Synaptic lipid signaling

significance of polyunsaturated fatty acids and platelet-activating factor
  • Nicolas G. Bazan
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Louisiana State University Neuroscience Center of Excellence and Department of Ophthalmology, Louisiana State University Health Sciences Center, New Orleans, LA
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      Neuronal cellular and intracellular membranes are rich in specialized phospholipids that are reservoirs of lipid messengers released by specific phospholipases and stimulated by neurotransmitters, neurotrophic factors, cytokines, membrane depolarization, ion channel activation, etc. Secretory phospholipases A2 may be both intercellular messengers and generators of lipid messengers. The highly networked nervous system includes cells (e.g., astrocytes, oligodendrocytes, microglial cells, endothelial microvascular cells) that extensively interact with neurons; several lipid messengers participate in these interactions. This review highlights modulation of postsynaptic membrane excitability and long-term synaptic plasticity by cyclooxygenase-2-generated prostaglandin E2, arachidonoyldiacylcylglycerol, and arachidonic acid-containing endocannabinoids. The peroxidation of docosahexaenoic acid (DHA), a critical component of excitable membranes in brain and retina, is promoted by oxidative stress. DHA is also the precursor of enzyme-derived, neuroprotective docosanoids. The phospholipid platelet-activating factor is a retrograde messenger of long-term potentiation, a modulator of glutamate release, and an upregulator of memory formation. Lipid messengers modulate signaling cascades and contribute to cellular differentiation, function, protection, and repair in the nervous system.
      Lipidomic neurobiology will advance our knowledge of the brain, spinal cord, retina, and peripheral nerve function and diseases that affect them, and new discoveries on networks of signaling in health and disease will likely lead to novel therapeutic interventions.
      The highly networked organization of the nervous system encompasses a variety of cells that display, as a distinctive feature, one of the largest membrane surface areas of all cells. This would be very clearly illustrated if the plasma membrane from a neuron (e.g., hippocampal CA1 or Purkinje cell of the cerebellum) could be spread out on a flat surface: the branching and complexities of dendrites that form the dendritic spines are very extensive, largely comprising postsynaptic elements. An important additional feature of neuronal dendrites is that they undergo dramatic changes in shape and length during development and learning; they are also affected in aging and in several pathologic conditions. Astrocytes, oligodendrocytes, and microglial cells have very large plasma membrane surface areas as well. To add a further level of complexity, all of these cells extensively interact with neurons. How membranes are organized has conceptually evolved from the lipid bilayer with embedded proteins to a highly dynamic, heterogeneous patchwork of microdomains that contain ion channels, receptors, transporters, and other proteins. Cellular membranes in the nervous system were divided in the past into relatively more fluid membranes (e.g., those of cells of gray matter) and relatively more rigid membranes (e.g., oligodendrocyte plasma membrane that spirals around the axon to form the myelin), according to the higher or lower content of PUFAs in phospholipids. Currently, in neurons, glia, and endothelial cells of the cerebrovasculature, several phospholipid pools are increasingly being recognized as reservoirs of lipid messengers. Specific lipid messengers are cleaved from these reservoir phospholipids by phospholipases upon stimulation by neurotransmitters, neurotrophic factors, cytokines, membrane depolarization, ion channel activation, etc. Lipid messengers regulate and interact with multiple other signaling cascades, contributing to the development, differentiation, function, protection, and repair of the cells of the nervous system
      To highlight the significance of PUFA and of a bioactive phospholipid, platelet-activating factor (PAF), we provide here examples of lipid signaling in synaptic plasticity and memory, as well as in pathologic conditions relevant to retinal degeneration, stroke, epilepsy, and Alzheimer's disease. This article describes certain aspects of a field that is rapidly growing and does not represent a complete account. Only some of the several contributions made to this theme are discussed: omission of papers was unavoidable due to the relative brevity of this article and the large scope of this field.

      CYCLOOXYGENASE-2-GENERATED PROSTAGLANDIN E2 MODULATES POSTSYNAPTIC MEMBRANE EXCITABILITY AND LONG-TERM SYNAPTIC PLASTICITY IN HIPPOCAMPAL NEURONS

      Brain contains various cyclooxygenases (COXs) for the enzymatic conversion of free arachidonic acid (AA) into PGH2, the precursor of prostaglandins, thromboxanes, and prostacyclin. COX-1 is thought to be constitutively expressed, whereas COX-2 is both constitutive as well as inducible. COX-3 is a third isoenzyme that is highly expressed in nervous tissue. This enzyme seems to be generated by COX-1 intron-1 retention. Partial COX-1a and b proteins were also reported as additional COXs (
      • Chandrasekharan N.V.
      • Dai H.
      • Roos K.L.
      • Evanson N.K.
      • Tomsik J.
      • Elton T.S.
      • Simmons D.L.
      COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression.
      ,
      • Bazan N.G.
      • Flower R.J.
      Lipids signals in pain control.
      ).
      Basal, “constitutive” COX-2 expression is regulated by synaptic activity and is up-regulated by high-frequency stimulation associated with long-term synaptic plasticity (
      • Yamagata K.
      • Andreasson K.I.
      • Kaufmann W.E.
      • Barnes C.A.
      • Worley P.F.
      Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids.
      ). Furthermore, COX-2 is enriched in dendritic spines in excitatory synapses (
      • Kaufmann W.E.
      • Worley P.F.
      • Pegg J.
