Acute effects of combining citalopram and pindolol on regional brain serotonin synthesis in sham operated and olfactory bulbectomized rats

The olfactory bulbectomized (OBX) rat is considered to be a good model of the pathology of human depression and also of the functional actions of antidepressant drug therapy. It has been proposed that antidepressant effects of selective serotonin reuptake inhibitors (SSRIs) can be accelerated by blocking 5-HT_1A/B autoreceptors with pindolol. The underlying mechanism is thought to involve acute unrestricting of 5-HT release and, consequently, relatively enhanced 5-HT turnover throughout the forebrain serotonergic networks. The effect of this combination on 5-HT turnover in sham operated or OBX rats can be assessed at the level of 5-HT synthesis, a very important presynaptic step in serotonergic neurotransmission, using the α-[¹⁴C]methyl-l-tryptophan autoradiography method. In sham rats, acute citalopram (20 mg/kg) treatment increased synthesis at almost all serotonergic terminal regions but slightly decreased synthesis at serotonergic cell body regions (i.e. dorsal and median (not significant) raphe; ～16%). Combining pindolol (10 mg/kg) with citalopram further increased synthesis at many regions in sham rats (relative to treatment with only citalopram). In OBX rats, citalopram decreased synthesis at a few terminal regions and greatly decreased synthesis at the dorsal and median raphe (～45%; relative to OBX rats treated with saline). Combining pindolol with citalopram greatly increased synthesis at almost all regions in OBX rats (relative to treatment with only citalopram). These results suggest that acute citalopram effects result in elevated terminal 5-HT synthesis, but these effects are restrained by 5-HT_1A/B autoreceptor feedback to different degrees in sham and OBX rats. Moreover, 5-HT_1A/B autoreceptor feedback is stronger in OBX rats and may underlie the delay of SSRI effects in OBX rats and, correspondingly, in human depression. Pindolol acceleration and augmentation of SSRI antidepressant therapy for human depression may be mediated by attenuation of 5-HT_1A/B autoreceptor feedback, permitting unhindered SSRI effects on serotonergic terminals.     

Chronic valproate treatment decreases the in vivo turnover of arachidonic acid in brain phospholipids: a possible common effect of mood stabilizers

Both (Li(+)) and valproic acid (VPA) are effective in treating bipolar disorder, but the pathway by which either works, and whether it is common to both drugs, is not agreed upon. We recently reported, using an in vivo fatty acid model, that Li(+) reduces the turnover rate of the second messenger arachidonic acid (AA) by 80% in brain phospholipids of the awake rat, without changing turnover rates of docosahexaenoic or palmitic acid. Reduced AA turnover was accompanied by down-regulation of gene expression and protein levels of an AA-specific cytosolic phospholipase A(2) (cPLA(2)). To see if VPA had the same effect on AA turnover, we used our in vivo fatty acid model in rats chronically administered VPA (200 mg/kg, i.p. for 30 days). Like Li(+), VPA treatment significantly decreased AA turnover within brain phospholipids (by 28-33%), although it had no effect on cPLA(2) protein levels. Thus, both mood stabilizers, Li(+) and VPA have a common action in reducing AA turnover in brain phospholipids, albeit by different mechanisms.     

Brain arachidonic and docosahexaenoic acid cascades are selectively altered by drugs, diet and disease

Metabolic cascades involving arachidonic acid (AA) and docosahexaenoic acid (DHA) within brain can be independently targeted by drugs, diet and pathological conditions. Thus, AA turnover and brain expression of AA-selective cytosolic phospholipase A(2) (cPLA(2)), but not DHA turnover or expression of DHA-selective Ca(2+)-independent iPLA(2), are reduced in rats given agents effective against bipolar disorder mania, whereas experimental excitotoxicity and neuroinflammation selectively increase brain AA metabolism. Furthermore, the brain AA and DHA cascades are altered reciprocally by dietary n-3 polyunsaturated fatty acid (PUFA) deprivation in rats. DHA loss from brain is slowed and iPLA(2) expression is decreased, whereas cPLA(2) and COX-2 are upregulated, as are brain concentrations of AA and its elongation product, docosapentaenoic acid (DPA). Positron emission tomography (PET) has shown that the normal human brain consumes 17.8 and 4.6 mg/day, respectively, of AA and DHA, and that brain AA consumption is increased in Alzheimer disease patients. In the future, PET could help to determine how human brain AA or DHA consumption is influenced by diet, aging or disease.     

