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Increased catecholamine levels in specific brain regions of a rat model of depression: normalization by chronic antidepressant treatment
Brain Research 824 Ž1999. 243–250
Research report
Increased catecholamine levels in specific brain regions of a rat model of depression: normalization by chronic antidepressant treatment Abraham Zangen a , David H. Overstreet b , Gal Yadid
a, )
a
b
Faculty of Life Sciences, Bar Ilan UniÕersity, Ramat-Gan, Israel Department of Psychiatry, UniÕersity of North Carolina, Chapel Hill, NC, USA Accepted 2 February 1999
Abstract Alterations in catecholamine levels and neurotransmission have been shown in depressive disorders. However, the exact sites of alterations and the relation between these alterations to the etiology of the disease and the effectiveness of antidepressant therapy are poorly understood. In this study, catecholamine levels and metabolism were measured in specific brain regions of a genetic rat model of depression wFlinders Sensitive Line ŽFSL. ratsx, and compared to normal Sprague–Dawley rats. Norepinephrine levels were found to be two to threefold higher in the nucleus accumbens, prefrontal cortex, hippocampus and median raphe nucleus of FSL rats as compared with control Sprague–Dawley rats. Dopamine levels were sixfold higher in the nucleus accumbens and twofold higher in the striatum, hippocampus and hypothalamus of FSL rats as compared with control Sprague–Dawley rats. After chronic treatment with the antidepressant desipramine, the immobility score in a swim test, as a measure of a behavioral deficit, as well as catecholamine levels of the FSL rats became normalized, but these parameters in the control rats did not change. The results indicate that the behavioral deficits expressed in the FSL model for depression correlate with increased catecholamine levels in specific brain sites, and further suggest the FSL rats as a model for elucidation of the molecular mechanism of clinically used antidepressant drugs. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Antidepressant; Catecholamines; Dopamine; Norepinephrine; Turnover
1. Introduction Effective treatment of depressive disorder will be advanced by the precise delineation of the neurochemical basis of the disease. Alterations in the catecholaminergic system during depression and upon antidepressant treatment have been reported. However, no study actually reported altered catecholamine levels in brain structures of depressed patients. In the cerebrospinal fluid ŽCSF., dopamine ŽDA. levels were reported to be higher in depressed patients as compared to healthy volunteers w13x. However, levels of the DA metabolite homovanillic acid ŽHVA. in CSF of depressed patients was reported to be decreased in most studies w2,25,26,33x, while plasma HVA levels were normal w13,27x. NE levels in CSF of depressed patients were not significantly different from healthy vol)
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unteers w13,21,25x, but plasma NE levels have been reported to be higher in depressed patients w10,24x. Brains from suicide victims, in which suicidal behavior rather than diagnosis of depression was studied, showed normal levels of NE, DA and HVA w4,6,20x. Chronic treatment of depressed patients with tricyclic antidepressants causes an increase in plasma levels of NE w15,34x, but serotonin reuptake blockers did not affect NE plasma levels w15x. The effect of antidepressants on CSF or brain levels of NE and DA in depressed patients, have not been reported yet, but CSF HVA levels were not significantly affected in patients chronically treated with antidepressants w7x. Studies with rats showed that chronic treatment with certain but not all antidepressants decreased NE levels in some brain regions, while DA levels were not affected in any brain region w8,28,30x. The genetically-selected Flinders Sensitive Line ŽFSL. rats exhibit behavioral features characteristic of depression, such as reduced locomotion, increased anhedonia in re-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 2 1 4 - 7
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A. Zangen et al.r Brain Research 824 (1999) 243–250
sponse to chronic mild stress, increased amount of rapid eye movement ŽREM. sleep, reduction in REM sleep onset, cognitive difficulties and reduced body weight Žfor review see Ref. w17x.. Chronic, but not acute, treatment of the FSL rats with antidepressants, such as desipramine, imipramine, sertraline and fluoxetine, diminishes, and in the case of desipramine, almost abrogates their behavioral manifestations of depression Žfor review see Refs. w17,18x.. Thus, the FSL rats appear to be an appropriate experimental model for studying the neurochemical mechanisms involved in depressive disorders and the mode of action of antidepressants. In our previous study we demonstrated high serotonin levels in limbic regions of FSL rats w37x. Chronic treatment with desipramine, a selective NE reuptake inhibitor, normalized their limbic 5-HT levels suggesting a possible role for NE on the 5-HT neurons. Therefore, in this study, the raphe nucleus, site of the 5-HT cell bodies, and other brain regions, where abnormal serotonin levels were previously observed, were assessed for catecholamine content. Desipramine was used to assess whether its effect on the behavioral manifestations of depression would also correlate with an ability to affect catecholamine levels in specific brain regions of FSL rats.