      • Bremer M.
      • Isakson P.
      COX-2, a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex.
      ). These findings implicate both constitutive and inducible COX-2 in synaptic plasticity.
      More direct evidence on the significance of COX-2 in synaptic plasticity was obtained using COX-2 inhibitors and prostaglandins in hippocampal dentate granule neurons (
      • Chen C.
      • Magee J.C.
      • Bazan N.G.
      Cyclooxygenase-2 regulates prostaglandin E2 signaling in hippocampal long-term synaptic plasticity.
      ). These studies demonstrated that selective COX-2 inhibitors, but not COX-1 inhibitors, decrease induction of long-term potentiation (LTP) in hippocampal dentate granule neurons. Prostaglandin E2 (PGE2), but not PGD2 or PGF2α, reverses COX-2 inhibitor-induced suppression of LTP. This is in agreement with the observation that COX-2 preferentially channels the conversion of AA toward PGE2 and prostacyclin (
      • Brock T.G.
      • McNish R.W.
      • Peters-Golden M.
      Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2.
      ). PGE2 receptors are expressed in hippocampus, implying that the action of COX-2 inhibition on LTP induction in dentate neurons results from blocking the synthesis of PGE2. Therefore, it is very likely that PGE2 is the effector of COX-2-mediated activity-dependent synaptic plasticity (
      • Chen C.
      • Magee J.C.
      • Bazan N.G.
      Cyclooxygenase-2 regulates prostaglandin E2 signaling in hippocampal long-term synaptic plasticity.
      ).
      The phospholipid messenger PAF has been proposed as a retrograde messenger of LTP (
      • Kato K.
      • Clark G.D.
      • Bazan N.G.
      • Zorumski C.F.
      Platelet activating factor as a potential retrograde messenger in Ca1 hippocampal long-term potentiation.
      ) and a presynaptic site of action has been evidenced (see below). The action of PGE2 on synaptic modification, however, is not on presynaptic endings, but rather on postsynaptic sites. PGE2 regulates membrane excitability by modulating K+, hyperpolarization-activated current, and tetrodotoxin-resistant Na+-channel currents in sensory neurons (
      • Gold M.S.
      • Levine J.D.
      • Correa A.M.
      Modulation of TTX-R INa by PKC and PKA and their role in PGE2-induced sensitization of rat sensory neurons in vitro.
      ,
      • Ingram S.L.
      • Williams J.T.
      Modulation of the hyperpolarization-activated current (Ih) by cyclic nucleotides in guinea-pig primary afferent neurons.
      ,
      • Nicol G.D.
      • Vasko M.R.
      • Evans A.R.
      Prostaglandins suppress an outward potassium current in embryonic rat sensory neurons.
      ). Moreover, spatiotemporal correlation of coincidence of postsynaptic firing and excitatory postsynaptic potentials are key in synaptic efficacy (
      • Bi G.Q.
      • Poo M.M.
      Synaptic modification by correlated activity: Hebb's postulate revisited.
      ,
      • Koester H.
      • Sakmann B.
      Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials.
      ,
      • Magee J.C.
      • Johnston D.
      A synaptically controlled, associative signal for Hebbian plasticity in hippocampal neurons.
      ,
      • Markram H.
      • Lübke J.
      • Frotscher M.
      • Sakmann B.
      Regulation of synaptic efficacy by coincidence of postsynaptic APs and EPSPs.
      ). In fact, decreasing postsynaptic neuron membrane excitability, which leads to a decrease in the number of postsynaptic action potentials during high-frequency stimulation of the presynaptic ending, decreases the probability of LTP induction. It was found that COX-2 inhibition attenuates postsynaptic membrane excitability. Thus, PGE2 derived from the tonic activity of constitutive COX-2 participates in neuronal membrane excitability. One other important factor in LTP is the active propagation of axonally generated action potentials back into the dendrites, where associated dendritic influx plays a significant role (
      • Magee J.C.
      • Johnston D.
      A synaptically controlled, associative signal for Hebbian plasticity in hippocampal neurons.
      ). Pharmacologic COX-2 blocking decreases back-propagating dendritic action potential-associated Ca2+ influx (
      • Chen C.
      • Magee J.C.
      • Bazan N.G.
      Cyclooxygenase-2 regulates prostaglandin E2 signaling in hippocampal long-term synaptic plasticity.
      ). Overall, constitutively inducible COX-2 regulates membrane excitability and activity-dependent LTP induction in hippocampus.
      Depolarizing concentrations of K+ promote active release of AA in cortical neurons (
      • Taylor A.L.
      • Hewett S.J.
      Potassium-evoked glutamate release liberates arachidonic acid from cortical neurons.
      ), retina (
      • Birkle D.L.
      • Bazan N.G.
      Effects of K+ depolarization on the synthesis of prostaglandins and hydroxyeicosatetra(5,8,ll,l4) enoic acids (HETE) in the rat retina. Evidence for esterification of l2-HETE in lipids.
      ), and synaptosomes (
      • Birkle D.L.
      • Bazan N.G.
      Effect of bicuculline-induced status epilepticus on prostaglandins and hydroxyeicosatetraenoic acids in rat brain subcellular fractions.
      ). One interpretation of these results is that high K+ promotes a depolarization-dependent Ca2+ influx through voltage-gated Ca2+ channels. This in turn activates presynaptic glutamate release that modulates excitatory glutamate receptors (
      • Taylor A.L.
      • Hewett S.J.