Regional brain serotonin synthesis is increased in the olfactory bulbectomy rat model of depression: an autoradiographic study

Serotonin synthesis rates were evaluated using alpha-[14C]methyl-l-tryptophan (alpha-MTrp) autoradiographic methods in olfactory bulbectomized (OBX) rats. They were significantly (p < 0.05) increased in the frontal (50%) and parietal (40%) cortices, superior olive (over 30%), and the substantia nigra (30%) in the OBX rats as compared to the sham operated animals. There were also increases in 5-hydroxytryptamine (5-HT) synthesis in some limbic areas: the cingulate (32%), the medial forebrain bundle (58%), the hippocampus (13-25%) and the thalamus (22-40%). The largest increase in 5-HT synthesis after OBX was observed in the sensory-motor cortex (67%). 5-HT synthesis rates were significantly decreased in the dorsal and medial raphe nuclei, but there was no significant change the ventral tegmental area and the locus coeruleus following OBX. These results indicate that olfactory bulbectomy causes an imbalance in 5-HT synthesis in some projection areas by disproportionally increasing 5-HT synthesis rates in specific brain regions and making more 5-HT available for neurotransmission. This imbalance in 5-HT synthesis and the subsequent elevation of tissue 5-HT may be responsible for the creation of non-physiological circuitry which may, in part, be reflected in the symptoms resembling human depression.     

Olfactory bulbectomy-induced changes in the reproductive behaviour of the south Indian gerbil, Tatera indica cuvieri

Male South Indian gerbils (T. indica cuvieri), both adults and weanlings, were olfactory bulbectomized (OBX) and changes in male reproductive behaviour were assessed. Consequent to OBX, majority of the adult males failed to ejaculate, and courtship behaviour has also been considerably reduced. All OBX weanlings were rendered incapable of ejaculation. However, maturational parameters, and organ weights (testes, epididymis and seminal vesicle) remained unchanged in OBX males.     

Mitochondrial dysfunction in neocortex and hippocampus of olfactory bulbectomized mice,a model of Alzheimer’s disease

Structural and functional impairments of mitochondria in brain tissues in the pathogenesis of Alzheimer’s disease (AD) cause energy deficiency, increased generation of reactive oxygen species (ROS), and premature neuronal death. However, the causal relations between accumulation of beta-amyloid (Aβ) peptide in mitochondria and mitochondrial dysfunction, as well as molecular mechanisms underlying deleterious effects of both these factors in sporadic AD, the most common form in humans, remain unknown. Here we used olfactory bulbectomized (OBX) mice of NMRI strain as a model for sporadic AD. Five weeks after surgery, the OBX mice developed major behavioral and biochemical features of AD neurodegeneration, including spatial memory loss, increased brain levels of Aβ, and energy deficiency. Mitochondria isolated from the neocortex and hippocampus of OBX mice displayed severe functional impairments, such as low NADH oxidation rate, reduced transmembrane potential, and decreased cytochrome c oxidase (complex IV) activity that correlated with high levels of soluble Aβ1-40. Mitochondria from OBX mice showed increased contents of lipid peroxidation products, indicative of the development of oxidative stress. We found that neurodegeneration caused by olfactory bulbectomy is accompanied by energy metabolism disturbances and oxidative stress in brain mitochondria similar to those occurring in transgenic animals–familial AD models and patients with sporadic AD. Therefore, OBX mice can serve as a valid AD model for investigating the mechanisms of AD neurodegeneration, drug testing, and development of therapeutic strategies for AD treatment.     

Olfactory bulbectomy attenuates cardiovascular sympathoexcitatory reflexes in rats

Bilateral removal of the olfactory lobes in rats produces a number of behavioral, endocrine, and neurochemical alterations in the brain. Little is known, however, regarding the effects of this treatment on cardiovascular function and autonomic reflexes. Male Sprague-Dawley rats underwent bilateral surgical ablation of the olfactory bulbs (n = 10) or were sham operated (n = 8). After 3 wk of recovery, animals were instrumented with femoral catheters and a lumbar sympathetic nerve recording electrode. After 24 h of recovery, cardiovascular responses to arterial baroreflex manipulation, air jet stress, and smoke exposure were recorded. Olfactory bulbectomized rats demonstrated attenuated sympathoexcitatory responses to hypotension, air jet stress, and smoke exposure, as well as elevated basal blood pressure, compared with sham-operated rats. These data indicate that the integrity of the olfactory bulbs in rats is important for the elicitation of normal cardiovascular and autonomic responses to a number of evocative stimuli.     