2. Materials and methods 2.1. Animals Male FSL, selectively bred from Sprague–Dawley rats w17x and control ŽSprague–Dawley. rats Ž230–260 g. were housed two per cage under conditions of constant temperature Ž228C. and humidity Ž50%., with a 12:12 h light:dark cycle. Food and water were provided ad libitum. All animal procedures were approved by the Bar-Ilan University Animal Care Committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Some rats were injected intraperitoneally with desipramine Ž5 mgrkg in 0.5 ml saline; Sigma. or saline once a day for 18 days. 2.2. Swim test A modified forced swim test protocol was conducted in a cylindrical tank Ž40 cm high and 18 cm in diameter; constructed at Bar-Ilan University., which contained enough water at 258C so that the rat could not touch the bottom with its hind paws. A rat was considered to have stopped swimming when both front paws were immobile. The animals were given a single 5-min exposure to the
Table 1 Immobility during the swim test of FSL and control rats untreated or chronically treated with desipramine or saline
Control FSL
Untreated
Saline
Desipramine
91"29 165"48 a
113"36 194"52 a
86"22 83"20 b
Mean"S.D. values of total immobility Žexpressed in seconds. are depicted from 10 rats in each group. a p- 0.01 FSL versus control. b p- 0.01 saline versus desipramine.
swim tank 24 h after the last treatment of desipramine or saline because previous studies w18,23x demonstrated that this protocol was effective in detecting the anti-immobility effects of classical antidepressants and in rejecting psychomotor stimulants such as amphetamine, which typically give false positive results in the standard forced swim test w5x. The swim tests and biochemical measurements were performed on different groups of animals in order to avoid the effects of stress on the biochemical measurements. 2.3. Brain dissection and extraction Rats were decapitated 24 h after the last injection and their brains were rapidly removed. The hypothalamus, not including the median eminencerpituitary stalk, was surgically dissected out using forceps and immediately frozen in liquid nitrogen. The brains were then placed in a rat brain mold Žconstructed at Bar-Ilan University. on ice and serial 0.5 mm sections were cut and placed on chilled microscope slides. Tissue punches of the nucleus accumbens, striatum, prefrontal cortex, hippocampus, dorsal raphe and median raphe Žwhich included the paramedian raphe. nucleus were rapidly taken using a stainless steel cannulas with an inner diameter of 1.1 mm, as described previously w19,37x, and included most of the desired regions. The tissue samples were immediately frozen in liquid nitrogen and stored at y708C until extraction. Extraction was achieved by thawing the punches and subjecting them to probe sonication Ž80 W for 5 s with a Sonifier B-12; Branson, Danbury, CN. in 0.5 ml of a perchlorate solution Ž0.1 M. containing EDTArethanol Ž0.02r1%. on ice. A sample Ž100 ml. was removed for protein analysis and the rest was subjected to centrifugation Ž2000 = g, 10 min, 48C.. The resulting supernatants Žthe tissue extracts. were filtered Ž0.45 mm Acrodisk, Gelman; Ann Arbor, MI. and stored at y708C until subjected to HPLC analysis to determine the catecholamine content. The HPLC analysis was performed within 2 months.
Fig. 1. Catecholamine levels in brain regions of FSL and control rats. Mean " S.D. depicted from 10 rats in each group. Values have been multiplied or divided by 10 as indicated in the graphs. Significance between groups was tested by independent two-tailed t-test. ) p - 0.05 vs. the corresponding control group. Note: the hypothalamus tissue sample did not include the median eminencerpituitary stalk and the median raphe tissue sample included the paramedian raphe.
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Table 2 Indexes of dopamine turnover in regions of the brains of FSL and control rats untreated or chronically treated with desipramine or saline Rat strain
Control
Treatment
–
Saline
Desipramine
–
Saline
Desipramine
n
10
6
6
10
6
6
Brain region Nucleus accumbens Prefrontal cortex Hippocampus Hypothalamus Striatum Median raphe nucleus Dorsal raphe nucleus
wDOPACxrwDAx ratio Žmean " S.D.. 0.84 " 0.19 0.80 " 0.16 4.26 " 1.17 4.58 " 1.62 1.48 " 0.27 1.18 " 0.13 0.46 " 0.17 0.36 " 0.08 0.98 " 0.21 0.95 " 0.12 2.25 " 0.47 3.09 " 0.75 4.06 " 0.95 4.51 " 1.29
0.81 " 0.17 5.50 " 1.34 1.44 " 0.40 0.36 " 0.07 1.01 " 0.19 2.36 " 0.73 3.64 " 0.83
0.76 " 0.13 7.34 " 2.03 a 1.29 " 0.21 0.39 " 0.11 0.52 " 0.07 a 1.89 " 0.59 3.82 " 0.55
0.64 " 0.13 7.63 " 2.31a 1.09 " 0.15 0.33 " 0.12 0.64 " 0.07 a 2.65 " 0.44 4.74 " 1.42
0.72 " 0.15 4.29 " 1.16 b 1.50 " 0.32 0.30 " 0.06 0.72 " 0.05 2.07 " 0.34 4.17 " 1.50
a b
FSL
p - 0.05 FSL versus control. p - 0.05 saline versus desipramine.