      Potassium-evoked glutamate release liberates arachidonic acid from cortical neurons.
      ). Thus Ca2+ influx through the N-methyl-d-aspartate (NMDA) receptor, and direct Ca2+ entrance through voltage-dependent calcium channels, activate phospholipase A2 (PLA2) to result in AA release via phospholipid hydrolysis. In addition, glutamate may activate metabotropic receptors that, through phospholipase C (PLC) activation, generate diacylglycerol.
      Neuronal COX-2 expression is also up-regulated in experimental epileptogenesis, when aberrant synaptic plasticity is thought to be activated. The experimental kindling model resembles aspects of mesial temporal lobe epilepsy (
      • Lothman E.W.
      • Williamson J.M.
      Rapid kindling with recurrent hippocampal seizures.
      ). Kindling epileptogenesis is triggered by repeated subconvulsive stimulation, which gradually results in intensified seizures. Both COX-2 and cPLA2 expression are activated, indicating that the free AA released is converted into prostaglandins during epileptogenesis (
      • Tu B.
      • Bazan N.G.
      Hippocampal kindling epileptogenesis upregulates neuronal cyclooxygenase-2 expression in neocortex.
      ). Nimesulide, a COX-2 blocker, decreases kindling epileptogenesis. The inability of nimesulide to completely inhibit kindling development suggests either a limited bioavailability of the drug to neuronal COX-2 to attain full blockade and/or a redundancy of the signaling involved. For example COX-1, which is not inhibited by nimesulide, may catalyze the synthesis of prostaglandins, minimizing the action of nimesulide. The exact mechanism by which COX-2 inhibition attenuates kindling epileptogenesis is not understood. Notwithstanding, COX-2 inhibition may diminish prostaglandin(s) and/or PAF synthesis, lipid messengers that are both involved in synaptic facilitation (
      • Bazan N.G.
      • Packard M.G.
      • Teather L.
      • Allan G.
      Bioactive lipids in excitatory neurotransmission and neuronal plasticity.
      ). Moreover, kindling epileptogenesis promotes selective neuronal COX-2 expression, initially in the hippocampus and subsequently in the neocortex. The stimulating electrode in this particular experiment was placed in the ventral hippocampus. Taken together, these studies suggest that the spreading of kindling-induced COX-2 expression from hippocampal neurons to neocortical neurons is a major event in the permanent facilitation of aberrant functional connectivity, and that COX-2 is a mediator (
      • Tu B.
      • Bazan N.G.
      Hippocampal kindling epileptogenesis upregulates neuronal cyclooxygenase-2 expression in neocortex.
      ). Kindling is also a model of bipolar disorders, and valproate, a mood stabilizer, when chronically administered to rats, down-regulates AA conversion into prostaglandins by decreasing COX brain expression (
      • Bosetti F.
      • Weerasinghe G.R.
      • Rosenberger T.A.
      • Rapoport S.I.
      Valproic acid down regulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase-1 and -2 in rat brain.
      ). Lithium, also used to treat bipolar disorders, potentiates brain AA signaling (
      • Basselin M.
      • Chang L.
      • Seemann R.
      • Bell J.M.
      • Rapoport S.I.
      Chronic lithium administration potentiates brain arachidonic acid signaling at rest and during cholinergic activation in awake rats.
      ).
      Astrocytes are also active in the synthesis of prostaglandins, particularly in response to interleukin-1β(IL-1β) (
      • O'Banion M.K.
      • Miller J.C.
      • Chang J.W.
      • Kaplan M.D.
      • Coleman P.D.
      Interleukin-1 β induces prostaglandin G/H synthase 2 (cyclooxygenases-2) in primary murine astrocyte cultures.
      ). Astrocytes form an intimate sheath around synapses and increase their intracellular Ca2+ content when excitatory neurotransmission is activated (
      • Nedergaard M.
      Direct signaling from astrocytes to neurons in cultures of mammalian brain cells.
      ). As a consequence, there is a calcium-dependent glutamate release from astrocytes (
      • Bezzi P.
      • Carmignoto G.
      • Pasti L.
      • Vesce S.
      • Rossi D.
      • Rizzini B.L.
      • Pozzan T.
      • Volterra A.
      Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.
      ), an event that is activated by prostaglandins (Fig. 1). Thus, these lipid messengers may be an intercellular signal of an integrated glutamatergic cross-talk between neurons and astrocytes (
      • Bezzi P.
      • Carmignoto G.
      • Pasti L.
      • Vesce S.
      • Rossi D.
      • Rizzini B.L.
      • Pozzan T.
      • Volterra A.
      Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.
      ). At present there are several evolving concepts on the nature of the signaling that allows neurons and astrocytes to communicate. In fact, this communication is bidirectional, and also has consequences on activity-dependent modulation of cerebral blood flow (
      • Anderson C.M.
      • Nedergaard M.
      Astrocyte-mediated control of cerebral microcirculation.
      ,
      • Zonta M.
      • Angulo M.C.
      • Gobbo S.
      • Rosengarten B.
      • Hossmann K.A.
      • Pozzan T.
      • Carmignoto G.
      Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation.
      ) through multiple astrocyte signaling, including possible cytochrome P450 epoxygenase, which synthesizes epoxyeicosatrienoic acids that in turn elicit vasodilatory properties on the cerebral microcirculation (
      • Harder D.R.
      • Alkayed N.J.
      • Lange A.R.