Intranasal administration of mitochondria improves spatial memory in olfactory bulbectomized mice

Here, we found that functionally active mitochondria isolated from the brain of NMRI donor mice and administrated intranasally to recipient mice penetrated the brain structures in a dose-dependent manner. The injected mitochondria labeled with the MitoTracker Red localized in different brain regions, including the neocortex and hippocampus, which are responsible for memory and affected by degeneration in patients with Alzheimer''s disease. In behavioral experiments, intranasal microinjections of brain mitochondria of native NMRI mice improved spatial memory in the olfactory bulbectomized (OBX) mice with Alzheimer’s type degeneration. Control OBX mice demonstrated loss of spatial memory tested in the Morris water maze. Immunocytochemical analysis revealed that allogeneic mitochondria colocalized with the markers of astrocytes and neurons in hippocampal cell culture. The results suggest that a non-invasive route intranasal administration of mitochondria may be a promising approach to the treatment of neurodegenerative diseases characterized, like Alzheimer''s disease, by mitochondrial dysfunction.     

A genetic rat model of depression,Flinders sensitive line,has a lower density of 5-HT1A receptors,but a higher density of 5-HT1B receptors,compared to control rats

Deficiencies in brain serotonergic neurotransmission, which is in part associated with the alteration of brain serotonin (5-HT) receptors, have been proposed as part of a neurochemical imbalance in affective disorders, including depression. The drugs used for the treatment of these disorders generally act through and/or on the serotonergic system. Different animal models of depression have provided researchers with tools to obtain a better understanding of drug actions and possibilities to obtain insight into the neurochemical bases of these disorders. The measurements of the 5-HT_1A and 5-HT_1B receptor densities in a rat model of depression, Flinders sensitive line (FSL) rats, and comparisons with Sprague–Dawley (SPD) and Flinders resistant line (FRL) rats, are reported here. The receptor sites were quantified by autoradiography in more than 25 distinct brain regions known to have relatively large densities of respective sites. Some brain regions (e.g., dental gyrus, septal nucleus) were divided into several parts, according to previously known subdivisions, because of a substantial heterogeneity of these receptors. The densities in the FSL rats (“depressed” rats) were compared statistically to those in the SPD rats. In addition, comparisons were made to the densities in the FRL rats (rats not showing depressive symptoms). Comparisons were performed with the SPD and FRL rats because both of these strains have been used as control animals in studies of FSL rats. The results show that the densities of 5-HT_1A receptors are not significantly different between the FSL and SPD rats, but they are significantly different from the FRL rats. 5-HT_1A receptor density is significantly higher in the FRL rats than the SPD rats. The 5-HT_1B receptors were significantly greater in the FSL rats than in either the SPD or FRL rats. In addition, the FRL rats have 5-HT_1B receptor densities significantly lower in many brain regions than the SPD rats. The data presented here, in addition to previously reported differences in regional synthesis between these strains and the effect of acute citalopram on synthesis, suggest that SPD rats are likely a more appropriate control than FRL rats, when studies of FSL rats are performed with drugs acting directly or indirectly on, or through, the brain serotonergic system. However, comparisons, particularly of neurochemical and/or biological parameters in FRL rats, may reveal new insight into the alterations of 5-HT neurotransmission in this animal model of depression and possibly human depression, as well as the elevation of symptoms with treatments. The data also suggest that there could be a different fraction of 5-HT_1A receptors in high and low affinity states in these strains, as well as the possibility of different intracellular signalling.     

Suppression of Neuroinflammatory and Apoptotic Signaling Cascade by Curcumin Alone and in Combination with Piperine in Rat Model of Olfactory Bulbectomy Induced Depression

Objectives

Bilateral destruction of the olfactory bulbs is known to cause behavioral changes analogous to symptoms of depression. Curcumin, a traditional Indian spice is currently being investigated in different psychiatric problems including depression. Dietary phytochemicals are currently used as an adjuvant therapy to accelerate their therapeutic efficacy. Therefore, the present study is an attempt to elucidate the neuroprotective mechanism of curcumin and its co-administration with piperine against olfactory bulbectomy induced depression in rats.

