BRAIN REGION-DEPENDENT CHANGES IN THE EXPRESSION
OF ENDOCANNABINOID-METABOLIZING ENZYMES
IN RATS FOLLOWING ANTIDEPRESSANT DRUGS

Depression is one of the most prevalent and debilitating psychiatric disorders, which is characterized by impairments in cognition, memory, motivation, emotional regulation and motor function (1). However, its etiology remains unclear. Possible pathophysiological mechanisms of depression include the involvement of the endocannabinoid (eCB) system in the pathogenesis of this disease (2). Preclinical pharmacological and genetic evidences for the implication of the eCB system in depression are well documented, but the role of this system in the mechanism of action of antidepressants has not been fully elucidated.

The eCBs (mainly anandamide and 2-arachidonoylglycerol (2-AG)) are the arachidonic acid derivatives. These compounds are synthesized ‘on demand’ in the postsynaptic neurons and are not stored in secretory vesicles (2). eCBs production in the postsynaptic neurons is induced by depolarization-induced calcium signaling, reduction of intracellular potassium concentration and/or activation of Gq/11-coupled receptors (3). Anandamide is produced from glycerophospholipid in a two-step procedure. Initially, Ca2+-dependent N-acyltransferase catalyzes N-acylation of phosphatidylethanolamine, what results in forming N-acylphosphatidylethanolamine (NAPE). Then, N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) releases anandamide from NAPE. Interestingly, it was shown that anandamide formation was also the process independent of NAPE-PLD, involving the phospholipase C and tyrosine phosphatase enzyme (PTPN22) (4) or the α/β-hydrolase 4 (Abh4) and phosphodiesterase (5). Anandamide is removed from the extracellular space through cellular reuptake and enzymatic degradation. Fatty acid amide hydrolase (FAAH) terminates the biological activity of anandamide by hydrolyzing this molecule to arachidonic acid and ethanolamine (6). On the other hand, cyclooxygenase-2 (COX-2) or several lipoxygenases (12-LOX and 15-LOX) are also involved in the anandamide degradation (7). At the same time, the generation of 2-AG is dependent on the diacylglycerol lipase (DAGL), which releases 2-AG through hydrolysis of diacylglycerol (DAG). DAG can be generated from phosphoinositides by phospholipase C (PLC) or from phosphatic acid (PA) by PA-phosphohydrolase in the cell membrane (8). 2-AG can be metabolized by either FAAH in the post-synaptic neurons or by monoacylglycerol lipase (MAGL) in the presynaptic neurons (9-14). Brain 2-AG, 15% of total content, is catalyzed by two serine hydrolases, α/β-hydrolase 6 (ABHD6) and α/β-hydrolase 12 (ABHD12) (15).

Preclinical studies in several models of depression showed that depressive-like behavior was associated with decreased brain eCBs levels in rats after chronic unpredictable stress (CUS) (16, 17), maternal deprivation (18) and olfactory bulbectomy (19, 20) and with down-regulation of CB1 receptors in stressed (16, 17) and bulbectomized rats (20). On the other hand, antidepressants evoked different changes in the eCB system. In fact, imipramine (IMI) administered chronically increased the level of eCBs in the dorsal striatum (21), escitalopram (ESC) increased eCB levels in the hippocampus and dorsal striatum or decreased their concentration in the cortical structures and cerebellum (21), while tianeptine (TIA) treatment produced an increase of the eCB levels in the hippocampus (anandamide), dorsal striatum (anandamide and 2-AG) and frontal cortex (2-AG) (21). Desipramine (22) and fluoxetine (23, 24) did not change the eCBs levels, while tranylcypromine decreased anandamide levels in the limbic areas and increased 2-AG concentration in the prefrontal cortex (23). The effects in the eCB system observed after antidepressant administration are often bi-directional and the mechanism of these changes still remains unclear. Additionally, little is known about the effects of antidepressants on enzymes involved in the eCB metabolism, which are potentially important targets for pharmaceutical development.