      • Gebremedhin D.
      • Roman R.J.
      Functional hyperemia in the brain: hypothesis for astrocyte-derived vasodilator metabolites.
      ). Astrocytes also release AA in response to oxidative stress by activation of cPLA2 and Ca2+-independent PLA2 (iPLA2) (
      • Xu J.
      • Sun A.Y.
      • Sun G.Y.
      Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2).
      ). These cells have complex regulatory mechanisms for cPLA2 activation involving protein kinase C (PKC) and MAP kinases (
      • Xu J.
      • Weng Y.I.
      • Simonyi A.
      • Krugh B.W.
      • Liao Z.
      • Weisman G.A.
      • Sun G.Y.
      • Simoni A.
      Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachidonic acid release in primary murine astrocytes.
      ). Moreover, the astrocytes' response to proinflammatory cytokines includes induction of secretory PLA2 (sPLA2) expression (
      • Tong W.
      • Shah D.
      • Xu J.
      • Diehl J.A.
      • Hans A.
      • Hannink M.
      • Sun G.Y.
      Involvement of lipid mediators on cytokine signaling and induction of secretory phospholipase A2 in immortalized astrocytes (DITNC).
      ).
      Figure thumbnail gr1
      Fig. 1Simplified cartoon of phospholipases A2 at the synapse. A depolarizing stimulus at the presynaptic terminal triggers glutamate release. Glutamate binds to the N-methyl-d-aspartate receptor and as a consequence an influx of calcium in the postsynaptic neuron occurs. Calcium-mediated activation of the cytoplasmic PLA2 results in the release of arachidonic acid (AA), docosahexaenoic acid (DHA), and lyso-platelet-activating factor (PAF), the PAF precursor. Although PAF has a very short biologic half-life, on repeated stimulus sufficient PAF accumulates to diffuse back across the synaptic cleft. Experimental evidence for this was provided by injecting PAF into the postsynaptic neuron and monitoring neurotransmitter release (
      • Kato K.
      • Clark G.D.
      • Bazan N.G.
      • Zorumski C.F.
      Platelet activating factor as a potential retrograde messenger in Ca1 hippocampal long-term potentiation.
      ). PAF binds to its presynaptic receptor and enhances glutamate exocytosis by an as yet undefined mechanism. During synaptic plasticity events, PAF may also activate gene expression that in turn is probably involved in long-term alterations of synaptic function (not shown in this diagram). Cell surface PAF-receptor antagonists confer neuroprotection during ischemia-reperfusion and inhibit PAF-induced glutamate release from hippocampal neurons and CA1 long-term potentiation formation, presumably through the same mechanism. The inhibitory effects of this antagonist on glutamate release could account in part for its neuroprotection in ischemia reperfusion. Secretory PLA2 (sPLA2) may be released from the presynaptic terminal (
      • Matsuzawa A.
      • Makoto M.
      • Atsumi G.
      • Imal K.
      • Prados P.
      • Inoue K.
      • Kudo I.
      Release of secretory phospholipase A2 from rat neuronal cells and its possible function in the regulation of catecholamine secretion.
      ): sPLA2 binding sites are present in neurons (
      • Lambeau G.
      • Barhanin J.
      • Schweitz H.
      • Qar J.
      • Lazdunski M.
      Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom.
      ,
      • Lambeau G.
      • Schmid-Alliana A.
      • Lazdunski M.
      • Barhanin J.
      Identification and purification of a very high affinity binding protein for toxic phospholipases A2 in skeletal muscle.
      ,
      • Lambeau G.
      • Ancian P.
      • Barhanin J.
      • Lazdunski M.
      Cloning and expression of a membrane receptor for secretory phospholipases A2.
      ,
      • Lambeau G.
      • Ancian P.
      • Nicolas J-P.
      • Beiboer S.H.W.
      • Moinier D.
      • Verheij H.
      • Lazdunski M.
      Structural elements of secretory phospholipases A2 involved in the binding to M-type receptors.
      ), and sPLA2 promotes active AA remodeling in neurons in culture (
      • Kolko M.
      • DeCoster M.A.
      • Rodriguez de Turco E.B.
      • Bazan N.G.
      Synergy by secretory phospholipase A2 and glutamate on inducing cell death and sustained arachidonic acid metabolic changes in primary cortical neuronal cultures.
      ) and may also promote DHA release. DHA may also be released by cPLA2. Free DHA may subsequently follow enzyme-mediated oxygenation pathways and lead to the synthesis of docosanoids, messengers made in the retina (
      • Bazan N.G.
      • Birkle D.L.
      • Reddy T.S.
      Docosahexaenoic acid (22:6, n 3) is metabolized to lipoxygenase reaction products in the retina.
      ) and brain (
      • Marcheselli V.L.
      • Hong S.
      • Lukiw W.J.
      • Tian X.H.
      • Gronert K.
      • Musto A.
      • Hardy M.
      • Gimenez J.M.
      • Chiang N.
      • Serhan C.N.
      • Bazan N.G.
      Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression.
      ). Free radicals would accumulate during oxidative stress. Downstream lipid signaling modulates neuronal function and survival. Synapses are intimately surrounded by astrocytes, which express glutamate transporters that remove the excitatory neurotransmitter from the vicinity of the synaptic cleft. Astrocytes also respond to prostaglandins by releasing glutamate through a Ca2+-dependent mechanism (
      • Bezzi P.
      • Carmignoto G.
      • Pasti L.