Methods

Rats undergone olfactory bulbs ablations were analyzed after post-surgical rehabilitation period of 2 weeks. Animals were then treated with different doses of curcumin (100, 200 and 400 mg/kg; p.o.), piperine (20 mg/kg; p.o.) and their combination daily for another 2 weeks. Imipramine (10 mg/kg; i.p.) served as a standard control. Various behavioral tests like forced swim test (FST), open field behaviour and sucrose preference test (SPT) were performed, followed by estimation of biochemical, mitochondrial, molecular and histopathological parameters in rat brain.

Results

Ablation of olfactory bulbs caused depression-like symptoms as evidenced by increased immobility time in FST, hyperactivity in open field arena, and anhedonic like response in SPT along with alterations in mitochondrial enzyme complexes, increased serum corticosterone levels and oxidative damage. These deficits were integrated with increased inflammatory cytokines (TNF-α) and apoptotic factor (caspase-3) levels along with a marked reduction in neurogenesis factor (BDNF) in the brain of olfactory bulbectomized (OBX) rats. Curcumin treatment significantly and dose-dependently restored all these behavioral, biochemical, mitochondrial, molecular and histopathological alterations associated with OBX induced depression. Further, co-administration of piperine with curcumin significantly potentiated their neuroprotective effects as compared to their effects alone.

Conclusions

The present study highlights that curcumin along with piperine exhibits neuroprotection against olfactory bulbectomy induced depression possibly by modulating oxidative-nitrosative stress induced neuroinflammation and apoptosis.     

Stimulation of the Sigma-1 Receptor by DHEA Enhances Synaptic Efficacy and Neurogenesis in the Hippocampal Dentate Gyrus of Olfactory Bulbectomized Mice

Dehydroepiandrosterone (DHEA) is the most abundant neurosteroid synthesized de novo in the central nervous system. We previously reported that stimulation of the sigma-1 receptor by DHEA improves cognitive function by activating calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C and extracellular signal-regulated kinase in the hippocampus in olfactory bulbectomized (OBX) mice. Here, we asked whether DHEA enhances neurogenesis in the subgranular zone of the hippocampal dentate gyrus (DG) and improves depressive-like behaviors observed in OBX mice. Chronic treatment with DHEA at 30 or 60 mg/kg p.o. for 14 days significantly improved hippocampal LTP impaired in OBX mice concomitant with increased CaMKII autophosphorylation and GluR1 (Ser-831) phosphorylation in the DG. Chronic DHEA treatment also ameliorated depressive-like behaviors in OBX mice, as assessed by tail suspension and forced swim tests, while a single DHEA treatment had no affect. DHEA treatment also significantly increased the number of BrdU-positive neurons in the subgranular zone of the DG of OBX mice, an increase inhibited by treatment with NE-100, a sigma-1 receptor antagonist. DHEA treatment also significantly increased phosphorylation of Akt (Ser-473), Akt (Ser-308) and ERK in the DG. Furthermore, GSK-3β (Ser-9) phosphorylation increased in the DG of OBX mice possibly accounting for increased neurogenesis through Akt activation. Finally, we confirmed that DHEA treatment of OBX mice increases the number of BrdU-positive neurons co-expressing β-catenin, a downstream GSK-3βtarget. Overall, we conclude that sigma-1 receptor stimulation by DHEA ameliorates OBX-induced depressive-like behaviors by increasing neurogenesis in the DG through activation of the Akt/GSK-3β/β-catenin pathway.     

Gabapentin's minimal action on markers of rat brain arachidonic acid metabolism agrees with its inefficacy against bipolar disorder

In rats, FDA-approved mood stabilizers used for treating bipolar disorder (BD) selectively downregulate brain markers of the arachidonic acid (AA) cascade, which are upregulated in postmortem BD brain. Phase III clinical trials show that the anticonvulsant gabapentin (GBP) is ineffective in treating BD. We hypothesized that GBP would not alter the rat brain AA cascade. Chronic GBP (10mg/kg body weight, injected i.p. for 30 days) compared to saline vehicle did not significantly alter brain expression or activity of AA-selective cytosolic phospholipase A(2) (cPLA(2)) IVA or secretory (s)PLA(2) IIA, activity of cyclooxygenase-2, or prostaglandin E(2) or thromboxane B(2) concentrations. Plasma esterified and unesterified AA concentration was unaffected. These results, taken with evidence of an upregulated AA cascade in the BD brain and that approved mood stabilizers downregulate the rat brain AA cascade, support the hypothesis that effective anti-BD drugs act by targeting the brain AA cascade whereas ineffective drugs (such as GBP) do not target this pathway, and suggest that the rat model might be used for screening new anti-BD drugs.     

