PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Psychiatry. Author manuscript; available in PMC 2010 June 1.
Published in final edited form as:
PMCID: PMC2874651
NIHMSID: NIHMS78032

Chronic imipramine but not bupropion increases arachidonic acid signaling in rat brain: is this related to ‘switching’ in bipolar disorder?

Abstract

Agents effective against mania in bipolar disorder are reported to decrease turnover of arachidonic acid (AA) in phospholipids and expression of calcium-dependent AA-selective cytosolic phospholipase A2 (cPLA2) in rat brain. In contrast, fluoxetine, an antidepressant that is reported to switch bipolar depressed patients to mania, increases cPLA2 expression and AA turnover in rat brain. We therefore hypothesized that antidepressants that increase switching to mania generally increase cPLA2 and AA turnover in brain. To test this hypothesis, adult male CDF-344 rats were administered imipramine and bupropion, with reported high and low switching rates, respectively, at daily doses of 10 and 30mgkg−1 i.p., respectively, or i.p. saline (control) for 21 days. Frontal cortex expression of different PLA2 enzymes and AA turnover rates in brain when the rats were unanesthetized were measured. Compared with chronic saline, chronic imipramine but not bupropion significantly increased cortex cPLA2 mRNA activity, protein and phosphorylation, expression of the cPLA2 transcription factor, activator protein-2α(AP-2α) and AA turnover in phospholipids. Protein levels of secretory phospholipase A2, calcium-independent phospholipase A2, cyclooxygenase (COX)-1 and COX-2 were unchanged, and prostaglandin E2 was unaffected. These results, taken with prior data on chronic fluoxetine in rats, suggest that antidepressants that increase the switching tendency of bipolar depressed patients to mania do so by increasing AA recycling and metabolism in brain. Mania in bipolar disorder thus may involve upregulated brain AA metabolism.

Keywords: arachidonic acid, bipolar disorder, bupropion, cPLA2, imipramine, switching

Introduction

Persons with bipolar disorder (BD) experience episodes of depression more frequently and for longer periods than episodes of mania.1 Bipolar depression is a major cause of psychiatric morbidity and mortality and poses a major public health concern. A challenge in treating bipolar depression is the tendency of many antidepressants, particularly tricyclic antidepressants and serotonin selective reuptake inhibitors, to induce episodes of mania or to increase cycle frequency or symptom intensity.2,3 The causes for the tendency of antidepressants to ‘switch’ depressed bipolar patients to mania are not understood.

Antimanic mood stabilizers, lithium, valproic acid and carbamazepine, when given chronically to rats to produce plasma concentrations therapeutically relevant to BD, reduce arachidonic acid (AA; 20:4n−6) turnover in phospholipids and expression of AA cascade enzymes in rat brain.4 AA is a polyunsaturated fatty acid found predominately in the stereo-specifically numbered (sn)-2 position of membrane phospholipids, and can be released from this position by certain phospholipase A2 (PLA2) enzymes.5 These enzymes can be activated through their coupling to certain neuroreceptors by a G-protein or by calcium,610 or in conditions of neuroinflammation through their coupling to cytokine receptors.1113 The unesterified AA that is released by a PLA2 can be converted to eicosanoids or other active metabolites, subjected to β-oxidation, or reesterified into the sn-2 position of membrane phospholipids via a long-chain acyl-CoA synthetase and an acyltransferase.1419 Released AA and its metabolites can influence a number of brain processes, including signal transduction, transcription, neuronal activity, apoptosis, inflammation and blood flow.2026

The agents lithium, carbamazepine (5H-dibenz[b, f]azepine-5-carboxamide; 5-carbamoyl-5H-dibenz[b, f]azepine) and valproic acid (2-propylpentanoic acid), which are effective in the manic phase of BD, are reported to decrease brain AA turnover rate in membrane phospholipids in unanesthetized rats without changing the turnover rate of docosahexaenoic acid (DHA; 22:6n−3),2731 another polyunsaturated fatty acid usually found in the sn-2 position. The effect of chronically administered lithium and carbamazepine on AA turnover was ascribed to a selective decrease in the mRNA, protein and activity levels of AA-selective Ca2+-dependent cytosolic PLA2 (cPLA2),32,33 whereas chronic valproate inhibited the activity of an AA-selective acyl-CoA synthetase that is involved in recycling AA back into membrane phospholipids.27,34

In contrast to the three antimanic agents, another mood stabilizer, lamotrigine (3,5-diamino-6-(2,3- dichlorophenyl)-astriazine), which is preferred for treating bipolar depression and rapid cycling, neither decreased AA turnover in brain phospholipids of unanesthetized rats35 nor changed cPLA2 expression, whereas it decreased transcription and protein of cyclooxygenase (COX)-2.36 Topiramate (2,3:4,5-bis-Oisopropylidene- β-d-fructopyranose sulfamate), which had been thought effective against BD37 and shown to reduce hyperactivity in a quinpirole rat model,38 did not significantly change expression of any of the measured enzymes in the AA cascade, including cPLA2, or alter AA or DHA turnover in rat brain phospholipids.39,40 Supporting this lack of effect, four double-blind placebo-controlled trials later demonstrated that topiramate was ineffective in BD,41 and it was withdrawn as a treatment.

A number of antidepressants when administered to bipolar depressed patients cause them to ‘switch’ to mania more frequently than expected.2,3,42,43 Because antimanic mood stabilizers when given chronically to rats downregulate brain AA turnover and AA enzymes, we thought it possible that the increased switching tendency caused by some of these anti-depressants might be due to their upregulating the brain AA cascade. This hypothesis is consistent with evidence that lamotrigine, which does not upregulate AA turnover but reduces COX-2 expression in rat brain,35,36 does not increase switching, whereas fluoxetine, which upregulates AA turnover and cPLA2 mRNA, protein and activity in rat brain,44,45 is reported to do so.42,46,47

To further test the hypothesis, in the present study, we examined brain AA metabolism and expression of brain PLA2 and COX enzymes in rats treated chronically with two additional antidepressants, imipramine (5-[3-(dimethylamino) propyl]-10,11- dihydro-5H-dibenz [b,f]-azepine monohydrochloride) and bupropion ((±)-1- (3-chlorophenyl)-2-[(1,1 dimethylethyl)amino]-1- propanone hydrochloride). Imipramine, a tricyclic antidepressant, is beneficial for unipolar depression, and is thought to act by inhibiting serotonin and noradrenalin reuptake transporters. At a clinically relevant dose (10mgkg−1 body weight), imipramine decreases depression scores in rodents, as measured by a variety of tests.4851 Bupropion is thought to act by inhibiting both noradrenalin and dopamine reuptake, but has no significant serotonergic effect.52 Acute or chronic bupropion given to rats had an antidepressant effect by reducing immobility time in the forced swim test,5355 and improved behavior in another animal model of depression.56 Because imipramine but not bupropion is reported to increase switching in bipolar depressed patients,43,47,5759 we hypothesized that chronic imipramine but not bupropion would increase markers of AA metabolism, including AA turnover in phospholipids and cPLA2 expression, in rat brain.