2.4. Analysis of the catecholamine and their metabolites content of the tissue punches
3. Results 3.1. BehaÕioral swim test for immobility
Quantitation of the NE, DA, L-DOPA, HVA and DOPAC content of the tissue punch extracts was performed as described previously w36x. Briefly, the filtered supernatants of each tissue extract were injected directly via a HPLC pump ŽModel 510, Waters, Milford, MA. onto a column ŽMerck C-18, 5 mm; 4.6 mm i.d.= 250 mm, at 308C. coupled to an electrochemical detector ŽModel 460 Waters. with an EiCOM CB-100 analytical cell ŽEiCOM; Kyoto, Japan. and the oxidation potential set to 0.77 V. The mobile phase Ž2 l of water containing 0.55 g heptane sulfonic acid, 0.2 g EDTA, 16 ml triethylamine, 12 ml 85% phosphoric acid, and 80 ml acetonitrile; pH s 2.6. was pumped at 0.8 mlrmin. All HPLC reagents were obtained from Fisher Scientific ŽFair Lawn, NJ.. The limit of detection for NE, DA, L-DOPA, HVA and DOPAC were about 10–20 fmol per sample. The catecholamine and metabolite concentrations were expressed in relation to the protein content of the samples, which were quantified with Bio-Rad Protein Assay Kits. 2.5. Statistical analysis Data were expressed as mean " S.D. values obtained from the indicated number of rats. Significance between groups was tested by independent two-tailed t-test or ANOVA with post-hoc Student Newman Keuls. A probability value of - 0.05 was considered significant.
During the swim test, the FSL rats were immobile 82% longer than the control Sprague–Dawley rats Žaverage immobility times of 165 " 48 versus 91 " 29 s, respectively, n s 10 per group, p - 0.01.. Chronic treatment with desipramine Ž5 mgrkg, once daily for 18 days. reduced the immobility of the FSL rats to values close to those of the control rats, but did not significantly affect the immobility of the control rats ŽTable 1.. 3.2. Catecholamine content of Õarious brain regions DA levels were sixfold higher in the nucleus accumbens and twofold higher in the striatum, hippocampus and hypothalamus of FSL rats than in the corresponding brain regions of the control Sprague–Dawley rats ŽFig. 1A; p - 0.01, n s 10 per group.. NE levels were two to threefold higher in the nucleus accumbens, prefrontal cortex, hippocampus and median raphe nucleus of FSL rats than in the corresponding brain regions of the control rats ŽFig. 1B; p - 0.01, n s 10 per group.. The dopamine precursor, L-DOPA, was threefold higher in the nucleus accumbens of FSL rats, but not different from the control rats in the other tested regions ŽFig. 1C; p - 0.01, n s 10 per group.. DOPAC and HVA levels were fivefold higher in the nucleus accumbens and twofold higher in the striatum and hypothalamus of FSL rats than in the corresponding brain
Fig. 2. ŽA–G.: Effect of chronic desipramine treatment on the catecholamine levels in brain regions of FSL and control rats. Rats were pretreated with desipramine or saline for 18 days. ŽI. control saline; Žopen square with diagonal lines. control desipramine; ŽB. FSL saline; Žopen square with horizontal lines. FSL desipramine. Mean " S.D. depicted from 6 rats in each group. Significantly different values were detected with ANOVA followed by post-hoc Student Newman Keuls. ) p - 0.05, FSL saline vs. FSL-desipramine or control groups. Note: Ž1. In the striatum ŽE., DOPAC and HVA levels in FSL-desipramine are not significantly different from FSL-saline. Ž2. The hypothalamus tissue sample did not include the median eminencerpituitary stalk and the median raphe tissue sample included the paramedian raphe.
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regions of the control rats ŽFig. 1D; p - 0.01, n s 10 per group.. DA turnover ŽwDOPACxrwDAx. was 47% lower in the striatum and 72% higher in the prefrontal cortex of the FSL rats as compared with the control rats. Significant differences in the DA turnover in the other regions of the brain was not observed ŽTable 2.. 3.3. Effect of desipramine on catecholamine content and turnoÕer Chronic treatment with desipramine reduced the catecholamine levels in specific brain regions of FSL rats close to those of the control Sprague–Dawley rats ŽFig. 2A–G.. However, this treatment only slightly decreased the abnormal DA levels in the striatum of the FSL rats. ŽFig. 2E.. Also, the chronic desipramine treatment mostly did not affect the catecholamine levels in the brain regions of FSL rats, where normal levels were observed without desipramine treatment ŽFig. 2A–G.. In all the tested brain regions of the control Sprague–Dawley rats, the catecholamine levels were not significantly affected by chronic desipramine treatment ŽFig. 2A–G.. Injections of saline did not significantly affect the catecholamine levels in the brains of control and FSL rats. Chronic treatment with desipramine normalized the increased DA turnover in the prefrontal cortex of the FSL rats, but did not significantly affect the decreased DA turnover in their striatum. The DA turnover was not affected in the other tested regions of the FSL rats, nor in any tested region of the control rats ŽTable 2..