The aim of the present study was to determine the effect of administration of antidepressant drugs on the expression of enzymes involved in the eCB metabolism. We set out to determine the effect of chronic or acute administration of antidepressant drugs on the expression of eCB synthesizing enzymes (for anandamide: NAPE-PLD and for 2-AG: diacylglycerol lipase α (DAGLα)) or eCB degrading enzymes (for anandamide: FAAH and for 2-AG: MAGL) as a potential mechanism of alterations in the eCB system after antidepressant treatment. We selected antidepressants with different mechanisms of action including IMI (a noradrenaline and serotonin reuptake inhibitor as well as a less potent antagonist of histamine (H1), alpha-1 (α1) adrenergic and muscarinic acetylcholine receptors), ESC (a selective serotonin reuptake inhibitor) and TIA (a selective serotonin reuptake enhancer).

MATERIAL AND METHODS

Animals

Male Wistar rats (weighing 280 – 300 g, 8 – 9 weeks old) were used. The animals were kept in air-conditioned rooms (22 ± 2ºC) in plastic cages with a natural light/night cycle and with free access to food and water. All experiments were carried out in accordance with the Directive 2010/63/EU and were approved by the Local Ethical Committee as compliant with the Polish Law (21 August 1997). Each group consisted of 8 animals.

Drugs

Imipramine hydrochloride (IMI; Sigma Aldrich, USA), escitalopram oxalate (ESC; Lundbeck, Denmark) and tianeptine sodium (TIA; Anpharm, Poland) were used. Antidepressants were dissolved in 0.9% NaCl (pH of an ESC solution was neutralized with 10% NaOH solution) and were administered (1 ml/kg) once per day between 9:00 – 12:00 intraperitoneally (ip), acutely or chronically (14 days). The doses for drugs were chosen based on effective doses used in our previous reports: IMI (15 mg/kg), ESC (10 mg/kg) and TIA (10 mg/kg) (21).

Isolation of brain structures

Rats were decapitated 24 hours after the last administration of antidepressant drugs and their brains were immediately removed. Rat brain structures (i.e., the prefrontal cortex, frontal cortex, hippocampus, dorsal striatum, nucleus accumbens and cerebellum) were isolated from two hemispheres according to The Rat Brain Atlas (25), rapidly frozen on dry ice and stored at –80ºC for Western blot analysis.

Western blot

Frozen brain structures were homogenized in a buffer (1 mM HEPES, 0.1 M DTT, 0.1 mM EGTA (pH 7.2 – 7.8), COMPLETE and sterile water) using a ball homogenizer (Bioprep-24, Allsheng, China) (10 s at 10000 rpm). Then homogenates were mixed with a loading buffer and denatured for 2 min at 85ºC, 2 min in ice, 5 min at 85ºC, and finally 2 min in ice. Protein concentration was measured in the brain homogenates using a bicinchoninic acid (BCA) protein assay kit (Serva, Germany). Protein samples (30 µg) were loaded on the gradient 8 – 16% SDS polyacrylamide gels (Bio-Rad Corp., Hercules, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in 3% non-fat dry milk prepared in TBST. Separate sets of membranes were incubated overnight at 4ºC with goat anti-NAPE-PLD polyclonal antibody (1:200; ab95397; Abcam, UK), goat anti-MAGL polyclonal antibody (1:200; ab24701; Abcam, UK), rabbit anti-DAGLα polyclonal antibody (1:200; ab81984; Abcam, UK), and mouse anti-FAAH monoclonal antibody (1:200; sc-100739; Santa Cruz Biotechnology, USA). Antibodies have been validated previously using knockout mice (20). The expressions of NAPE-PLD, FAAH, DAGLα or MAGL were evaluated relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using rabbit anti-GAPDH polyclonal antibody (1:1500, sc-25778, Santa Cruz Biotechnology, USA). Blots were washed in TBST and incubated with donkey anti-goat secondary antibody (1:6000; 926-68074; Li-cor, USA), goat anti-rabbit secondary antibody (1:6000; 926- 68071; Li-cor, USA) or goat anti-mouse secondary antibody (1:6000; 926-32210; Li-cor, USA). The expression of particular proteins was analyzed with a fluorescence detection kit Odyssey Clx (Li-cor, USA). Analysis was performed using the Image Studio v.2.1 software package. All data were expressed as % of control.