Materials and methods

Animals

Chemicals and reagents, including bupropion and imipramine, were purchased from Sigma Chemicals (St Louis, MO, USA) unless otherwise indicated. The study was conducted following the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (Publication no. 80-23) and was approved by the Animal Care and Use Committee of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Animal Care and Use Committee. Male CDF-344 rats, weighing 180–190 g (Charles River Laboratories; Wilmington, MA, USA), were acclimatized for 1 week in an animal facility with controlled temperature, humidity and light cycle, ad libitum access to food (NIH-31) and water. Rats were assigned randomly to an acute or a chronic treatment (imipramine and bupropion) group or to a control group. Chronic drug-treated rats received 10mgkg−1 day−1 imipramine or 30mgkg−1 day−1 bupropion dissolved in saline (0.9%) once daily for 21 days intraperitoneally (i.p.), whereas controls received the same volume of saline once daily i.p., also for 21 days. For acute studies (to separate chronic from acute drug action), rats were randomized into three treatment groups that received a single i.p. injection of imipramine (10mgkg−1) or bupropion (30mgkg−1) or an equivalent volume of saline 3 h before a study.

The chronic dosing regimens for the two drugs are reported to decrease depression scores in rodents, as measured by a variety of behavioral and biochemical tests.4851,60 On the last day of a dosing regimen, a rat was injected with its appropriate treatment 3 h before its brain was removed for enzyme, protein, prostaglandin E2 (PGE2) or kinetic analysis. For molecular analysis, 3 h after the last injection, the rat was anesthetized with CO2 and decapitated. The frontal cortex was rapidly dissected and frozen in 2-methylbutane at −50 °C, then stored at −80 °C until use. For PGE2 measurements, the rat was lightly anesthetized with sodium pentobarbital (50mgkg−1; Abbott Laboratories, Chicago, IL, USA) and subjected to head-focused microwave irradiation to inactivate enzymes and stop brain metabolism (5.5kW, 4.8 s; Cober Electronics, Stamford, CT, USA).61,62

For the kinetic radiotracer study, the rat was anesthetized with 1–3% halothane, and polyethylene catheters were inserted into the femoral artery and vein as reported.31 The rat was allowed to recover from surgery for 3 h, with its hindquarters loosely wrapped and taped to a wooden block. During recovery, arterial blood pressure was monitored, and temperature was maintained at 37 °C by means of a rectal probe and a heating element (Indicating Temperature Controller; Yellow Springs Instruments, Yellow Springs, OH, USA). Blood pH, pCO2 and pO2 were monitored using a Rapidlab 248 pH/blood gas analyzer (Bayer, East Walpole, MA, USA), and heart rate and blood pressure were analyzed using a CyberSense monitor (Model BPM01 CyQ 302, Nicholasville, KY, USA).

Infusion of [1-14C] arachidonic acid and tissue sampling

[1-14C]AA (50 mCimmol−1, >98% pure, Moravek Biochemicals, Brea, CA, USA) was prepared in saline containing 50mgml−1 fatty acid-free bovine serum albumin (Sigma) as described previously.31,61 Three hours after the last injection, unanesthetized rats were infused intravenously (i.v.) for 5 min with 1ml containing 170 μCi kg−1 AA at a rate of 0.223 (1−e−1.92t) ml min−1 with a computer-controlled variable rate infusion pump (No. 22; Harvard Apparatus, South Natick, MA, USA) to achieve a steady-state plasma-specific activity within 1 min.30,31,63 Arterial blood samples were collected at 0, 15, 30, 45, 90, 180, 240 and 300 s during infusion to determine radioactive and cold concentrations of non-esterified AA in plasma. Five minutes after starting infusion, the rat was anesthetized with sodium pentobarbital (50mgkg−1, i.v.) and subjected to head-focused microwave irradiation to stop brain metabolism (5.5kW, 4.8 s; Cober Electronics, Norwalk, CT, USA).64,65 The brain was excised, bisected sagittally and stored at −80 °C for further analysis.

Brain lipid extraction and chromatography

Total lipids were extracted from frozen plasma and from one brain hemisphere by the method of Folch.66 Heptadecanoic acid (17:0) was added as an internal standard to plasma before extraction. The extracts were separated by thin layer chromatography on silica gel plates (Whatman, Clifton, NJ, USA). Unesterified fatty acids were separated using a mixture of heptane:diethylether:glacial acetic acid (60:40:2 by volume),29 and phospholipids (choline glycerol-phospholipid, ChoGpl; phosphatidylserine, PtdSer; phosphatidylinositol, PtdIns; ethanolamine glycerophospholipid, EtnGpl) were separated in chloroform:- methanol:H2O:glacial acetic acid (60:50:4:1 by volume)67 and identified with unlabeled standards in separate lanes. Phospholipid and standard bands were visualized with 6-p-toluidine-2-naphthalenesulfonic acid (Acros, Fairlawn, NJ, USA) under ultraviolet light. Each band was removed and analyzed for radioactivity by liquid scintillation counting. Phospholipid bands were also individually scraped and 200 μl toluene was added with a known amount of di-17:0-PtdCho for quantification before methylation. Fatty acid methyl esters were formed by heating the phospholipid scrapes in 1% H2SO4 in methanol at 70 °C for 3 h.68 The methyl esters were separated on a 30m×25mm inner diameter capillary column (SP-2330; Supelco, Bellefonte, PA, USA), using gas chromatography with a flame ionization detector (Model 6890N; Agilent Technologies, Palo Alto, CA, USA). Runs were initiated at 80 °C, with a temperature gradient to 160 °C (10 °Cmin−1) and 230 °C (3°Cmin−1) in 31 min, and held at 230 °C for 10 min. Peaks were identified by retention times of fatty acid methyl ester standards (Nu-Chek-Prep, Elysian, MN). Fatty acid concentrations (nmol g−1 brain or nmol ml−1 plasma) were calculated by proportional comparison of gas chromatography peak areas to that of the 17:0 internal standard. Tracer identification and separation were performed on fatty acid methyl esters of pooled plasma samples (at the end of the infusion) and pooled brain total lipid extracts as described above. The fatty acid methyl esters were separated as described previously with slight modifications69 using a high-performance liquid chromatography (HPLC) (Beckman, Fullerton, CA, USA) equipped with an in-line UV/vis detector (λ= Gilson, Middleton, WI, USA) and an in-line scintillation counter (β-RAM; IN/US System, Tampa, FL, USA) with a Luna C18 column (Phenomenex, Torrance, CA, USA). Initial conditions were set to a 1 ml min−1 gradient system composed of (A) 100% H2O and (B) 100% acetonitrile. The gradient started with 85% B, for 30 min, and then increased to 100% B over 10 min where it was held for 20 min before returning to 85% B over 5 min.