      • Vesce S.
      • Rossi D.
      • Rizzini B.L.
      • Pozzan T.
      • Volterra A.
      Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.
      ). For the sake of simplicity, endocannabinoids and the CB1 receptor are not included.

      20:. 4-DAG IN SYNAPTIC PLASTICITY

      Another example of lipid signaling in synaptic function involves the messengers released from inositol lipids. The excitatory neurotransmitter glutamate, in addition to activating PLA2, activates PLC through G-protein-linked glutamate metabotropic receptors. As a consequence, the pool size of DAG increases as does that of inositol-1,4,5-trisphosphate (IP3). The DAG are complex in terms of their fatty acid composition because their pool size is also modulated by another phospholipase (PLD)-mediated degradation of phosphatidylcholine (PC) to phosphatidic acid (PA), followed by lipid phosphate phosphatase-mediated generation of DAG. Now we know that cellular compartments are made up also by scaffold/anchoring proteins that contain anchoring sites for multiple enzymes of convergent as well as divergent signal-transduction pathways. Thus, the fatty acid composition analyzed in a tissue or cell extract does not represent the specificities of subcellular signaling. Inositol lipid-derived DAG display, as the prevalent molecular species, arachidonoyl-stearoyl-glycerol. This composition also corresponds to the major molecular species of the membrane reservoir, phosphatidylinositol-4,5-bisphosphate (PIP2), critical in inositol lipid signaling.
      Inositol lipid synthesis in neurons is maintained by a de novo route through PA, and the DAG kinase step contributes to their replenishment (by its product PA) after synaptic activity-induced PIP2 hydrolysis. Thus, the DAG∊ (specific for 20:4-DAG, see below) becomes critical for the synthesis of PIP2 containing 20:4.
      Diacylglycerols are messengers that modulate certain PKC isoforms, whereas IP3 releases Ca2+ from intracellular stores. DAG targets may discern DAG fatty acid composition (
      • Pettitt T.R.
      • Wakelam M.J.O.
      Diacylglycerol kinase epsilon, but not zeta, selectively removes polyunsaturated diacylglycerol, inducing altered protein kinase C distribution in vivo.
      ). Thus, DAG generated from PC through PLD has different fatty acid composition from that generated from PIP2. PC at sn-2 is mainly oleate, 18:1,n-3 (
      • Hodgkin M.N.
      • Pettitt T.R.
      • Martin A.
      • Michell R.H.
      • Pemberton A.J.
      • Wakelam M.J.
      Diacylglycerols and phosphatidates: which molecular species are intracellular messengers?.
      ), as compared with arachidonate, which is the major sn-2 acyl chain in PIP2. During seizures, the main DAG that transiently accumulates in brain is 20:4-DAG (
      • Bazan N.G.
      Effects of ischemia and electroconvulsive shock on free fatty acid pool in the brain.
      ,
      • Aveldaño M.I.
      • Bazan N.G.
      Rapid production of diacylglycerols enriched in arachidonate and stearate during early brain ischemia.
      ,
      • Aveldaño de Caldironi M.I.
      • Bazan N.G.
      Alpha-Methyl-p-Tyrosine inhibits the production of free arachidonic acid and diacylglycerols in brain after a single electroconvulsive shock.
      ); however, early and late DAG peaks display different fatty acid composition, strongly implying sources other than PIP2. Thus, it is clear that the fatty acid composition of inositol lipids is important in the generation of 20:4-DAG. To achieve enrichment in 20:4 in PIP2, enzymes for inositol lipid synthesis selectively employ 20:4-containing lipids as substrates (
      • Prescott S.M.
      • Majerus P.W.
      The fatty acid composition of phosphatidylinositol from thrombin-stimulated human platelets.
      ). A critical enzyme is DAG kinase, which catalyzes the phosphorylation of DAG to PA. There are nine genes encoding these enzymes. DGK∊, which selectively phosphorylates 20:4-DAG, when deleted has helped to dissect several of these biochemical and neurobiological issues (
      • Rodriguez de Turco E.B.
      • Tang W.
      • Tophan M.K.
      • Sakane F.
      • Marcheselli V.L.
      • Chen C.
      • Taketomi A.
      • Prescott S.M.
      • Bazan N.G.
      Diacylglycerol kinase e regulates seizure susceptibility and long-term potentiation through arachidonoyl-inositol lipid signaling.
      ).
      DGK∊ brain cellular distribution as mGluR1 is localized in Purkinje cerebellar neurons, mitral cells of the olfactory bulb, hippocampal interneurons, and neurons of the thalamus and substantia nigra (
      • Petralia R.S.
      • Wang Y.X.
      • Singh S.
      • Wu C.
      • Shi L.
      • Wei J.
      • Wenthold R.J.
      A monoclonal antibody shows discrete cellular and subcellular localizations of mGluR1 alpha metabotrophic glutamate receptors.
      ,
      • Berthele A.
      • Laurie D.J.
      • Platzer S.
      • Zieglgansberger W.
      • Tolle T.R.
      • Sommer B.
      Differential expression of rat and human type I metabotrophic glutamate receptor splice variant messenger RNAs.
      ). The neurotransmitter glutamate activates G-protein-coupled mGluR1, promoting PIP2 degradation by PLC and the subsequent activation of PKCβ. DGK∊-deficient mice displayed a marked down-regulation of 20:4-inositol lipid signaling when exposed to electroconvulsive shock (ECS). This was reflected by lower PIP2 degradation and lower accumulation of DAG, free arachidonic, and stearic acids after ECS. These studies on DGK∊ suggest that this signaling pathway participates in synaptic function, long-term potentiation, seizures, and neuroprotection.

