21 Sheline YI. 3D MRI studies of neuroanatomic changes in unipolar major depression: the role of stress and medical comorbidity. Biol Psychiatry 2000;48:791–800. Crossref Medline ISIGoogle Scholar

  • 22 Haydon PG. Glia: listening and talking to the synapse. Nat Rev Neurosci 2001;2:185–93. Crossref Medline ISIGoogle Scholar

  • 23 Brambilla P, Perez J, Barale F, Schettini G, Soares JC. GABAergic dysfunction in mood disorders. Mol Psychiatry 2003;8:721–37. Crossref Medline ISIGoogle Scholar

  • 24 Farchione TR, Moore GJ, Rosenberg DR. Proton magnetic resonance spectroscopic imaging in pediatric major depression. Biol Psychiatry 2002;52:86–92. Crossref Medline ISIGoogle Scholar

  • 25 Brambilla P, Stanley JA, Nicoletti MA, Sassi RB, Mallinger AG, Frank E, et al. 1H magnetic resonance spectroscopy study of dorsolateral prefrontal cortex in unipolar mood disorder patients. Psychiatry Res Neuroimaging 2005;138:131–9. Crossref Medline ISIGoogle Scholar

  • 26 Maeda F, Keenan JP, Pascual-Leone A. Interhemispheric asymmetry of motor cortical excitability in major depression as measured by transcranial magnetic stimulation. Br J Psychiatry 2000;177:169–73. Crossref Medline ISIGoogle Scholar

  • 27 Borsini F, Mancinelli A, D'Aranno V, Evangelista S, Meli A. On the role of endogenous GABA in the forced swimming test in rats. Pharmacol Biochem Behav 1988;29:275–9. Crossref Medline ISIGoogle Scholar

  • 28 Michael N, Erfurth A, Ohrmann P, Arolt V, Heindel W, Pfleiderer B. Metabolic changes within the left dorsolateral prefrontal cortex occurring with electroconvulsive therapy in patients with treatment resistant unipolar depression. Psychol Med 2003;33:1277–84. Crossref Medline ISIGoogle Scholar

  • 29 de Graff RA, Brown PB, McIntyre S, Nixon TW, Behar KL, Rothman DL. High magnetic field water and metabolite proton T1 and T2 relaxation in rat brain in vivo. Mag Reson Med 2006;56:386–94. Crossref Medline ISIGoogle Scholar

Volume 82, Issue 979July 2009
Pages: 529-e147

© The British Institute of Radiology


History

  • RevisedMay 15,2008
  • ReceivedMarch 31,2008
  • AcceptedJune 12,2008
  • Published onlineFebruary 13,2014

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This study was supported by grants from the Seoul R&BD Program (10550), the Korea Health 21 R&D Project, the Ministry of Health & Welfare, Republic of Korea (02-PJ3-PG6-EV07-0002) (A081057) and the Purpose Basic Research Grant of the KOSEF (R01-2007-000-20782-0).