Quantification of labeled and unlabeled acyl-CoA

Acyl-CoA species were isolated from the remaining half brain by the method of Deutsch.70 Weighed brain and a known amount of 17:0-CoA as an internal standard were placed in a 15ml conical vial before sonication of the brain in 25mM potassium phosphate. Isopropanol (2 ml) was added to the vial and the homogenate was sonicated again. Saturated ammonium sulfate (0.25 ml) was added and the sample was lightly shaken by hand. Acetonitrile (4 ml) was added and the sample was vortexed for 10 min before centrifugation. The upper phase was extracted and 10 ml of 25mM potassium phosphate was added. Each sample was run three times through an activated oligonucleotide purification cartridge (Applied Biosystems, Foster City, CA, USA), washed with 10 ml of 25mM potassium phosphate and eluted with 400 μl isopropanol:1mM glacial acetic acid (75:25 by volume). Samples were dried under nitrogen and reconstituted in 100 μl isopropanol:1mM glacial acetic acid (75:25 by volume) for HPLC analysis. Acyl-CoA species were separated using HPLC (Beckman) with a Symmetry C-18, 5 μm column (250×.6mm; Waters-Millipore Corp., Milford, MA, USA). Conditions were set to a 1 ml min−1 gradient system composed of (A) 75mM potassium phosphate and (B) 100% acetonitrile. The gradient started with 44% B, increased to 49% over 25 min and then to 70% over 5 min, remained at 70% for 9 min and returned to 44% over 4 min and was held there for 4 min (end of run). Concentrations of acyl-CoA species and their associated radioactivity were measured using peak area analysis from HPLC chromatograms relative to 17:0-CoA and liquid scintillation counting. These values were used to calculate the specific activities of arachidonoyl-CoA.

Calculations

The model for determining in vivo kinetics of brain fatty acids in rats is described in detail elsewhere.18,30 Briefly, unidirectional incorporation coefficients, ki (ml s−1 g−1) of [1-14C]AA, representing incorporation from plasma into brain phospholipid compartments i, were calculated as:

ki=cbr,i(T)0Tcpldt
(1)

cbr,i(T)(nCig1) is radioactivity of brain lipid i at time T = 5 min (time of termination of experiment), t is time after beginning infusion and cp1(nCiml1) is the plasma concentration of labeled AA during infusion. Integrals of plasma radioactivity were determined by trapezoidal integration. The synthesis of AA from its dietary precursor linoleic acid (18:2n−6) represents less than 0.5% of the plasma AA flux into brain.71 Thus, net rates of incorporation of non-esterified AA from plasma into brain phospholipid i, Jin,i, and from brain arachidonoyl-CoA, JFA,i, were calculated as:

Jin,i=Kicpl
(2)

JFA,i=Jin,iλ
(3)

cpl (nmol ml−1) is the concentration of unlabeled non-esterified AA in plasma. A ‘dilution factor’ λ is defined as the steady-state ratio during [1-14C]AA infusion of specific activity of the brain arachidonoyl- CoA pool to plasma unesterified AA specific activity,

λ=cbr,acylCoA/cbr,acylCoAcplasma/cplasma
(4)

The steady state is reached within 1 min after infusion begins.63,72 The fractional turnover rate of AA within phospholipid i, FFA,i (%h−1), is defined as:

FFA,i=JFA,icbr,i
(5)

Preparation of cytoplasmic and nuclear extracts

Cytoplasmic and nuclear extracts were prepared from rat frontal cortex, as previously described.45 Briefly, the frontal cortex was homogenized in 10mM HEPES, pH 7.9, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 10mM KCl, buffer with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) using a Teflon-glass homogenizer. After adding 0.5% NP-40, five additional strokes were performed. The suspension was incubated for 30 min on ice, and then centrifuged in a microcentrifuge at 13 000 g for 1 h at 4 °C. To the nuclear pellet, solution B (20mM HEPES, pH 7.9, 1mM EDTA, 1mM EGTA, 1mM DTT, 0.4M NaCl) and a protease inhibitor cocktail (Roche) were added. Tubes were mixed and placed on a small rotatory shaker for 30 min. Finally, the mixture was centrifuged at 13 000 g for 1 h at 4 °C. The supernatant containing the proteins from the nuclear extracts was transferred to a fresh tube. Protein concentrations of cytoplasmic and nuclear extracts were determined using Bio-Rad Protein Reagent (Bio-Rad, Hercules, CA, USA). The composition of extracts from each fraction was confirmed using specific antibodies (data not shown).

Phospholipase A2 activity and prostaglandin E2 concentration

Supernatants corresponding to the cytosolic fraction were assayed for cPLA2 activity, using appropriate assay kits (Cayman, Ann Arbor, MI, USA). PGE2 was extracted according to the method of Radin.73 A portion of the extract was dried under nitrogen and assayed for PGE2 using a polyclonal enzyme-linked immunosorbent assay according to the manufacturer’s instructions (Oxford Biomedical Research, Product No. EA 02, Oxford, MI, USA).

Western blot analysis

Proteins from cytoplasmic (50 μg) and nuclear (50 μg) extracts were separated on 4–20% SDS-polyacrylamide gels (PAGE) (Bio-Rad). Following SDS-PAGE, the proteins were electrophoretically transferred to a nitrocellulose membrane. Protein blots were incubated overnight at 4 °C in TBS buffer, containing 5% nonfat dried milk and 0.1% Tween-20, with specific primary antibodies (1:1000 dilution) for the group IVA cPLA2, group IIA secretory phospholipase A2 (sPLA2), group VIA calcium-independent phospholipase A2 (iPLA2),5 COX-1 (1:1000) (Santa Cruz Biotech, Santa Cruz, CA, USA), activator protein-2α (AP-2α), AP-2β, NFκB p65, p50 and COX-2 (1:1000) (Cell Signaling, Beverly, MA, USA). Protein blots were incubated with appropriate HRP-conjugated secondary antibodies (Cell Signaling) and visualized using a chemiluminescence reaction (Amersham, Piscataway, NJ, USA) on X-ray film (XAR-5, Kodak). Optical densities of immunoblot bands were measured using Alpha Innotech Software (Alpha Innotech, San Leandro, CA, USA) and were normalized to β-actin (Sigma) to correct for unequal loading. All the experiments were carried out three times with 10 independent samples per group. Values are expressed as percent of control.