      ARACHIDONIC ACID-CONTAINING MESSENGERS IN THE PSYCHOACTIVE ACTIONS OF ENDOCANNABINOIDS

      N-arachidonoyl ethanolamine and 2-arachidonoyl glycerol are endogenous ligands (endocannabinoids) of the G-protein-coupled CB1 receptor, to which the main psychoactive component of marijuana, tetrahydrocannabinol, binds. Interestingly, the cellular distribution in brain of the membrane-associated enzyme fatty acid amide hydrolase is complementary to that of the CB1 receptor (
      • Egertova M.
      • Giang D.K.
      • Cravatt B.F.
      • Elphick M.R.
      A new perspective on cannabinoid signaling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain.
      ,
      • Tsou K.
      • Nogueron M.
      • Muthian S.
      • Sanudo-Pena M.
      • Hillard C.
      • Deutsch D.
      • Walker J.
      Fatty acid amide hydrolase is located preferentially in large neurons in the rat central nervous system as revealed by immunohistochemistry.
      ). This accounts for the fact that the half-life of N-arachidonyl ethanolamine (anandamide) is only a few minutes (
      • Willoughby K.A.
      • Moore S.F.
      • Martin B.R.
      • Ellis E.F.
      The biodisposition and metabolism of anandamide in mice.
      ). Thus, this hydrolytic enzyme degrades a potent lipid messenger that modulates neuronal function, as is also the case with PAF acetylhydrolase (discussed below).
      Endocannabinoids are postulated to signal another mode of transynaptic interaction, the “depolarization-induced suppression of inhibition” (
      • Ohno-Shosaku T.
      • Maejima T.
      • Kano M.
      Endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals.
      ,
      • Wilson R.I.
      • Nicoll R.A.
      Endogenous cannabinoids mediate retrograde signaling at hippocampal synapses.
      ,
      • Wilson R.I.
      • Nicoll R.A.
      Endocannabinoid signaling in the brain.
      ). This is a retrograde mode of communication, where endocannabinoids disinhibit neuronal activity by blocking neurotransmitter release from gamma aminobutyric acid (GABA) ergic neurons (
      • Katona I.
      • Sperlagh B.
      • Sik A.
      • Kafalvi A.
      • Vizi E.S.
      • Mackie K.
      • Freoud T.F.
      Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons.
      ). Consequently, the outcome is stimulation of neurotransmission. In different neuronal circuits, endocannabinoids may exert excitatory or inhibitory modulatory actions (
      • Wilson R.I.
      • Nicoll R.A.
      Endocannabinoid signaling in the brain.
      ,
      • Clement A.B.
      • Hawkins E.G.
      • Lichtman A.H.
      • Cravatt B.F.
      Increased seizure susceptibility and proconvulsant activity of anandamide in mice lacking fatty acid amide hydrolase.
      ).

      sPLA2: AN INTERCELLULAR MESSENGER

      We refer to PLA2 in several parts of this article. These enzymes represent a growing family of enzymes that overall include the higher molecular weight enzymes, the cPLA2s, the lower molecular weight secretory sPLA2, and the Ca2+-iPLA2 (
      • Rosenberger T.A.
      • Villacreses N.E.
      • Contreras M.A.
      • Bonventre J.V.
      • Rapoport S.I.
      Brain lipid metabolism in the cPLA2 knockout mouse.
      ,
      • Six D.A.
      • Dennis E.A.
      The expanding superfamily of phospholipase A2 enzymes: classification and characterization.
      ,
      • Molloy G.Y.
      • Rattray M.
      • Williams R.J.
      Genes encoding multiple forms of phospholipase A2 are expressed in rat brain.
      ,
      • Dennis E.A.
      Diversity of group types, regulation, and function of phospholipase A2.
      ). At present, there is still an incomplete understanding of the properties and significance of these enzymes in the nervous system. When the cPLA2 was deleted, there was an increased resistance to cerebral ischemia, indicating an important function of this enzyme in ischemic neuronal damage (
      • Bonventre J.V.
      • Huang Z.
      • Taher M.R.
      • O'Leary E.
      • Li E.
      • Moskowitz M.A.
      • Saperstein A.
      Reduced fertility and post-ischaemic brain injury in mice deficient in cytosolic phospholipase A2.
      ). Newer, noninvasive imaging approaches to study brain PLA2 have recently linked the activation of this enzyme with 5-HT(2a) receptors (
      • Qu Y.
      • Chang L.
      • Klaff J.
      • Balbo A.
      • Rapoport S.I.
      Imaging brain phospholipase A2 activation in awake rats in response to the 5–HT2A/2C agonist (+/−)2,5-dimenthoxy-4-iodophenyl-2-aminopropane (DOI).
      ).
      sPLA2s binding to N (neural)- and M (muscle)-type receptors were discovered by using various purified snake and bee venom sPLA2s as ligands (
      • Lambeau G.
      • Barhanin J.
      • Schweitz H.
      • Qar J.
      • Lazdunski M.
      Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom.
      ,
      • Lambeau G.
      • Schmid-Alliana A.
      • Lazdunski M.
      • Barhanin J.