4. Discussion The present study indicates that increased catecholamine levels in specific brain regions might reflect depressive disorder. In FSL rats, chronic treatment with desipramine, which improves their behavioral deficits, also normalizes their altered catecholamine levels. The most striking differences between FSL and control rats were observed in the DA levels of the nucleus accumbens. This region is an important limbic site, where the release of DA is associated with motivation and reward w16,29,31,35x. The increased tissue content of catecholamines measured in some brain regions of FSL rats might reflect either increased synthesis or decreased elimination of catecholamines and metabolites. In FSL rats, it is unlikely that a reduction in the elimination of DA could solely be due to a decreased activity of monoamine oxidase, since the HVA and DOPAC levels are also increased in these rats Žand the wDOPACxrwDAx ratios are not lower except for in the striatum.. However, a decreased elimination could be caused by a decreased release of catecholamines and
metabolites from nerve terminals, since elimination from brain tissue requires exposure of the compounds to the extracellular space. Also, a decreased DA or NE release from nerve terminals may cause increased synthesis via a compensatory feedback mechanism w11,12x. A decreased release of DA in the nucleus accumbens of FSL rats may explain their behavioral deficit regarding motivation and reward. The lower DA turnover ŽwDOPACxrwDAx ratios. in the striatum of FSL rats can also be explained by a decreased release of DA, since DOPAC and HVA are generated mainly from DA that upon release was taken up by the cells and metabolized intracellularly w9x. Previously we reported increased tyrosine hydroxylase mRNA levels in the ventral tegmental area Žthe main location of dopaminergic cells that project to the limbic regions. of FSL rats as compared with control Sprague–Dawley rats. In the substantia nigra tyrosine hydroxylase mRNA levels were similar in both strains w32x. These previous observations correlate with the present data that show increased levels of L-DOPA and DA in the limbic regions and suggest an increased synthesis of catecholamines in brain regions innervated by the ventral tegmental area, since tyrosine hydroxylase is the rate limiting enzyme for catecholamine biosynthesis. However, in regions innervated mainly by dopaminergic cells located in the substantia nigra, i.e., the striatum, an increased catecholamine synthesis would not be expected in FSL rats. Indeed, in the striatum, the L-DOPA levels are not increased in FSL rats ŽFig. 1C.. Also in the nucleus accumbens, the L-DOPA levels were increased only by 267% in FSL rats while DA levels increased by 581% ŽFig. 1A,C.. Therefore, it is suggested that not only an increased synthesis, but also a decreased release may cause the net increase in DA levels in this specific brain region of FSL rats. In the raphe nucleus, DA levels were normal in the FSL rats, however, NE levels were increased in the median but not in the dorsal raphe of the FSL rats, as compared to the control rats ŽFig. 1B.. An inhibitory effect of NE on 5-HT neurons in the raphe nucleus was previously shown w3x. Since the 5-HT cell bodies in the median raphe innervates mainly limbic areas and those in the dorsal raphe innervates mainly non-limbic areas w14x, it is suggested that the increased tissue levels of NE observed in the median raphe of FSL rats could be involved in the abnormal 5-HT levels specifically observed in their limbic regions w37x. A key behavioral abnormality exhibited by FSL rats is an exaggerated immobility during exposure to stressors like forced swimming. In previous studies, the immobility of FSL rats in a 5-min swim test paradigm was 2–3 times higher than that of ‘normal’ rats w18,23,37x. This behavioral abnormality was normalized by chronic, but not acute, antidepressant treatment, with desipramine being the most effective antidepressant w18,23x. Moreover, we reported that desipramine also affected the serotonergic neurochemistry in FSL rats w37x. In this study, chronic treatment with desipramine, which improved the behav-
A. Zangen et al.r Brain Research 824 (1999) 243–250
ioral deficit of FSL rats in the swim test paradigm ŽTable 1. also normalized the catecholamine levels in most brain regions of the FSL rats, except the striatum which is the only non-limbic related region, of all tested regions ŽFig. 2.. Thus, the increased catecholamine levels in limbic regions of FSL rats appear to correspond to the observed behavioral deficit. Chronic treatment with desipramine affected the levels of catecholamines only in the brain regions of FSL rats where abnormal catecholamine levels were observed and did not alter catecholamine levels in any region of the brains of the control Sprague–Dawley rats. These findings indicate a site-specific action of the drug and the importance of using depressed rather than normal subjects for studying the mode of action of antidepressants. Since desipramine is mainly a NE reuptake inhibitor, its effect on NE levels may be explained by a feedback mechanism i.e., decreased synthesis caused by the increased extracellular levels affecting NE autoreceptors. However, its effect on DA levels in some brain regions, indicates a possible interaction between noradrenergic and dopaminergic terminals in those regions. In postmortem samples taken from depressed suicides, in which suicidal behavior rather than diagnosis was studied, normal levels of NE, DA and HVA were observed w4,6,20x. However suicide victims suffering from depression represent only a specific subtype of depressive disorder Ž15% of all depressed patients., that usually has an aggressive component w1,22x. Previous studies with rats showed that chronic treatment with desipramine Žand other antidepressants. did not affect DA levels in various brain regions w8,28,30x. These studies are consistent with our observations with the control rats. Clinically, antidepressant treatment does not affect mood and behavior of non-depressives, therefore, the interpretations of results obtained from normal rats has limited relevance to the neurochemical abnormalities observed in depression and in the therapeutic effect of antidepressant drugs. On the other hand, the parallel normalization of both behavioral and neurochemical abnormalities in FSL rats after chronic treatment with desipramine indicates the validity of the model for studying the mode of action of antidepressants and suggests that decreasing catecholamine levels in specific regions is important for the therapeutic effect of antidepressants. Such a decrease in catecholamine levels could be due to a direct decrease in catecholamine synthesis, an increase in catecholamine release, or an increase in the post synaptic responses to DA and NE in specific regions, especially the nucleus accumbens. Since we have already reported an affect of desipramine on abnormal 5-HT levels in FSL rats, we suggest that more than one monoaminergic system and perhaps more than one brain region is involved with the etiology of depressive disorders. Further studies are needed to elucidate the specific interactions between these monoaminergic systems.