Functional recovery after olfactory bulbectomy in rats: effect of embryonal brain grafts

Regeneration of olfactory receptor neurones after bulbectomy can lead to formation of extrabulbar synapses, the functional significance of which remains controversial. Adult hooded rats (n = 27) were bilaterally bulbectomized under pentobarbital anaesthesia. Small pieces of brain tissues (1-2 mm3; OB: olfactory bulb; St: corpus striatum) were obtained from embryos of the same strain and placed into the bulbectomy-produced cavity in contact with the exposed brain surface. Smell was tested at 2- to 3-week intervals from 7 weeks to 7 months after the operation. The latency to find hidden food gradually improved and attained the intact control level in bulbectomized rats without grafts, but remained poor in the OB and St transplanted groups. Seven to ten months after transplantation, spontaneous unit activity and unit reactions to amyl acetate vapours were examined with a carbon fibre microelectrode. Unit responses in the transplants resembled those in the normal OB, but were less pronounced. Morphological examination of the transplant and of its connections with the olfactory receptor neurones and with the host brain suggested that functional recovery was mediated by the connections of the olfactory axons with the remnants of the OB, with the anterior olfactory nucleus and/or with the frontal cortex. The adverse effect of OB and St transplants was probably due to their poor connectivity with the host brain which prevented the regenerating olfactory axons from reaching higher olfactory centres.     

Effect of immobilization stress on serotonin content and turnover in regions of the rat brain.

Sprague-Dawley rats were stressed by immobilization from 30 to 300 minutes and the effects on serotonin (5-HT) and 5-hydroxy-indoleacetic acid (5-HIAA) content were determined in the cerebral cortex, diencephalon, striatum, hippocampus and the brain stem. In a subsequent study 5-HT turnover rate in these brain areas was estimated by measuring 5-HIAA accumulation 0, 30, 60 and 90 minutes after probenecid. The content of 5-HIAA and the turnover rate of 5-HT were significantly increased in the cerebral cortex shortly after the onset of immobilization. The content of 5-HIAA in the brainstem was increased by immobilization although 5-HT turnover rate was not increased. Short term increases in 5-HIAA content were observed in the striatum and hippocampus. However, no significant changes in 5-HT turnover rate were observed in either of these 2 brain areas. Immobilization did not affect 5-HIAA content or 5-HT turnover in the diencephalon. The sensitivity of the serotonergic system in the cerebral cortex to immobilization stress suggests that this brain region could be used in future studies of the interrelationships between stress and the brain serotonergic system.     

Decreased calcium/calmodulin-dependent protein kinase II and protein kinase C activities mediate impairment of hippocampal long-term potentiation in the olfactory bulbectomized mice

Olfactory bulbectomized (OBX) mice showed significant impairment of learning and memory-related behaviors 14 days after olfactory bulbectomy, as measured by passive avoidance and Y-maze tasks. We here observed a large impairment of hippocampal long-term potentiation (LTP) in the OBX mice. Concomitant with decreased acetylcholinesterase expression, protein kinase C (PKC)alpha autophosphorylation and NR1(Ser-896) phosphorylation significantly decreased in the hippocampal CA1 region of OBX mice. Both PKCalpha and NR1(Ser-896) phosphorylation significantly increased following LTP in the control mice, whereas increases were not observed in OBX mice. Like PKC activities, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in the hippocampal CA1 region of OBX mice as compared with that of control mice. In addition, increased CaMKII autophosphorylation following LTP was not observed in OBX mice. Finally, the impairment of CaMKII autophosphorylation was closely associated with reduced pGluR1(Ser-831) phosphorylation, without change in synapsin I (site 3) phosphorylation in the hippocampal CA1 region of OBX mice. Taken together, in OBX mice NMDA receptor hypofunction, possibly through decreased PKCalpha activity, underlies decreased CaMKII activity in the post-synaptic regions, thereby impairing LTP induction in the hippocampal CA1 region. Both decreased PKC and CaMKII activities with concomitant LTP impairment account for the learning disability observed in OBX mice.     

