Effects of chronic clozapine administration on markers of arachidonic acid cascade and synaptic integrity in rat brain

Abstract

Background: The mode of action of clozapine, an atypical antipsychotic approved for treating schizophrenia (SZ) and used for bipolar disorder (BD) mania, remains unclear. We tested for overlap with the actions of the mood stabilizers, lithium, carbamazepine and valproate, which downregulate arachidonic acid (AA) cascade markers in rat brain and upregulate BDNF. AA cascade markers are upregulated in BD and SZ postmortem BD brain in association with neuroinflammation and synaptic loss, while BDNF is decreased.

Methods: Rats were injected intraperitoneally with a therapeutically relevant dose of clozapine (10 mg/kg/day) or with saline for 30 days, and AA cascade and synaptic markers and BDNF were measured in the brain.

Results: Compared with saline-injected rats, chronic clozapine increased brain activity, mRNA and protein levels of docosahexaenoic acid (DHA)-selective calcium-independent phospholipase A₂ type VIA (iPLA₂), mRNA and protein levels of BDNF and of the postsynaptic marker, drebrin, while decreasing cyclooxygenase (COX) activity and concentration of prostaglandin E₂ (PGE₂), a proinflammatory AA metabolite. Activity and expression of AA-selective calcium-dependent cytosolic cPLA₂ type IVA and of secretory sPLA₂ Type II were unchanged.

Conclusions: These results show overlap with effects of mood stabilizers with regard to downregulation of COX activity and PGE₂ and to increased BDNF and suggest a common action against the reported neuropathology of BD and SZ. The increased iPLA₂ expression following clozapine suggests increased production of anti-inflammatory DHA metabolites, and, with increased BDNF and drebrin, clear neuroprotective action.

Conflict of interest statement

Conflict of interest

Authors have no conflict of interest.

Figures

Figure 1. mRNA, protein and activity levels of PLA2 enzymes in brains of rats treated with clozapine

mRNA, protein l and activity levels of iPLA2 (A–C), cPLA2 (D–F) and sPLA2 (G–I). mRNA data are expressed as the relative level of PLA2 normalized to the endogenous control (â-globulin) using the ÄÄCT method. Protein levels are the ratios of optical density of PLA2 to â–actin, expressed as percent of control. Values are mean ± SD (n = 8 for both groups). *p < 0.05, **p < 0.01. C, control; CZ, clozapine.

Figure 1. mRNA, protein and activity levels of PLA2 enzymes in brains of rats treated with clozapine

mRNA, protein l and activity levels of iPLA2 (A–C), cPLA2 (D–F) and sPLA2 (G–I). mRNA data are expressed as the relative level of PLA2 normalized to the endogenous control (â-globulin) using the ÄÄCT method. Protein levels are the ratios of optical density of PLA2 to â–actin, expressed as percent of control. Values are mean ± SD (n = 8 for both groups). *p < 0.05, **p < 0.01. C, control; CZ, clozapine.

Figure 1. mRNA, protein and activity levels of PLA2 enzymes in brains of rats treated with clozapine

mRNA, protein l and activity levels of iPLA2 (A–C), cPLA2 (D–F) and sPLA2 (G–I). mRNA data are expressed as the relative level of PLA2 normalized to the endogenous control (â-globulin) using the ÄÄCT method. Protein levels are the ratios of optical density of PLA2 to â–actin, expressed as percent of control. Values are mean ± SD (n = 8 for both groups). *p < 0.05, **p < 0.01. C, control; CZ, clozapine.

Figure 2. mRNA and protein levels of COX-1 and -2 in the rat brain

mRNA and protein levels of COX-1 (A and C) and COX-2 (B and D). mRNA data are expressed as relative level of COXs normalized to endogenous control (â-globulin) using the ÄÄCT method. The protein level is the ratio of optical density of COX to â–actin, expressed as percent of control. Values are mean ± SD (n = 8 for both groups). **p < 0.01. C, control; CZ, clozapine.

Figure 3. Enzyme activity of COXs in the brains of rats treated with clozapine

COX activity was measured in the brain extracts of control rats or rats treated with clozapine. Data are expressed as percent of control. Values are mean ± SD (n = 8 for both groups). *p

Figure 4. mRNA and protein levels of 5-, 12-, and 15-LOX in the rat brain

mRNA and protein levels of 5-LOX (A and D), 12-LOX (B and E) and 15-LOX (C and F). mRNA data are expressed as the relative level of LOXs normalized to the endogenous control (â-globulin) using the ÄÄCT method. The protein level is the ratio of optical density of LOXs to â–actin, expressed as percent of control. Values are mean ± SD (n = 8 for both groups). *p < 0.05, ***p < 0.001. C, control; CZ, clozapine.

Figure 5. Levels of PGE2, TXB2 and LTB4 in rat brains

PGE2, TXB2, and LTB4 were extracted according to the Radin’s method (Radin, 1981) and analyzed using a polyclonal enzyme-linked immunosorbent assay. Values are mean ± SD (n = 8 for both groups). *p < 0.05, **p < 0.01.

Figure 6. mRNA and protein levels of BDNF in rat brains

mRNA data are expressed as relative levels of BDNF normalized to the endogenous control (â-globulin) using the ÄÄCT method. The protein levels were measured by an ELISA kit. Values are mean ± SD (n = 8 for both groups). *p < 0.05, **p < 0.01.

Figure 7. mRNA and protein levels of synaptophysin and drebrin in the rat brain

mRNA and protein levels of synaptophysin (A and C) and (B and D). mRNA data are expressed as relative levels of synaptophysin or drebrin normalized to the endogenous control (â-globulin) using the ÄÄCT method. The protein level is the ratio of optical density of synaptophysin or drebrin to â–actin, expressed as percent of control. Values are mean ± SD (n = 8 for both groups). *p < 0.05. C, control; CZ, clozapine.