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Acknowledgements This research was performed as part of A. Zangen’s PhD thesis and supported by Bar-Ilan Research Foundation Žno. 2520..
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Research report
Increased catecholamine levels in specific brain regions of a rat model of depression: normalization by chronic antidepressant treatment Abraham Zangen a , David H. Overstreet b , Gal Yadid
a, )
a
b
Faculty of Life Sciences, Bar Ilan UniÕersity, Ramat-Gan, Israel Department of Psychiatry, UniÕersity of North Carolina, Chapel Hill, NC, USA Accepted 2 February 1999
Abstract Alterations in catecholamine levels and neurotransmission have been shown in depressive disorders. However, the exact sites of alterations and the relation between these alterations to the etiology of the disease and the effectiveness of antidepressant therapy are poorly understood. In this study, catecholamine levels and metabolism were measured in specific brain regions of a genetic rat model of depression wFlinders Sensitive Line ŽFSL. ratsx, and compared to normal Sprague–Dawley rats. Norepinephrine levels were found to be two to threefold higher in the nucleus accumbens, prefrontal cortex, hippocampus and median raphe nucleus of FSL rats as compared with control Sprague–Dawley rats. Dopamine levels were sixfold higher in the nucleus accumbens and twofold higher in the striatum, hippocampus and hypothalamus of FSL rats as compared with control Sprague–Dawley rats. After chronic treatment with the antidepressant desipramine, the immobility score in a swim test, as a measure of a behavioral deficit, as well as catecholamine levels of the FSL rats became normalized, but these parameters in the control rats did not change. The results indicate that the behavioral deficits expressed in the FSL model for depression correlate with increased catecholamine levels in specific brain sites, and further suggest the FSL rats as a model for elucidation of the molecular mechanism of clinically used antidepressant drugs. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Antidepressant; Catecholamines; Dopamine; Norepinephrine; Turnover
1. Introduction Effective treatment of depressive disorder will be advanced by the precise delineation of the neurochemical basis of the disease. Alterations in the catecholaminergic system during depression and upon antidepressant treatment have been reported. However, no study actually reported altered catecholamine levels in brain structures of depressed patients. In the cerebrospinal fluid ŽCSF., dopamine ŽDA. levels were reported to be higher in depressed patients as compared to healthy volunteers w13x. However, levels of the DA metabolite homovanillic acid ŽHVA. in CSF of depressed patients was reported to be decreased in most studies w2,25,26,33x, while plasma HVA levels were normal w13,27x. NE levels in CSF of depressed patients were not significantly different from healthy vol)
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unteers w13,21,25x, but plasma NE levels have been reported to be higher in depressed patients w10,24x. Brains from suicide victims, in which suicidal behavior rather than diagnosis of depression was studied, showed normal levels of NE, DA and HVA w4,6,20x. Chronic treatment of depressed patients with tricyclic antidepressants causes an increase in plasma levels of NE w15,34x, but serotonin reuptake blockers did not affect NE plasma levels w15x. The effect of antidepressants on CSF or brain levels of NE and DA in depressed patients, have not been reported yet, but CSF HVA levels were not significantly affected in patients chronically treated with antidepressants w7x. Studies with rats showed that chronic treatment with certain but not all antidepressants decreased NE levels in some brain regions, while DA levels were not affected in any brain region w8,28,30x. The genetically-selected Flinders Sensitive Line ŽFSL. rats exhibit behavioral features characteristic of depression, such as reduced locomotion, increased anhedonia in re-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 2 1 4 - 7
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sponse to chronic mild stress, increased amount of rapid eye movement ŽREM. sleep, reduction in REM sleep onset, cognitive difficulties and reduced body weight Žfor review see Ref. w17x.. Chronic, but not acute, treatment of the FSL rats with antidepressants, such as desipramine, imipramine, sertraline and fluoxetine, diminishes, and in the case of desipramine, almost abrogates their behavioral manifestations of depression Žfor review see Refs. w17,18x.. Thus, the FSL rats appear to be an appropriate experimental model for studying the neurochemical mechanisms involved in depressive disorders and the mode of action of antidepressants. In our previous study we demonstrated high serotonin levels in limbic regions of FSL rats w37x. Chronic treatment with desipramine, a selective NE reuptake inhibitor, normalized their limbic 5-HT levels suggesting a possible role for NE on the 5-HT neurons. Therefore, in this study, the raphe nucleus, site of the 5-HT cell bodies, and other brain regions, where abnormal serotonin levels were previously observed, were assessed for catecholamine content. Desipramine was used to assess whether its effect on the behavioral manifestations of depression would also correlate with an ability to affect catecholamine levels in specific brain regions of FSL rats.