      Identification and purification of a very high affinity binding protein for toxic phospholipases A2 in skeletal muscle.
      ,
      • Lambeau G.
      • Ancian P.
      • Barhanin J.
      • Lazdunski M.
      Cloning and expression of a membrane receptor for secretory phospholipases A2.
      ,
      • Lambeau G.
      • Ancian P.
      • Nicolas J-P.
      • Beiboer S.H.W.
      • Moinier D.
      • Verheij H.
      • Lazdunski M.
      Structural elements of secretory phospholipases A2 involved in the binding to M-type receptors.
      ). The activation of sPLA2 receptors in primary neurons in culture, using purified sPLA2 as a ligand, promotes a synergy with glutamate, inducing neuronal cell death (
      • Kolko M.
      • DeCoster M.A.
      • Rodriguez de Turco E.B.
      • Bazan N.G.
      Synergy by secretory phospholipase A2 and glutamate on inducing cell death and sustained arachidonic acid metabolic changes in primary cortical neuronal cultures.
      ). Moreover, AA metabolism (
      • Kolko M.
      • DeCoster M.A.
      • Rodriguez de Turco E.B.
      • Bazan N.G.
      Synergy by secretory phospholipase A2 and glutamate on inducing cell death and sustained arachidonic acid metabolic changes in primary cortical neuronal cultures.
      ), COX-2 activation (
      • Kolko M.
      • Rodriguez de Turco E.B.
      • Diemer N.H.
      • Bazan N.B.
      Neuronal damage by secretory phospholipase A2: modulation by cytosolic phospholipase A2, platelet-activating factor, and cyclooxygenase-2 in neuronal cells in culture.
      ), ischemic neuronal survival (
      • Kolko M.
      • Bruhn T.
      • Christensen T.
      • Lazdunski M.
      • Lambeau G.
      • Bazan N.G.
      • Diemer N.H.
      Secretory phospholipase A2 potentiates glutamate-induced rat striatal neuronal cell death in vivo.
      ), and calcium influx (
      • DeCoster M.A.
      • Lambeau G.
      • Lazdunski M.
      • Bazan N.G.
      Secreted phospholipase A2 potentiates glutamate-induced calcium increase and cell death in primary neuronal cultures.
      ) are clearly engaged in this response. Figure 1 outlines the potential involvement of sPLA2 at the synapse. It has been suggested that sPLA2 is coreleased along with neurotransmitters (
      • Matsuzawa A.
      • Makoto M.
      • Atsumi G.
      • Imal K.
      • Prados P.
      • Inoue K.
      • Kudo I.
      Release of secretory phospholipase A2 from rat neuronal cells and its possible function in the regulation of catecholamine secretion.
      ). This issue needs to be further explored in relation to specific synapses. At any rate sPLA2 in the intercellular space is able to generate lipid mediators, and the enzyme protein itself, when secreted, becomes a messenger. It is of interest that the consequences of sPLA2 acting on cells is the release of lipid messengers, either on the cell surface or intercellularly. It is not clear yet how sPLA2s may act on astrocytes, microglia, and neurons, or whether they are restricted to some cells.
      Another remarkable case of a lipid-metabolizing enzyme that is present in the extracellular space is the major component of the cerebrospinal fluid, prostaglandin D synthase (
      • Beuckmann C.T.
      • Gordon W.C.
      • Kanaoka Y.
      • Eguchi N.
      • Marcheselli V.L.
      • Gerashchenko D.Y.
      • Urade Y.
      • Hayaishi O.
      • Bazan N.G.
      Lipocalin-type prostaglandin D synthase (β-trace) is located in pigment epithelial cells of rat retina and accumulates within interphotoreceptor matrix.
      ,
      • Geraschenko D.Y.
      • Beuckmann C.T.
      • Marcheselli V.L.
      • Gordon W.C.
      • Kanaoka Y.
      • Eguchi N.
      • Urade Y.
      • Hayaishi O.
      • Bazan N.G.
      Localization of lipocalin-type prostaglandin D synthase (β-trace) in iris, ciliary body and eye fluids.
      ,
      • Geraschenko D.Y.
      • Beuckmann C.T.
      • Kanaoka Y.
      • Eguchi N.
      • Gordon W.C.
      • Urade Y.
      • Bazan N.G.
      • Hayaishi O.
      Dominant expression of rat prostanoid DP receptor mRNA in leptomeninges, inner segments of photoreceptor cells, iris epithelium, and ciliary processes.
      ).
      sPLA2 receptor activation in neurons induces sustained modifications in neuronal metabolism of arachidonoyl-phospholipids, although the specific messengers formed are not yet defined. sPLA2 receptor activation and glutamate synergize AA mobilization and neurotoxicity. Therefore, excitotoxicity may not only involve glutamate, as often assumed, but also sPLA2 (
      • Kolko M.
      • DeCoster M.A.
      • Rodriguez de Turco E.B.
      • Bazan N.G.
      Synergy by secretory phospholipase A2 and glutamate on inducing cell death and sustained arachidonic acid metabolic changes in primary cortical neuronal cultures.
      ). It is also possible that sPLA2 may have modulatory physiological actions at the synapse and other sites of the nervous system.