Statistical analysis

All data were expressed as the mean ± SEM. Statistical analyses covered a one-way analysis of variance (ANOVA), followed by the Dunnett’s test to examine differences between group means. P < 0.05 was considered statistically significant.

RESULTS

Anandamide metabolizing enzymes

1. N-acyl phosphatidylethanolamine phospholipase D

IMI induced a change in NAPE-PLD protein expression in the prefrontal cortex (F(2.23) = 11.01; P = 0.0008), where an increase (P < 0.05) in the expression of this protein was seen after acute administration of IMI (Fig. 1).

Fig. 1. Changes in the expression of anandamide synthesizing enzyme NAPE-PLD in the brain structures following acute and chronic administration of antidepressants. Abbreviations: NAPE-PLD, N-acyl phosphatidylethanolamine phospholipase D; PFCTX, prefrontal cortex; FCTX, frontal cortex; HIP, hippocampus; DSTR, dorsal striatum; NAc, nucleus accumbens; CER, cerebellum; IMI (15), imipramine (15 mg/kg); ESC (10), escitalopram (10 mg/kg); TIA (10), tianeptine (10 mg/kg). All data are expressed as the mean ± SEM. N = 8 rats/group. *P < 0.05; **P < 0.01 versus VEH (vehicle).

Administration of ESC induced changes in NAPE-PLD protein expression in the hippocampus (F(2.23) = 7.789; P = 0.0037) and dorsal striatum (F(2.23) = 6.472; P = 0.0081). A post hoc analysis revealed an increase in NAPE-PLD protein expression in the hippocampus (P < 0.01) and dorsal striatum (P < 0.05) after chronic ESC treatment (Fig. 1).

After TIA treatment an alteration in NAPE-PLD protein expression was observed in the prefrontal cortex (F(2.23) = 5.042; P = 0.0182). Higher levels of NAPE-PLD protein expression (P < 0.05) in this brain structure were noted after acute and chronic administration of TIA (Fig. 1).

2. Fatty acid amide hydrolase

IMI produced changes in FAAH protein expression in the prefrontal cortex (F(2.23) = 6.069; P = 0.0097) and dorsal striatum (F(2.23) = 6.766; P = 0.0064). FAAH protein expression was either increased in the prefrontal cortex after acute IMI administration or decreased in the dorsal striatum (P < 0.01) after chronic treatment with this drug (Fig. 2).

Fig. 2. Changes in the expression of anandamide degrading enzyme FAAH in the brain structures following acute and chronic administration of antidepressants. Abbreviations: FAAH, fatty acid amide hydrolase; PFCTX, prefrontal cortex; FCTX, frontal cortex; HIP, hippocampus; DSTR, dorsal striatum; NAc, nucleus accumbens; CER, cerebellum; IMI (15), imipramine (15 mg/kg); ESC (10), escitalopram (10 mg/kg); TIA (10), tianeptine (10 mg/kg). All data are expressed as the mean ± SEM. N = 8 rats/group. *P < 0.05; **P < 0.01 versus VEH (vehicle).

Administration of ESC resulted in a change in FAAH protein expression in the dorsal striatum (F(2.23) = 6.755; P = 0.0065), where a decrease in this protein expression (P < 0.005) was observed after chronic drug administration (Fig. 2).

TIA produced a shift in FAAH protein expression in the prefrontal cortex (F(2.23) = 8.747; P = 0.0022), where an increase in this protein expression was detected after acute

(P < 0.01) and chronic (P < 0.05) administration of TIA (Fig. 2).