2. Materials and methods 2.1. Animals Male FSL, selectively bred from Sprague–Dawley rats w17x and control ŽSprague–Dawley. rats Ž230–260 g. were housed two per cage under conditions of constant temperature Ž228C. and humidity Ž50%., with a 12:12 h light:dark cycle. Food and water were provided ad libitum. All animal procedures were approved by the Bar-Ilan University Animal Care Committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Some rats were injected intraperitoneally with desipramine Ž5 mgrkg in 0.5 ml saline; Sigma. or saline once a day for 18 days. 2.2. Swim test A modified forced swim test protocol was conducted in a cylindrical tank Ž40 cm high and 18 cm in diameter; constructed at Bar-Ilan University., which contained enough water at 258C so that the rat could not touch the bottom with its hind paws. A rat was considered to have stopped swimming when both front paws were immobile. The animals were given a single 5-min exposure to the
Table 1 Immobility during the swim test of FSL and control rats untreated or chronically treated with desipramine or saline
Control FSL
Untreated
Saline
Desipramine
91"29 165"48 a
113"36 194"52 a
86"22 83"20 b
Mean"S.D. values of total immobility Žexpressed in seconds. are depicted from 10 rats in each group. a p- 0.01 FSL versus control. b p- 0.01 saline versus desipramine.
swim tank 24 h after the last treatment of desipramine or saline because previous studies w18,23x demonstrated that this protocol was effective in detecting the anti-immobility effects of classical antidepressants and in rejecting psychomotor stimulants such as amphetamine, which typically give false positive results in the standard forced swim test w5x. The swim tests and biochemical measurements were performed on different groups of animals in order to avoid the effects of stress on the biochemical measurements. 2.3. Brain dissection and extraction Rats were decapitated 24 h after the last injection and their brains were rapidly removed. The hypothalamus, not including the median eminencerpituitary stalk, was surgically dissected out using forceps and immediately frozen in liquid nitrogen. The brains were then placed in a rat brain mold Žconstructed at Bar-Ilan University. on ice and serial 0.5 mm sections were cut and placed on chilled microscope slides. Tissue punches of the nucleus accumbens, striatum, prefrontal cortex, hippocampus, dorsal raphe and median raphe Žwhich included the paramedian raphe. nucleus were rapidly taken using a stainless steel cannulas with an inner diameter of 1.1 mm, as described previously w19,37x, and included most of the desired regions. The tissue samples were immediately frozen in liquid nitrogen and stored at y708C until extraction. Extraction was achieved by thawing the punches and subjecting them to probe sonication Ž80 W for 5 s with a Sonifier B-12; Branson, Danbury, CN. in 0.5 ml of a perchlorate solution Ž0.1 M. containing EDTArethanol Ž0.02r1%. on ice. A sample Ž100 ml. was removed for protein analysis and the rest was subjected to centrifugation Ž2000 = g, 10 min, 48C.. The resulting supernatants Žthe tissue extracts. were filtered Ž0.45 mm Acrodisk, Gelman; Ann Arbor, MI. and stored at y708C until subjected to HPLC analysis to determine the catecholamine content. The HPLC analysis was performed within 2 months.
Fig. 1. Catecholamine levels in brain regions of FSL and control rats. Mean " S.D. depicted from 10 rats in each group. Values have been multiplied or divided by 10 as indicated in the graphs. Significance between groups was tested by independent two-tailed t-test. ) p - 0.05 vs. the corresponding control group. Note: the hypothalamus tissue sample did not include the median eminencerpituitary stalk and the median raphe tissue sample included the paramedian raphe.
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Table 2 Indexes of dopamine turnover in regions of the brains of FSL and control rats untreated or chronically treated with desipramine or saline Rat strain
Control
Treatment
–
Saline
Desipramine
–
Saline
Desipramine
n
10
6
6
10
6
6
Brain region Nucleus accumbens Prefrontal cortex Hippocampus Hypothalamus Striatum Median raphe nucleus Dorsal raphe nucleus
wDOPACxrwDAx ratio Žmean " S.D.. 0.84 " 0.19 0.80 " 0.16 4.26 " 1.17 4.58 " 1.62 1.48 " 0.27 1.18 " 0.13 0.46 " 0.17 0.36 " 0.08 0.98 " 0.21 0.95 " 0.12 2.25 " 0.47 3.09 " 0.75 4.06 " 0.95 4.51 " 1.29
0.81 " 0.17 5.50 " 1.34 1.44 " 0.40 0.36 " 0.07 1.01 " 0.19 2.36 " 0.73 3.64 " 0.83
0.76 " 0.13 7.34 " 2.03 a 1.29 " 0.21 0.39 " 0.11 0.52 " 0.07 a 1.89 " 0.59 3.82 " 0.55
0.64 " 0.13 7.63 " 2.31a 1.09 " 0.15 0.33 " 0.12 0.64 " 0.07 a 2.65 " 0.44 4.74 " 1.42
0.72 " 0.15 4.29 " 1.16 b 1.50 " 0.32 0.30 " 0.06 0.72 " 0.05 2.07 " 0.34 4.17 " 1.50
a b
FSL
p - 0.05 FSL versus control. p - 0.05 saline versus desipramine.
2.4. Analysis of the catecholamine and their metabolites content of the tissue punches
3. Results 3.1. BehaÕioral swim test for immobility
Quantitation of the NE, DA, L-DOPA, HVA and DOPAC content of the tissue punch extracts was performed as described previously w36x. Briefly, the filtered supernatants of each tissue extract were injected directly via a HPLC pump ŽModel 510, Waters, Milford, MA. onto a column ŽMerck C-18, 5 mm; 4.6 mm i.d.= 250 mm, at 308C. coupled to an electrochemical detector ŽModel 460 Waters. with an EiCOM CB-100 analytical cell ŽEiCOM; Kyoto, Japan. and the oxidation potential set to 0.77 V. The mobile phase Ž2 l of water containing 0.55 g heptane sulfonic acid, 0.2 g EDTA, 16 ml triethylamine, 12 ml 85% phosphoric acid, and 80 ml acetonitrile; pH s 2.6. was pumped at 0.8 mlrmin. All HPLC reagents were obtained from Fisher Scientific ŽFair Lawn, NJ.. The limit of detection for NE, DA, L-DOPA, HVA and DOPAC were about 10–20 fmol per sample. The catecholamine and metabolite concentrations were expressed in relation to the protein content of the samples, which were quantified with Bio-Rad Protein Assay Kits. 2.5. Statistical analysis Data were expressed as mean " S.D. values obtained from the indicated number of rats. Significance between groups was tested by independent two-tailed t-test or ANOVA with post-hoc Student Newman Keuls. A probability value of - 0.05 was considered significant.