Total RNA isolation and real time RT-PCR

Total RNA and cDNA were prepared as described27 from frontal cortex and measured by real time quantitative RT-PCR, using the ABI PRISM 7000 sequence detection system (Applied Biosystems). Specific primers and probes for cPLA2, purchased from TaqMan gene expression assays (Applied Biosystems), consisted of a 20× mix of unlabeled PCR primers and Taqman minor groove binder (MGB) probe (FAM dye-labeled). The fold change in gene expression was determined using the ΔΔCT method.74 Data are expressed as the relative level of the target gene (cPLA2) in the chronic imipramine- or bupropion- administered animals normalized to the endogenous control (β-globulin) and relative to the control rats (saline injected) (calibrator), as described previously. 32,45 All the experiments were carried out twice in triplicate with 10 independent samples per group.

Data and statistics

A one-way analysis of variance with Tukey’s pairwise post hoc test was used to compare means between rats administered chronic bupropion, chronic imipramine or chronic saline (controls) (SAS 9.1.3, Cary, NC, USA). Statistical significance was taken as P≤0.05. Data are presented as means±standard deviation (s.d.) of independent measurements. Western blot and real time RT-PCR data are presented as means ±s.e.m. of independent measurements.

Results

Body weight

Rats chronically administered imipramine weighed 14% and 11% less than did controls or rats given bupropion, respectively (261±14, 224±12, 253±14 g for control, imipramine and bupropion, respectively, P < 0.001). There was no significant difference in body temperature, blood pressure, heart rate, arterial blood pH, pCO2 or pO2 between treatment groups and controls.

Plasma and brain fatty acids

As in our previous reports,28,35,40,44,62,64,75 HPLC separation of fatty acid methyl ester derivatives from total lipid extracts of pooled plasma confirmed that greater than 98% of radioactivity represented labeled AA after 5 min of infusion of [1-14C]AA across treatment groups. HPLC separation of fatty acid methyl ester derivatives from pooled brain total lipid extracts also showed that greater than 95% of total brain radioactivity was in the form of [1-14C]AA across treatment groups (data not shown). Percent radioactivities from plasma and brain total lipid extracts were comparable between the treated and control rats, and are similar to published data.28,35,40,44,62,64,75

Chronic or acute imipramine and bupropion did not significantly change the plasma concentration of unlabeled unesterified AA (Table 1), or of any other measured plasma unesterified fatty acid (data not shown), compared to control. There was no significant difference in the concentration of brain arachidonoyl- CoA or of any other measured acyl-CoA species between treatment groups and controls (Table 1). There was no significant difference in the unlabeled esterified fatty acid concentration including AA in brain phospholipids between treatment groups and controls (Table 1).

Table 1
Arterial plasma unesterified arachidonic acid and brain arachidonoyl-CoA and esterified arachidonic acid concentrations in control, imipramine, and bupropion treated rats

Kinetics

The integral of plasma radioactivity (denominator of Equation (1)) after the 5 min of [1-14C]AA infusion was significantly higher in chronic and acute imipramine-treated rats (217 072±98 701, 156 648±15 886 nCi s−1 ml−1, respectively) compared to chronic and acute bupropion-treated rats (125 064±13 459, 134 865±15 792 nCi s−1 ml−1, respectively) and chronic and acute controls (125 166±14 417, 121 588±12 547 nCi s−1 ml−1, respectively). Chronic imipramine, but not bupropion, increased ki (Equation (1)) of AA in ChoGpl, PtdIns, EtnGpl and total phospholipids by 24%, 26%, 15% and 21% compared to controls, respectively (Table 2). Chronic imipramine, but not chronic bupropion, increased Jin,i (Equation (2)) in ChoGpl, PtdIns, EtnGpl and total phospholipids by 38%, 39%, 29% and 35% compared to controls, respectively (Table 2). λ (Equation (3)) was not significantly different between imipramine and bupropion-treated rats and controls (Table 3). Chronic imipramine, but not chronic bupropion, increased JFA,i (Equation (4)) in ChoGpl, PtdSer, PtdIns, EtnGpl and total phospholipids by 71%, 41%, 72%, 58% and 66% compared to controls, respectively (Table 3). The turnover rate of AA (FFA,i, Equation (5)) was significantly higher within brain ChoGpl (80%), PtdSer (38%), PtdIns (63%), EtnGpl (47%) and total phospholipids (59%) in chronic imipramine-treated rats compared to controls (Table 3). In contrast, acute imipramine and bupropion did not alter any measured kinetics of AA (Tables 13).

Table 2
Incorporation coefficients (ki > *), and net rates of incorporation (Jin,i >) from plasma of unesterified arachidonic acid, into brain phospholipid classes in control, imipramine, and bupropion treated rats
Table 3
The net rates of arachidonic acid incorporation from brain arachidonoyl-CoA (JFA,i >) and of turnover (FFA,i >) in brain phospholipid classes, in control, imipramine, and bupropion treated rats

mRNA, protein and activity of cPLA2

Chronic imipramine, but not bupropion, significantly increased brain cPLA2 mRNA, protein and phosphorylated protein by 46%, 78% and 103%, respectively, compared to control levels (Figures 1a–c). Chronic imipramine, but not chronic bupropion, significantly increased cPLA2 activity by 26% compared to control (Figure 1d). sPLA2 and iPLA2 protein levels did not differ significantly between the groups (Figure 2).

Figure 1
Cytosolic phospholipase A2 (cPLA2) mRNA (a), protein (b), phosphorylated protein (c) and enzyme activity (d) in brains of rats chronically administered saline (control), imipramine (10mgkg−1) or bupropion (30mgkg−1) for 21 days. Data are ...
Figure 2
Secretory phospholipase A2 (sPLA2) (a) and calcium-independent phospholipase A2 (iPLA2) (b) protein levels in brains of rats chronically administered saline (control), imipramine (10mgkg−1) or bupropion (30mgkg−1) for 21 days. Data are ...

Cyclooxygenase protein levels and prostaglandin E2 concentration

Cyclooxygenase-1 and COX-2 protein levels and the concentration of PGE2, one of their major metabolites, were not changed significantly after chronic imipramine or bupropion, compared to control (Figure 3).

Figure 3
Cyclooxygenase-1 (COX-1) (a) and cyclooxygenase-2 (COX-2) (b) protein levels and prostaglandin E2 (PGE2) concentration (c) in brains of rats chronically administered saline (control), imipramine (10mgkg−1) or bupropion (30mgkg−1) for 21 ...

Cytosolic phospholipase A2-regulating transcription factors

Activator protein (AP)-2 and nuclear factor-kappa B (NF-κB) are recognized on the promoter region of cPLA2 and regulate cPLA2 transcription.76,77 Chronic imipramine, but not chronic bupropion, significantly increased the brain level of AP-2α protein by 48% (Figure 4a). AP-2β, NF-κB p65 and NF-κB p50 protein levels did not differ significantly between the groups (Figures 4b–d). The increase in AP-2 may have been responsible in part for the increased cPLA2 mRNA.