2-AG metabolizing enzymes

1. Diacylglycerol lipase α

IMI evoked changes in DAGLα protein expression in the frontal cortex (F(2.23) = 16.57; P < 0.0001) and cerebellum (F(2.23) = 9.116; P = 0.0018). IMI administered acutely and chronically increased DAGLα protein expression in the frontal cortex (P < 0.001), while a decrease in this protein expression was seen in the cerebellum (P < 0.01) after chronic IMI administration (Fig. 3).

Fig. 3. Changes in the expression of 2-AG synthesizing enzyme DAGLα in the brain structures following acute and chronic administration of antidepressants. Abbreviations: 2-AG, 2-arachidonoylglycerol; DAGLα, diacylglycerol lipase α; PFCTX, prefrontal cortex; FCTX, frontal cortex; HIP, hippocampus; DSTR, dorsal striatum; NAc, nucleus accumbens; CER, cerebellum; IMI (15), imipramine (15 mg/kg); ESC (10), escitalopram (10 mg/kg); TIA (10), tianeptine (10 mg/kg). All data are expressed as the mean ± SEM. N = 8 rats/group. *P < 0.05; **P < 0.01; ***P < 0.001 versus VEH (vehicle).

Administration of ESC resulted in the changes in DAGLα protein expression in the hippocampus (F(2.23) = 11.08; P = 0.0007) and dorsal striatum (F(2.23) = 7.314; P = 0.0047). Chronic ESC treatment induced an increase in DAGLα protein expression in these structures (P < 0.01) in rats (Fig. 3).

TIA caused a shift in DAGLα protein expression in the prefrontal cortex (F(2.23) = 5.340; P = 0.0151), frontal cortex (F(2.23) = 17.30; P < 0.0001) and dorsal striatum (F(2.23) = 4.075; P = 0.0347). Higher levels of DAGLα protein expression were found in the prefrontal (P < 0.05) and frontal (P < 0.001) cortex after acute administration of TIA. At the same time, chronic TIA administration induced an increase in DAGLα protein expression in the prefrontal cortex (P < 0.05), frontal cortex (P < 0.01) and dorsal striatum (P < 0.05) in rats (Fig. 3).

2. Monoacylglycerol lipase

IMI induced a change in MAGL protein expression in the frontal cortex (F(2.23) = 13.97; P = 0.0002), and a post hoc test revealed a decrease in this protein expression (P < 0.05) after chronic IMI administration (Fig. 4).

Fig. 4. Changes in the expression of 2-AG degrading enzyme MAGL in the brain structures following acute and chronic administration of antidepressants. Abbreviations: 2-AG, 2-arachidonoylglycerol; MAGL, monoacylglycerol lipase; PFCTX, prefrontal cortex; FCTX, frontal cortex; HIP, hippocampus; DSTR, dorsal striatum; NAc, nucleus accumbens; CER, cerebellum; IMI (15), imipramine (15 mg/kg); ESC (10), escitalopram (10 mg/kg); TIA (10), tianeptine (10 mg/kg). All data are expressed as the mean ± SEM. N = 8 rats/group. *P < 0.05 versus VEH (vehicle).

ESC treatment evoked a change of MAGL protein expression in the prefrontal cortex (F(2.23) = 6.435; P = 0.0078), where an increase in this protein expression (P < 0.05) was seen after chronic administration of ESC (Fig. 4).

Administration of TIA altered MAGL protein expression in the prefrontal cortex (F(2.23) = 4.171; P = 0.0325), with higher levels of MAGL protein expression (P < 0.05) being observed after acute and chronic TIA administration to rats (Fig. 4).

DISCUSSION

This research was aimed at analyzing the enzymes involved in the eCB metabolism in ex vivo rat brain tissue following acute and chronic administration of antidepressant drugs, what extends our previous research on the effects of the same treatment strategies on alterations in the eCB levels in the rat brain structures (21). Our current study indicates for the first time that antidepressant drugs modulate the eCB metabolizing enzymes expression in rats.