During the swim test, the FSL rats were immobile 82% longer than the control Sprague–Dawley rats Žaverage immobility times of 165 " 48 versus 91 " 29 s, respectively, n s 10 per group, p - 0.01.. Chronic treatment with desipramine Ž5 mgrkg, once daily for 18 days. reduced the immobility of the FSL rats to values close to those of the control rats, but did not significantly affect the immobility of the control rats ŽTable 1.. 3.2. Catecholamine content of Õarious brain regions DA levels were sixfold higher in the nucleus accumbens and twofold higher in the striatum, hippocampus and hypothalamus of FSL rats than in the corresponding brain regions of the control Sprague–Dawley rats ŽFig. 1A; p - 0.01, n s 10 per group.. NE levels were two to threefold higher in the nucleus accumbens, prefrontal cortex, hippocampus and median raphe nucleus of FSL rats than in the corresponding brain regions of the control rats ŽFig. 1B; p - 0.01, n s 10 per group.. The dopamine precursor, L-DOPA, was threefold higher in the nucleus accumbens of FSL rats, but not different from the control rats in the other tested regions ŽFig. 1C; p - 0.01, n s 10 per group.. DOPAC and HVA levels were fivefold higher in the nucleus accumbens and twofold higher in the striatum and hypothalamus of FSL rats than in the corresponding brain
Fig. 2. ŽA–G.: Effect of chronic desipramine treatment on the catecholamine levels in brain regions of FSL and control rats. Rats were pretreated with desipramine or saline for 18 days. ŽI. control saline; Žopen square with diagonal lines. control desipramine; ŽB. FSL saline; Žopen square with horizontal lines. FSL desipramine. Mean " S.D. depicted from 6 rats in each group. Significantly different values were detected with ANOVA followed by post-hoc Student Newman Keuls. ) p - 0.05, FSL saline vs. FSL-desipramine or control groups. Note: Ž1. In the striatum ŽE., DOPAC and HVA levels in FSL-desipramine are not significantly different from FSL-saline. Ž2. The hypothalamus tissue sample did not include the median eminencerpituitary stalk and the median raphe tissue sample included the paramedian raphe.
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regions of the control rats ŽFig. 1D; p - 0.01, n s 10 per group.. DA turnover ŽwDOPACxrwDAx. was 47% lower in the striatum and 72% higher in the prefrontal cortex of the FSL rats as compared with the control rats. Significant differences in the DA turnover in the other regions of the brain was not observed ŽTable 2.. 3.3. Effect of desipramine on catecholamine content and turnoÕer Chronic treatment with desipramine reduced the catecholamine levels in specific brain regions of FSL rats close to those of the control Sprague–Dawley rats ŽFig. 2A–G.. However, this treatment only slightly decreased the abnormal DA levels in the striatum of the FSL rats. ŽFig. 2E.. Also, the chronic desipramine treatment mostly did not affect the catecholamine levels in the brain regions of FSL rats, where normal levels were observed without desipramine treatment ŽFig. 2A–G.. In all the tested brain regions of the control Sprague–Dawley rats, the catecholamine levels were not significantly affected by chronic desipramine treatment ŽFig. 2A–G.. Injections of saline did not significantly affect the catecholamine levels in the brains of control and FSL rats. Chronic treatment with desipramine normalized the increased DA turnover in the prefrontal cortex of the FSL rats, but did not significantly affect the decreased DA turnover in their striatum. The DA turnover was not affected in the other tested regions of the FSL rats, nor in any tested region of the control rats ŽTable 2..