Figure 4
Activator protein-2α (AP-2α) (a), AP-2β (b), nuclear factor-kappa B p65 (NF-κB p65) (c) and NF-κB p50 (d) protein levels in brains of rats chronically administered saline (control), imipramine (10mgkg−1 ...

Acute effect of imipramine and bupropion

Acute imipramine and bupropion did not significantly change brain protein levels of cPLA2, sPLA2, iPLA2, COX-1 or COX-2 compared to the control group (Figure 5).

Figure 5
Cytosolic phospholipase A2 (cPLA2) (a), secretory phospholipase A2 (sPLA2) (b), calcium-independent phospholipase A2 (iPLA2) (c), cyclooxygenase-1 (COX-1) (d) and cyclooxygenase-1 (COX-2) (e) protein levels in brains of rats acutely administered saline ...

Discussion

Chronically administered imipramine, but not bupropion, increased AA incorporation and turnover in rat brain phospholipids and increased brain activity, protein, and mRNA levels of AA-selective cPLA2 78 as well as the level of its transcription factor, AP-2α. Activity and protein levels of sPLA2 and iPLA2 were not significantly affected by either drug. None of these changes was observed at 3 h following a single acute injection of imipramine or bupropion, indicating that they reflected the effect of chronic imipramine administration.

These findings, as well as published evidence that drug-induced changes in AA incorporation and turnover in rat brain phospholipids correlate with altered mRNA, protein and activity levels of cPLA2,4,62,79 and expression of its AP-2 transcription factor, suggest that the elevated AA incorporation and turnover rates following chronic imipramine were caused by increased cPLA2 activity. Similar correlated increases in cPLA2 and AA turnover follow chronic administration to rats of the antidepressant, fluoxetine.44

Phosphorylation of cPLA2 protein by mitogenactivated protein kinase (MAPK) can activate cPLA2,80,81 and imipramine is reported to activate the MAPK pathway in hippocampus-derived neuronal stem cells.82 Thus, MAPK activation may have accounted for the increased cPLA2 phosphorylation by chronic imipramine. The increased cPLA2 protein caused by chronic imipramine can be ascribed to an increase in cPLA2 mRNA and increased levels of the transcription factor AP-2α. Earlier studies also reported that increased AP-2 protein and binding activity were associated with increased cPLA2 mRNA and activity following chronic administration of N-methyl-D-aspartate (NMDA) to rats to produce excitotoxicity.62,79 Chronic fluoxetine in rats increased brain cPLA2 mRNA by increasing the RNA stabilizing protein AUF rather than by increasing AP-2.45

Neither chronic imipramine nor bupropion changed COX protein or PGE2 in the rat frontal cortex. Chronic fluoxetine, which increased rat frontal cortex cPLA2 activity and protein, also did not change brain COX expression or PGE2 concentration.44,45 On the other hand, lithium, carbamazepine and valproic acid, when given chronically to rats, decreased brain COX-2 protein and PGE2, and (for lithium and carbamazepine) cPLA2 expression, and lamotrigine decreased COX-2 transcription.4,83,84

Because chronic imipramine, like chronic fluoxetine, increased rat brain cPLA2 expression and AA turnover, without changing COX-2 or PGE2 levels, AA signaling may be related more directly to mood regulation than COX-2 activity or PGE2 formation. The observations that fluoxetine and imipramine increased AA signaling and cPLA2 expression, and effects of the antimanic agents in reducing these parameters, also may mean that bipolar mania is correlated with increased release of AA and increased AA metabolism through non-COX pathways.

Imipramine and fluoxetine, but not bupropion, increase the tendency of bipolar depressed patients to ‘switch’ to mania.42,43,47,5759,85 Imipramine, which inhibits synaptic reuptake of norepinephrine, serotonin and dopamine, is reported to cause an exceptionally high rate of manic and hypomanic reactions.43,57,86 Bupropion, which inhibits dopamine and norepinephrine but not serotonin reuptake, has a low switch rate,52,87 whereas fluoxetine, which primarily inhibits serotonin reuptake, produces a high switch rate.

The data in this paper, taken with published data on fluoxetine administration, suggest that antidepressant agents that increase switching of bipolar depressed patients to a manic state do so by increasing cPLA2 activity and AA turnover in brain phospholipids. The increased switching tendency may be related to the ability of the drugs to reduce synaptic serotonin uptake and increase serotonin in the synaptic cleft. cPLA2 activation and AA release from phospholipid can be initiated by activation of postsynaptic serotonergic 5-HT2A/2C receptors.69 Studies with additional antidepressants could test this suggestion.

Increased brain AA turnover and metabolism may be associated with bipolar mania. In rats that have been subjected to dietary deprivation of n−3 poly-unsaturated acids, brain cPLA2 activity, protein and mRNA are upregulated and scores on tests of aggression and depression, BD-like behaviors, are elevated compared with animals on an adequate diet.88,89 Further, gene expression of cPLA2, sPLA2 and COX- 2 appears increased in postmortem frontal cortex from BD compared to control subjects.90

Acknowledgments

This work was entirely supported by the Intramural Research Program of the National Institute on Aging, National Institutes of Health.