In the present study, an increase in NAPE-PLD and FAAH protein expression was evidenced in rats after TIA given acutely and chronically or IMI administered acutely. However, these changes did not induce the alteration in the anandamide levels in the prefrontal cortex (21). Moreover, IMI and TIA modulated the expression of either anandamide synthesizing or degrading enzymes, so changes in the tissue levels of anandamide may not occur in this case. It should be noted that NAPE-PLD and FAAH are also involved in the metabolism of other N-acylethanolamines (NAEs), and the observed changes in the expression of these enzymes may produce fluctuations in the NAEs levels (26). Interestingly, studies in animal models of depression documented an increase in FAAH levels with increased anandamide levels in the prefrontal cortex of bulbectomized rats (20), what may suggest that higher levels of FAAH protein expression are more characteristic for depression-like behavior.

Increased levels of NAPE-PLD protein expression after chronic ESC treatment were also observed in the hippocampus. In our previous research, the hippocampal anandamide levels were increased after chronic administration of all investigated antidepressants (21). The elevated anandamide levels were probably caused by the increased NAPE-PLD protein expression after chronic ESC treatment. Opposite changes were present in animal models of depression, where a reduction in the anandamide level caused by higher FAAH protein expression was seen in the hippocampus of Wistar Kyoto rats (27), bulbectomized rats (20) and rats subjected to chronic mild stress (CMS) (28). Therefore, chronic treatment with ESC induces changes in the eCB synthesizing enzymes and this modification is probably responsible for an antidepressant effect of this drug. The letter changes in the NAPE-PLD protein expression via reinforced eCB signaling may assert influence on the hippocampal neurogenesis (29) and reduction of excitotoxic damage, as well as they may have protective properties on the stress axis (30). On the other hand, the increased serotonin neurotransmission was seen after chronic ESC treatment with enhanced tonic activation of postsynaptic 5-HT1A receptors in the CA3 hippocampal pyramidal neurons and downregulation of postsynaptic 5-HT2A/C receptors (31). These adaptations link the fortnight antidepressant treatment with the onset of an antidepressant effect (32). Electrophysiological data showed that either URB597 (a FAAH inhibitor) or citalopram (a selective serotonin reuptake inhibitor) altered the functional states of 5-HT1A and 5-HT2A/C receptors in the hippocampus (33). Thus, the higher anandamide synthesis observed after ESC treatment in the hippocampus could reinforce the antidepressant-like effect of this drug. Additionally, it was shown that either direct CB1 receptor agonists or FAAH inhibitors increased neural serotonin activity (34-38) and serotonin efflux in the medial prefrontal cortex and the hippocampus (36). In the earlier study both acute and chronic IMI administration increased hippocampal anandamide levels (21). However, in this study no changes were observed in the hippocampal NAPE-PLD nor FAAH expression after IMI treatment. It should be emphasized that in the present research the expression, but not the activity, of enzymes was measured and thereby this discrepancy could be due to enzymatic activity changes.

In our research, chronic administration of IMI and ESC induced a reduction in the FAAH protein expression, while ESC administered chronically evoked an increase in NAPE-PLD protein expression in the dorsal striatum. Opposite changes, i.e. higher striatal FAAH levels, were seen in Wistar Kyoto rats (20). The latter changes probably might lead to an increase in the striatal anandamide levels after chronic administration of antidepressant drugs, which have been observed previously (21). Facilitating the eCB system within the striatum by chronic antidepressants administration may modulate the cortical symptom of depression, anhedonia, characterized by lower striatal anandamide level (17).