The behavioral and biochemical sequelae of the removal of the olfactory bulbs in C57Bl/6j mice]

Ablation of the olfactory bulbs in mice C57Bl/6j was accompanied by motor and orienting-exploratory activity augmentation in the "open field" test and deterioration of the learning ability to active and passive avoidance. The most expressed behavioural changes developed in four weeks after the surgery. Chronic administration of antidepressants (amitriptyline, 20 mg/kg; trazodone, 20 mg/kg; imipramine, 10 mg/kg; intraperitoneally) normalized behaviour of bulbectomized animals, trazodone being the most effective. In the biochemical studies the brainstem serotonin level was found to be decreased and the density of 5HT2-receptors in the cerebral cortex increased in bulbectomized mice. Only trazodone was able to correct the biochemical indices. The state of the bulbectomized mice is supposed to serve a model of a depression with hypo-function of serotonergic system of the brain.     

Arachidonic acid as a retrograde signal controlling growth and dynamics of retinotectal arbors

In the developing visual system, correlated presynaptic activity between neighboring retinal ganglion cells (RGC) stabilizes retinotopic synapses via a postsynaptic NMDAR (N-methyl-D-aspartate receptor)-dependent mechanism. Blocking NMDARs makes individual axonal arbors larger, which underlies an unsharpened map, and also increases branch turnover, as if a stabilizing factor from the postsynaptic partner is no longer released. Arachidonic acid (AA), a candidate retrograde stabilizing factor, is released by cytoplasmic phospholipase A2 (cPLA2) after Ca(2+) entry through activated NMDARs, and can activate presynaptic protein kinase C to phosphorylate various substrates such as GAP43 to regulate cytoskeletal dynamics. To test the role of cPLA2 in the retinotectal system of developing zebrafish, we first used PED6, a fluorescent reporter of cPLA2 activity, to show that 1-3 min of strobe flashes activated tectal cPLA2 by an NMDAR-dependent mechanism. Second, we imaged the dynamic growth of retinal arbors during both local inhibition of tectal cPLA2 by a pharmacological inhibitor, arachidonic tri-fluoromethylketone, and its suppression by antisense oligonucleotides (both injected intraventricularly). Both methods produced larger arbors and faster branch dynamics as occurs with blocking NMDARs. In contrast, intraocular suppression of retinal cPLA2 with large doses of antisense oligos produced none of the effects of tectal cPLA2 inhibition. Finally, if AA is the retrograde messenger, the application of exogenous AA to the tectum should reverse the increased branch turnover caused by blocking either NMDARs or cPLA2. In both cases, intraventricular injection of AA stabilized the overall branch dynamics, bringing rates down below the normal values. The results suggest that AA generated postsynaptically by cPLA2 downstream of Ca(2+) entry through NMDARs acts as a retrograde signal to regulate the dynamic growth of retinal arbors.     

Hydrogen peroxide generated at the level of mitochondria in response to peroxynitrite promotes U937 cell death via inhibition of the cytoprotective signalling mediated by cytosolic phospholipase A2

We have studied the relationships existing between delayed formation of H2O2 and activation of cytosolic phospholipase A2 (cPLA2), events respectively promoting toxicity or survival in U937 cells exposed to peroxynitrite. The outcome of an array of different approaches using phospholipase A2 inhibitors, or cPLA2 antisense oligonucleotides, as well as specific respiratory chain inhibitors and respiration-deficient cells led to the demonstration that H2O2 does not mediate toxicity by producing direct molecular damage. Rather, the effects of H2O2 were found to be upstream to the arachidonic acid (AA)-mediated cytoprotective signalling and in fact causally linked to inhibition of cPLA2. Thus, it appears that U937 cells exposed to nontoxic concentrations of peroxynitrite are nevertheless committed to death, which however is normally prevented by the activation of parallel pathways resulting in cPLA2-dependent release of AA. A rapid necrotic response, however, takes place when high concentrations of peroxynitrite promote formation of H2O2 at levels impairing the cPLA2 cytoprotective signalling.     

The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50-60% decrease in mRNA and protein levels of COX-2, but no change in these levels in COX-1 or in PGE synthase. Brain 5-lipoxygenase (5-LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+-dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX-2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX-2-derived eicosanoid production in mouse brain.