References

1. Judd LL, Akiskal HS, Schettler PJ, Coryell W, Endicott J, Maser JD, et al. A prospective investigation of the natural history of the long-term weekly symptomatic status of bipolar II disorder. Arch Gen Psychiatry. 2003;60:261–269. [PubMed]
2. Boerlin HL, Gitlin MJ, Zoellner LA, Hammen CL. Bipolar depression and antidepressant-induced mania: a naturalistic study. J Clin Psychiatry. 1998;59:374–379. [PubMed]
3. Settle EC, Jr, Settle GP. A case of mania associated with fluoxetine. Am J Psychiatry. 1984;141:280–281. [PubMed]
4. Rao JS, Lee HJ, Rapoport SI, Bazinet RP. Mode of action of mood stabilizers: is the arachidonic acid cascade a common target? Mol Psychiatry. 2008;13:585–596. [PubMed]
5. Lucas KK, Dennis EA. Distinguishing phospholipase A2 types in biological samples by employing group-specific assays in the presence of inhibitors. Prostaglandins Other Lipid Mediat. 2005;77:235–248. [PubMed]
6. Felder CC, Kanterman RY, Ma AL, Axelrod J. Serotonin stimulates phospholipase A2 and the release of arachidonic acid in hippocampal neurons by a type 2 serotonin receptor that is independent of inositolphospholipid hydrolysis. Proc Natl Acad Sci USA. 1990;87:2187–2191. [PubMed]
7. Garcia MC, Kim HY. Mobilization of arachidonate and docosahexaenoate by stimulation of the 5-HT2A receptor in rat C6 glioma cells. Brain Res. 1997;768:43–48. [PubMed]
8. Qu Y, Villacreses N, Murphy DL, Rapoport SI. 5-HT2A/2C receptor signaling via phospholipase A2 and arachidonic acid is attenuated in mice lacking the serotonin reuptake transporter. Psychopharmacology (Berl) 2005;180:12–20. [PubMed]
9. Stout BD, Clarke WP, Berg KA. Rapid desensitization of the serotonin (2C) receptor system: effector pathway and agonist dependence. J Pharmacol Exp Ther. 2002;302:957–962. [PubMed]
10. Basselin M, Chang L, Bell JM, Rapoport SI. Chronic lithium chloride administration to unanesthetized rats attenuates brain dopamine D2-like receptor-initiated signaling via arachidonic acid. Neuropsychopharmacology. 2005;30:1064–1075. [PubMed]
11. Kol S, Ben-Shlomo I, Ando M, Payne DW, Adashi EY. Interleukin- 1 beta stimulates ovarian phosphoipase A2 (PLA2) expression and activity: up-regulation of both secretory and cytosolic PLA2. Endocrinology. 1997;138:314–321. [PubMed]
12. McHowat J, Liu S. Interleukin-1beta stimulates phospholipase A2 activity in adult rat ventricular myocytes. Am J Physiol. 1997;272(2 Part 1):C450–C456. [PubMed]
13. Adibhatla RM, Hatcher JF. Secretory phospholipase A2 IIA is upregulated by TNF-alpha and IL-1alpha/beta after transient focal cerebral ischemia in rat. Brain Res. 2007;1134:199–205. [PMC free article] [PubMed]
14. Cunnane SC, Ryan MA, Nadeau CR, Bazinet RP, Musa-Veloso K, McCloy U. Why is carbon from some polyunsaturates extensively recycled into lipid synthesis? Lipids. 2003;38:477–484. [PubMed]
15. Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science. 2001;294:1871–1875. [PubMed]
16. Lands WEM, Crawford CG. Enzymes of Membrane Phospholipid Metabolism. Plenum; New York: 1976. pp. 3–85.
17. MacDonald JI, Sprecher H. Phospholipid fatty acid remodeling in mammalian cells. Biochim Biophys Acta. 1991;1084:105–121. [PubMed]
18. Robinson PJ, Noronha J, DeGeorge JJ, Freed LM, Nariai T, Rapoport SI. A quantitative method for measuring regional in vivo fatty-acid incorporation into and turnover within brain phospholipids: review and critical analysis. Brain Res Brain Res Rev. 1992;17:187–214. [PubMed]
19. Shimizu T, Wolfe LS. Arachidonic acid cascade and signal transduction. J Neurochem. 1990;55:1–15. [PubMed]
20. Bazan NG. Lipid signaling in neural plasticity, brain repair, and neuroprotection. Mol Neurobiol. 2005;32:89–103. [PubMed]
21. Devchand PR, Keller H, Peters JM, Vazquez M, Gonzalez FJ, Wahli W. The PPARalpha-leukotriene B4 pathway to inflammation control. Nature. 1996;384:39–43. [PubMed]
22. Hertz R, Magenheim J, Berman I, Bar-Tana J. Fatty acyl-CoA thioesters are ligands of hepatic nuclear factor-4alpha. Nature. 1998;392:512–516. [PubMed]
23. Kuehl FA, Jr, Humes JL, Tarnoff J, Cirillo VJ, Ham EA. Prostaglandin receptor site: evidence for an essential role in the action of luteinizing hormone. Science. 1970;169:883–886. [PubMed]
24. Mulligan SJ, MacVicar BA. Calcium transients in astrocyte endfeet cause cerebrovascular constrictions. Nature. 2004;431:195–199. [PubMed]
25. Samuelsson B, Dahlen SE, Lindgren JA, Rouzer CA, Serhan CN. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science. 1987;237:1171–1176. [PubMed]
26. Serhan CN, Savill J. Resolution of inflammation: the beginning programs the end. Nat Immunol. 2005;6:1191–1197. [PubMed]
27. Bazinet RP, Rao JS, Chang L, Rapoport SI, Lee HJ. Chronic valproate does not alter the kinetics of docosahexaenoic acid within brain phospholipids of the unanesthetized rat. Psychopharmacology (Berl) 2005;182:180–185. [PubMed]
28. Bazinet RP, Rao JS, Chang L, Rapoport SI, Lee HJ. Chronic carbamazepine decreases the incorporation rate and turnover of arachidonic acid but not docosahexaenoic acid in brain phospholipids of the unanesthetized rat: relevance to bipolar disorder. Biol Psychiatry. 2006;59:401–407. [PubMed]
29. Chang MC, Bell JM, Purdon AD, Chikhale EG, Grange E. Dynamics of docosahexaenoic acid metabolism in the central nervous system: lack of effect of chronic lithium treatment. Neurochem Res. 1999;24:399–406. [PubMed]
30. Chang MC, Contreras MA, Rosenberger TA, Rintala JJ, Bell JM, Rapoport SI. Chronic valproate treatment decreases the in vivo turnover of arachidonic acid in brain phospholipids: a possible common effect of mood stabilizers. J Neurochem. 2001;77:796–803. [PubMed]
31. Chang MC, Grange E, Rabin O, Bell JM, Allen DD, Rapoport SI. Lithium decreases turnover of arachidonate in several brain phospholipids. Neurosci Lett. 1996;220:171–174. [PubMed]
32. Ghelardoni S, Tomita YA, Bell JM, Rapoport SI, Bosetti F. Chronic carbamazepine selectively downregulates cytosolic phospholipase A2 expression and cyclooxygenase activity in rat brain. Biol Psychiatry. 2004;56:248–254. [PubMed]
33. Rintala J, Seemann R, Chandrasekaran K, Rosenberger TA, Chang L, Contreras MA, et al. 85 kDa cytosolic phospholipase A2 is a target for chronic lithium in rat brain. Neuroreport. 1999;10:3887–3890. [PubMed]