In this paper, we focused also on another eCB present in the central nervous system, 2-AG and its metabolism. An increase in MAGL protein expression was observed after chronic ESC treatment with higher 2-AG levels in the prefrontal cortex (21). MAGL protein expression was also increased in this structure after acute and chronic administration of TIA, with no change in the levels of 2-AG in the prefrontal cortex. However, the lack of changes in the 2-AG levels was provoked by an increase in the expression of the synthesizing enzyme DAGLα in this structure. Higher MAGL protein expression in the prefrontal cortex after antidepressants may provoke different modification of memory processes (39) or stress coping behaviors (40).

In addition, a decrease in MAGL protein expression and an increase in DAGLα protein expression were observed in the prefrontal cortex after acute and chronic administration of IMI, which might also induce a rise in the synthesis and reduction in the degradation of this eCB and its higher tissue levels (21). Additionally, TIA treatment evoked an increase in the DAGLα protein expression in the frontal cortex, which led to higher 2-AG tissue levels (21). eCB signal weakening within the frontal cortex has been shown in mood disorders (41). Therefore, the influence of IMI and TIA on the frontocortical enzymes involved in eCB catabolism may represent a potential antidepressant-like mechanism.

An increase in DAGLα protein expression with increased 2-AG levels (21) was seen after ESC treatment in the hippocampus as well as elevated DAGLα protein levels were observed in the dorsal striatum after chronic administration of ESC and TIA. The latter changes probably might lead to an increase in the striatal eCB levels after chronic administration of antidepressant drugs (21). Increased expression of DAGLα protein may produce antidepressant-like effects through enhancement of eCB-mTOR signaling (42).

A decrease in the DAGLα protein expression was shown after chronic administration of IMI, what probably induced a fall in the 2-AG levels in the cerebellum (21). This alteration may be related to a normalizing effect of IMI on long-term depression (LTD) in the cerebellum (43). DAGLα is a major enzyme responsible for 2-AG synthesis in nervous tissue, which was shown in DAGLα and DAGLβ knockout mice. However, both DAGLα and DAGLβ isoforms are expressed in the central nervous system (8, 44). DAGLβ is critical for the regulation of 2-AG levels in immune cells, and the effect of antidepressant drugs on the DAGLβ is unknown (8). Further research is needed to validate the relationship between antidepressants, DAGLβ, immune system dysregulation and depression.

In summary, the present study determined the effect of antidepressant drug administration on the eCB system biomarkers, i.e. enzymes involved in the eCB metabolism. The changes in the expression of eCBs metabolizing enzymes seem to be dependent on brain structure and/or properties of a drug. Our data must be interpreted with caution because antidepressants were given to naive rats without signs of depression. Thus, changes in the expression of eCB-metabolizing enzymes following antidepressant drugs may be used to provide pharmacological assessment of antidepressants under basal conditions. Further studies using animal models of depression, taking into account the correlation between eCB enzymatic changes and the antidepressant-like effect, will be required to ascertain whether similar results occur. However, this is the first report showing a potential role of antidepressant drugs in the modulation of the eCB metabolizing enzymes expression in rats. Collectively, these data provide a compelling argument that eCB signaling represents an important central player, which may be involved in the mechanism of action of antidepressants.

Abbreviations: 2-AG, 2-arachidonoylglycerol; CB, cannabinoid; CER, cerebellum; DAGLα, diacylglycerol lipase α; DSTR, dorsal striatum; eCB, endocannabinoid; ESC, escitalopram; FAAH, fatty acid amide hydrolase; FCTX, frontal cortex; HIP, hippocampus; IMI, imipramine; ip, intraperitoneal; MAGL, monoacylglycerol lipase; NAc, nucleus accumbens; NAPE-PLD, N-acyl phosphatidylethanolamine phospholipase D; PFCTX, prefrontal cortex; TIA, tianeptine

Acknowledgement: This study was supported by the research grant UMO-2012/05/B/NZ7/02589 from the National Science Centre, Cracow, Poland and by the statutory funds of the Maj Institute of Pharmacology, Polish Academy of Sciences (Cracow). Escitalopram was funded by Lundbeck.

Conflict of interest: None declared.

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