4. Discussion The present study indicates that increased catecholamine levels in specific brain regions might reflect depressive disorder. In FSL rats, chronic treatment with desipramine, which improves their behavioral deficits, also normalizes their altered catecholamine levels. The most striking differences between FSL and control rats were observed in the DA levels of the nucleus accumbens. This region is an important limbic site, where the release of DA is associated with motivation and reward w16,29,31,35x. The increased tissue content of catecholamines measured in some brain regions of FSL rats might reflect either increased synthesis or decreased elimination of catecholamines and metabolites. In FSL rats, it is unlikely that a reduction in the elimination of DA could solely be due to a decreased activity of monoamine oxidase, since the HVA and DOPAC levels are also increased in these rats Žand the wDOPACxrwDAx ratios are not lower except for in the striatum.. However, a decreased elimination could be caused by a decreased release of catecholamines and
metabolites from nerve terminals, since elimination from brain tissue requires exposure of the compounds to the extracellular space. Also, a decreased DA or NE release from nerve terminals may cause increased synthesis via a compensatory feedback mechanism w11,12x. A decreased release of DA in the nucleus accumbens of FSL rats may explain their behavioral deficit regarding motivation and reward. The lower DA turnover ŽwDOPACxrwDAx ratios. in the striatum of FSL rats can also be explained by a decreased release of DA, since DOPAC and HVA are generated mainly from DA that upon release was taken up by the cells and metabolized intracellularly w9x. Previously we reported increased tyrosine hydroxylase mRNA levels in the ventral tegmental area Žthe main location of dopaminergic cells that project to the limbic regions. of FSL rats as compared with control Sprague–Dawley rats. In the substantia nigra tyrosine hydroxylase mRNA levels were similar in both strains w32x. These previous observations correlate with the present data that show increased levels of L-DOPA and DA in the limbic regions and suggest an increased synthesis of catecholamines in brain regions innervated by the ventral tegmental area, since tyrosine hydroxylase is the rate limiting enzyme for catecholamine biosynthesis. However, in regions innervated mainly by dopaminergic cells located in the substantia nigra, i.e., the striatum, an increased catecholamine synthesis would not be expected in FSL rats. Indeed, in the striatum, the L-DOPA levels are not increased in FSL rats ŽFig. 1C.. Also in the nucleus accumbens, the L-DOPA levels were increased only by 267% in FSL rats while DA levels increased by 581% ŽFig. 1A,C.. Therefore, it is suggested that not only an increased synthesis, but also a decreased release may cause the net increase in DA levels in this specific brain region of FSL rats. In the raphe nucleus, DA levels were normal in the FSL rats, however, NE levels were increased in the median but not in the dorsal raphe of the FSL rats, as compared to the control rats ŽFig. 1B.. An inhibitory effect of NE on 5-HT neurons in the raphe nucleus was previously shown w3x. Since the 5-HT cell bodies in the median raphe innervates mainly limbic areas and those in the dorsal raphe innervates mainly non-limbic areas w14x, it is suggested that the increased tissue levels of NE observed in the median raphe of FSL rats could be involved in the abnormal 5-HT levels specifically observed in their limbic regions w37x. A key behavioral abnormality exhibited by FSL rats is an exaggerated immobility during exposure to stressors like forced swimming. In previous studies, the immobility of FSL rats in a 5-min swim test paradigm was 2–3 times higher than that of ‘normal’ rats w18,23,37x. This behavioral abnormality was normalized by chronic, but not acute, antidepressant treatment, with desipramine being the most effective antidepressant w18,23x. Moreover, we reported that desipramine also affected the serotonergic neurochemistry in FSL rats w37x. In this study, chronic treatment with desipramine, which improved the behav-
A. Zangen et al.r Brain Research 824 (1999) 243–250
ioral deficit of FSL rats in the swim test paradigm ŽTable 1. also normalized the catecholamine levels in most brain regions of the FSL rats, except the striatum which is the only non-limbic related region, of all tested regions ŽFig. 2.. Thus, the increased catecholamine levels in limbic regions of FSL rats appear to correspond to the observed behavioral deficit. Chronic treatment with desipramine affected the levels of catecholamines only in the brain regions of FSL rats where abnormal catecholamine levels were observed and did not alter catecholamine levels in any region of the brains of the control Sprague–Dawley rats. These findings indicate a site-specific action of the drug and the importance of using depressed rather than normal subjects for studying the mode of action of antidepressants. Since desipramine is mainly a NE reuptake inhibitor, its effect on NE levels may be explained by a feedback mechanism i.e., decreased synthesis caused by the increased extracellular levels affecting NE autoreceptors. However, its effect on DA levels in some brain regions, indicates a possible interaction between noradrenergic and dopaminergic terminals in those regions. In postmortem samples taken from depressed suicides, in which suicidal behavior rather than diagnosis was studied, normal levels of NE, DA and HVA were observed w4,6,20x. However suicide victims suffering from depression represent only a specific subtype of depressive disorder Ž15% of all depressed patients., that usually has an aggressive component w1,22x. Previous studies with rats showed that chronic treatment with desipramine Žand other antidepressants. did not affect DA levels in various brain regions w8,28,30x. These studies are consistent with our observations with the control rats. Clinically, antidepressant treatment does not affect mood and behavior of non-depressives, therefore, the interpretations of results obtained from normal rats has limited relevance to the neurochemical abnormalities observed in depression and in the therapeutic effect of antidepressant drugs. On the other hand, the parallel normalization of both behavioral and neurochemical abnormalities in FSL rats after chronic treatment with desipramine indicates the validity of the model for studying the mode of action of antidepressants and suggests that decreasing catecholamine levels in specific regions is important for the therapeutic effect of antidepressants. Such a decrease in catecholamine levels could be due to a direct decrease in catecholamine synthesis, an increase in catecholamine release, or an increase in the post synaptic responses to DA and NE in specific regions, especially the nucleus accumbens. Since we have already reported an affect of desipramine on abnormal 5-HT levels in FSL rats, we suggest that more than one monoaminergic system and perhaps more than one brain region is involved with the etiology of depressive disorders. Further studies are needed to elucidate the specific interactions between these monoaminergic systems.
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Acknowledgements This research was performed as part of A. Zangen’s PhD thesis and supported by Bar-Ilan Research Foundation Žno. 2520..
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