34. Bazinet RP, Weis MT, Rapoport SI, Rosenberger TA. Valproic acid selectively inhibits conversion of arachidonic acid to arachidonoyl- CoA by brain microsomal long-chain fatty acyl-CoA synthetases: relevance to bipolar disorder. Psychopharmacology (Berl) 2006;184:122–129. [PubMed]
35. Lee HJ, Rao JS, Chang L, Rapoport SI, Bazinet RP. Chronic lamotrigine does not alter the turnover of arachidonic acid within brain phospholipids of the unanesthetized rat: implications for the treatment of bipolar disorder. Psychopharmacology (Berl) 2007;193:467–474. [PubMed]
36. Lee HJ, Ertley RN, Rapoport SI, Bazinet RP, Rao JS. Chronic administration of lamotrigine downregulates COX-2 mRNA and protein in rat frontal cortex. Neurochem Res. 2008;33:861–866. [PubMed]
37. Erfurth A, Kuhn G. Topiramate monotherapy in the maintenance treatment of bipolar I disorder: effects on mood, weight and serum lipids. Neuropsychobiology. 2000;42(Suppl 1):50–51. [PubMed]
38. Shaldubina A, Einat H, Szechtman H, Shimon H, Belmaker RH. Preliminary evaluation of oral anticonvulsant treatment in the quinpirole model of bipolar disorder. J Neural Transm. 2002;109:433–440. [PubMed]
39. Ghelardoni S, Bazinet RP, Rapoport SI, Bosetti F. Topiramate does not alter expression in rat brain of enzymes of arachidonic acid metabolism. Psychopharmacology (Berl) 2005;180:523–529. [PubMed]
40. Lee HJ, Ghelardoni S, Chang L, Bosetti F, Rapoport SI, Bazinet RP. Topiramate does not alter the kinetics of arachidonic or docosahexaenoic acid in brain phospholipids of the unanesthetized rat. Neurochem Res. 2005;30:677–683. [PubMed]
41. Kushner SF, Khan A, Lane R, Olson WH. Topiramate monotherapy in the management of acute mania: results of four double-blind placebo-controlled trials. Bipolar Disord. 2006;8:15–27. [PubMed]
42. Ghaemi SN, Hsu DJ, Soldani F, Goodwin FK. Antidepressants in bipolar disorder: the case for caution. Bipolar Disord. 2003;5:421– 433. [PubMed]
43. Post RM, Altshuler LL, Leverich GS, Frye MA, Nolen WA, Kupka RW, et al. Mood switch in bipolar depression: comparison of adjunctive venlafaxine, bupropion and sertraline. Br J Psychiatry. 2006;189:124–131. [PubMed]
44. Lee HJ, Rao JS, Ertley RN, Chang L, Rapoport SI, Bazinet RP. Chronic fluoxetine increases cytosolic phospholipase A(2) activity and arachidonic acid turnover in brain phospholipids of the unanesthetized rat. Psychopharmacology (Berl) 2007;190:103–115. [PubMed]
45. Rao JS, Ertley RN, Lee HJ, Rapoport SI, Bazinet RP. Chronic fluoxetine upregulates activity, protein and mRNA levels of cytosolic phospholipase A2 in rat frontal cortex. Pharmacogenomics J. 2006;6:413–420. [PubMed]
46. Tohen M, Vieta E, Calabrese J, Ketter TA, Sachs G, Bowden C, et al. Efficacy of olanzapine and olanzapine-fluoxetine combination in the treatment of bipolar I depression. Arch Gen Psychiatry. 2003;60:1079–1088. [PubMed]
47. Leverich GS, Altshuler LL, Frye MA, Suppes T, McElroy SL, Keck PE, Jr, et al. Risk of switch in mood polarity to hypomania or mania in patients with bipolar depression during acute and continuation trials of venlafaxine, sertraline, and bupropion as adjuncts to mood stabilizers. Am J Psychiatry. 2006;163:232–239. [PubMed]
48. Daniel W, Adamus A, Melzacka M, Szymura J, Vetulani J. Cerebral pharmacokinetics of imipramine in rats after single and multiple dosages. Naunyn Schmiedebergs Arch Pharmacol. 1981;317:209– 213. [PubMed]
49. Maj J, Melzacka M, Mogilnicka E, Daniel W. Different pharmacokinetic and pharmacological effects following acute and chronic treatment with imipramine. J Neural Transm. 1982;54:219–228. [PubMed]
50. Nestler EJ, McMahon A, Sabban EL, Tallman JF, Duman RS. Chronic antidepressant administration decreases the expression of tyrosine hydroxylase in the rat locus coeruleus. Proc Natl Acad Sci USA. 1990;87:7522–7526. [PubMed]
51. Rogoz Z, Skuza G, Wojcikowski J, Daniel WA. Effects of combined treatment with imipramine and metyrapone in the forced swimming test in rats. Behavioral and pharmacokinetic studies. Pol J Pharmacol. 2003;55:993–999. [PubMed]
52. Fava M, Rush AJ, Thase ME, Clayton A, Stahl SM, Pradko JF, et al. 15 years of clinical experience with bupropion HCl: from bupropion to bupropion SR to bupropion XL. Prim Care Companion J Clin Psychiatry. 2005;7:106–113. [PubMed]
53. Kitamura Y, Fujitani Y, Kitagawa K, Miyazaki T, Sagara H, Kawasaki H, et al. Effects of imipramine and bupropion on the duration of immobility of ACTH-treated rats in the forced swim test: involvement of the expression of 5-HT2A receptor mRNA. Biol Pharm Bull. 2008;31:246–249. [PubMed]
54. Torregrossa MM, Folk JE, Rice KC, Watson SJ, Woods JH. Chronic administration of the delta opioid receptor agonist (+)BW373U86 and antidepressants on behavior in the forced swim test and BDNF mRNA expression in rats. Psychopharmacology (Berl) 2005;183:31–40. [PMC free article] [PubMed]
55. Torregrossa MM, Isgor C, Folk JE, Rice KC, Watson SJ, Woods JH. The delta-opioid receptor agonist (+)BW373U86 regulates BDNF mRNA expression in rats. Neuropsychopharmacology. 2004;29:649–659. [PubMed]
56. Jiao X, Pare WP, Tejani-Butt SM. Antidepressant drug induced alterations in binding to central dopamine transporter sites in the Wistar Kyoto rat strain. Prog Neuropsychopharmacol Biol Psychiatry. 2006;30:30–41. [PubMed]
57. Calabrese JR, Rapport DJ, Kimmel SE, Shelton MD. Controlled trials in bipolar I depression: focus on switch rates and efficacy. Eur Neuropsychopharmacol. 1999;9(Suppl 4):S109–S112. [PubMed]
58. Prien RF, Caffey EM, Jr, Klett CJ. Prophylactic efficacy of lithium carbonate in manic-depressive illness. Report of the Veterans Administration and National Institute of Mental Health collaborative study group. Arch Gen Psychiatry. 1973;28:337–341. [PubMed]
59. Prien RF, Kupfer DJ, Mansky PA, Small JG, Tuason VB, Voss CB, et al. Drug therapy in the prevention of recurrences in unipolar and bipolar affective disorders. Report of the NIMH Collaborative Study Group comparing lithium carbonate, imipramine, and a lithium carbonate-imipramine combination. Arch Gen Psychiatry. 1984;41:1096–1104. [PubMed]
60. Suckow RF, Smith TM, Perumal AS, Cooper TB. Pharmacokinetics of bupropion and metabolites in plasma and brain of rats, mice, and guinea pigs. Drug Metab Dispos. 1986;14:692–697. [PubMed]
61. DeGeorge JJ, Noronha JG, Bell J, Robinson P, Rapoport SI. Intravenous injection of [1-14C]arachidonate to examine regional brain lipid metabolism in unanesthetized rats. J Neurosci Res. 1989;24:413–423. [PubMed]
62. Lee HJ, Rao JS, Chang L, Rapoport SI, Bazinet RP. Chronic Nmethyl- D-aspartate administration increases the turnover of arachidonic acid within brain phospholipids of the unanesthetized rat. J Lipid Res. 2008;49:162–168. [PubMed]
63. Washizaki K, Smith QR, Rapoport SI, Purdon AD. Brain arachidonic acid incorporation and precursor pool specific activity during intravenous infusion of unesterified arachidonate in the anesthetized rat. J Neurochem. 1994;63:727–736. [PubMed]
64. Bazinet RP, Lee HJ, Felder CC, Porter AC, Rapoport SI, Rosenberger TA. Rapid high-energy microwave fixation is required to determine the anandamide (N-arachidonoylethanolamine) concentration of rat brain. Neurochem Res. 2005;30:597–601. [PubMed]
65. Deutsch J, Rapoport SI, Purdon AD. Relation between free fatty acid and acyl-CoA concentrations in rat brain following decapitation. Neurochem Res. 1997;22:759–765. [PubMed]
66. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226:497–509. [PubMed]
67. Skipski VP, Good JJ, Barclay M, Reggio RB. Quantitative analysis of simple lipid classes by thin-layer chromatography. Biochim Biophys Acta. 1968;152:10–19. [PubMed]
68. Makrides M, Neumann MA, Byard RW, Simmer K, Gibson RA. Fatty acid composition of brain, retina, and erythrocytes in breastand formula-fed infants. Am J Clin Nutr. 1994;60:189–194. [PubMed]
69. Aveldano MI, VanRollins M, Horrocks LA. Separation and quantitation of free fatty acids and fatty acid methyl esters by reverse phase high pressure liquid chromatography. J Lipid Res. 1983;24:83–93. [PubMed]
70. Deutsch J, Grange E, Rapoport SI, Purdon AD. Isolation and quantitation of long-chain acyl-coenzyme A esters in brain tissue by solid-phase extraction. Anal Biochem. 1994;220:321–323. [PubMed]
71. DeMar JC, Jr, Lee HJ, Ma K, Chang L, Bell JM, Rapoport SI, et al. Brain elongation of linoleic acid is a negligible source of the arachidonate in brain phospholipids of adult rats. Biochim Biophys Acta. 2006;1761:1050–1059. [PubMed]
72. Grange E, Deutsch J, Smith QR, Chang M, Rapoport SI, Purdon AD. Specific activity of brain palmitoyl-CoA pool provides rates of incorporation of palmitate in brain phospholipids in awake rats. J Neurochem. 1995;65:2290–2298. [PubMed]
73. Radin NS. Extraction of tissue lipids with a solvent of low toxicity. Methods Enzymol. 1981;72:5–7. [PubMed]
74. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–408. [PubMed]
75. Rao JS, Bazinet RP, Rapoport SI, Lee HJ. Chronic administration of carbamazepine down-regulates AP-2 DNA-binding activity and AP-2alpha protein expression in rat frontal cortex. Biol Psychiatry. 2007;61:154–161. [PubMed]
76. Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochem J. 1994;302(Part 3):723–727. [PubMed]
77. Morri H, Ozaki M, Watanabe Y. 5′-flanking region surrounding a human cytosolic phospholipase A2 gene. Biochem Biophys Res Commun. 1994;205:6–11. [PubMed]
78. Clark JD, Lin LL, Kriz RW, Ramesha CS, Sultzman LA, Lin AY, et al. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991;65:1043–1051. [PubMed]
79. Rao JS, Ertley RN, Rapoport SI, Bazinet RP, Lee HJ. Chronic NMDA administration to rats up-regulates frontal cortex cytosolic phospholipase A2 and its transcription factor, activator protein-2. J Neurochem. 2007;102:1918–1927. [PubMed]
80. Borsch-Haubold AG, Bartoli F, Asselin J, Dudler T, Kramer RM, Apitz-Castro R, et al. Identification of the phosphorylation sites of cytosolic phospholipase A2 in agonist-stimulated human platelets and HeLa cells. J Biol Chem. 1998;273:4449–4458. [PubMed]
81. Hefner Y, Borsch-Haubold AG, Murakami M, Wilde JI, Pasquet S, Schieltz D, et al. Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases. J Biol Chem. 2000;275:37542–37551. [PubMed]
82. Peng CH, Chiou SH, Chen SJ, Chou YC, Ku HH, Cheng CK, et al. Neuroprotection by Imipramine against lipopolysaccharide-induced apoptosis in hippocampus-derived neural stem cells mediated by activation of BDNF and the MAPK pathway. Eur Neuropsychopharmacol. 2008;18:128–140. [PubMed]
83. Bosetti F, Rintala J, Seemann R, Rosenberger TA, Contreras MA, Rapoport SI, et al. Chronic lithium downregulates cyclooxygenase- 2 activity and prostaglandin E(2) concentration in rat brain. Mol Psychiatry. 2002;7:845–850. [PubMed]
84. Bosetti F, Weerasinghe GR, Rosenberger TA, Rapoport SI. Valproic acid down-regulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase-1 and -2 in rat brain. J Neurochem. 2003;85:690–696. [PubMed]
85. Goodwin GM. Evidence-based guidelines for treating bipolar disorder: recommendations from the British Association for Psychopharmacology. J Psychopharmacol. 2003;17:149–173. 147. [PubMed]
86. Nemeroff CB, Evans DL, Gyulai L, Sachs GS, Bowden CL, Gergel IP, et al. Double-blind, placebo-controlled comparison of imipramine and paroxetine in the treatment of bipolar depression. Am J Psychiatry. 2001;158:906–912. [PubMed]
87. Richelson E. Synaptic effects of antidepressants. J Clin Psychopharmacol. 1996;16(3 Suppl 2):1S–7S. 7S–9S. [PubMed]
88. Rao JS, Ertley RN, DeMar JC, Jr, Rapoport SI, Bazinet RP, Lee HJ. Dietary n-3 PUFA deprivation alters expression of enzymes of the arachidonic and docosahexaenoic acid cascades in rat frontal cortex. Mol Psychiatry. 2007;12:151–157. [PubMed]
89. Rao JS, Ertley RN, Lee HJ, DeMar JC, Jr, Arnold JT, Rapoport SI, et al. n-3 polyunsaturated fatty acid deprivation in rats decreases frontal cortex BDNF via a p38 MAPK-dependent mechanism. Mol Psychiatry. 2007;12:36–46. [PubMed]
90. Rao JS, Kim H-W, Rapoport SI. Up-Regulated Arachidonic Acid Cascade Enzymes and their Transcription Factors in Post-Mortem Frontal Cortex from Bipolar Disorder Patients. Abstr. Soc Neurosci; San Diego, CA, USA. 2007. p. 707.5.