      DOCOSAHEXAENOIC ACID IN NEURONS AND PHOTORECEPTORS

      Docosahexaenoic acid (DHA) is a major PUFA in the central nervous system. Brain and retina contain the highest content of DHA in any tissues. There is still a void in knowledge about the significance of DHA. Here we highlight some aspects of DHA in brain and retina that may clarify its participation in cell function and in neurodegenerations. DHA is used continuously for the biogenesis and maintenance of neuronal and photoreceptor membranes. This system is supported by the liver, which supplies DHA incorporated into plasma lipoproteins, and is directed to the retina through choriocapillaries (
      • Scott B.L.
      • Bazan N.G.
      Membrane docosahexanoate is supplied to the developing brain and retina by the liver.
      ,
      • Li J.
      • Wetzel M.G.
      • O'Brien P.J.
      Transport of n-3 fatty acids from the intestine to the retina in rats.
      ,
      • Wang N.
      • Anderson R.E.
      Synthesis of docosahexaenoic acid by retina and retinal pigment epithelium.
      ,
      • Wang N.
      • Anderson R.E.
      Transport of 22:6n-3 in plasma and uptake into retinal pigment epithelium and retina.
      ,
      • Martin R.E.
      • Rodriguez de Turco E.B.
      • Bazan N.G.
      Developmental maturation of hepatic n-3 polyunsaturated fatty acid metabolism: supply of docosahexaenoic acid to retina and brain.
      ). The uptake of DHA by the retina involves the retinal pigment epithelium (RPE) and its delivery to photoreceptors (
      • Li J.
      • Wetzel M.G.
      • O'Brien P.J.
      Transport of n-3 fatty acids from the intestine to the retina in rats.
      ,
      • Wang N.
      • Anderson R.E.
      Synthesis of docosahexaenoic acid by retina and retinal pigment epithelium.
      ,
      • Wang N.
      • Anderson R.E.
      Transport of 22:6n-3 in plasma and uptake into retinal pigment epithelium and retina.
      ,
      • Gordon W.C.
      • Bazan N.G.
      Visualization of [3H]docosahexaenoic acid trafficking through photoreceptors and retinal pigment epithelium by electron microscopic autoradiography.
      ,
      • Bazan N.G.
      • Gordon W.C.
      • B Rodriguez de Turco E.
      The uptake, metabolism and conservation of docosahexaenoic acid (22:6-3) in brain and retina: alterations in liver and retina 22:6 metabolism during inherited progressive retinal degeneration.
      ,
      • Rodriguez de Turco E.B.
      • Jackson F.R.
      • Parkins N.
      • Gordon W.C.
      Strong association of unesterified [3H]docosahexaenoic acid and [3H-docosahexaenoyl]phosphatidate to rhodopsin during in vivo labeling of frog retinal rod outer segments.
      ). During the daily photoreceptor-renewal process, an active recycling of phagosome-derived DHA by RPE via the interphotoreceptor matrix retains DHA within photoreceptors (
      • Bazan N.G.
      • Gordon W.C.
      • B Rodriguez de Turco E.
      The uptake, metabolism and conservation of docosahexaenoic acid (22:6-3) in brain and retina: alterations in liver and retina 22:6 metabolism during inherited progressive retinal degeneration.
      ,
      • Gordon W.C.
      • Rodriguez de Turco E.B.
      • Bazan N.B.
      Retinal pigment epithelial cells play a central role in the conservation of docosahexaenoic acid by photoreceptor cells after shedding and phagocytosis.
      ,
      • Bazan N.G.
      • Rodriguez de Turco E.B.
      • Gordon W.C
      Docosahexaenoic acid supply to the retina and its conservation in photoreceptor cells by active retinal pigment epithelium-mediated recycling.
      ). Endothelial cells and RPE (
      • Wang N.
      • Anderson R.E.
      Synthesis of docosahexaenoic acid by retina and retinal pigment epithelium.
      ,
      • Wang N.
      • Anderson R.E.
      Transport of 22:6n-3 in plasma and uptake into retinal pigment epithelium and retina.
      ) are able to synthesize DHA from dietary precursors, and levels of n-3 fatty acids, such as 18:3n-3 and 20:5n-3, are very low in plasma; therefore, the retina must rely largely on DHA supplied by the liver, either derived from dietary sources or synthesized in the liver from 18:3n-3. A signal generated in brain or retina to control DHA delivery from the liver has been postulated (
      • Scott B.L.
      • Bazan N.G.
      Membrane docosahexanoate is supplied to the developing brain and retina by the liver.
      ); this would allow activation of DHA export during development, when DHA is required for active synapse and photoreceptor outer segment formation. This activation may also occur when injury or neurodegenerations result in loss of DHA from excitable membranes and replenishment of DHA is needed (
      • Scott B.L.
      • Bazan N.G.
      Membrane docosahexanoate is supplied to the developing brain and retina by the liver.
      ,
      • Martin R.E.
      • Rodriguez de Turco E.B.
      • Bazan N.G.
      Developmental maturation of hepatic n-3 polyunsaturated fatty acid metabolism: supply of docosahexaenoic acid to retina and brain.
      ).
      Rhodopsin in photoreceptors is immersed in a lipid environment highly enriched in phospholipids containing DHA. Some DHA is found in close association with rhodopsin (
      • Rodriguez de Turco E.B.
      • Jackson F.R.
      • Parkins N.
      • Gordon W.C.
      Strong association of unesterified [3H]docosahexaenoic acid and [3H-docosahexaenoyl]phosphatidate to rhodopsin during in vivo labeling of frog retinal rod outer segments.
      ,
      • Aveldaño M.I.
      Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes.
      ) and DHA is essential for rhodopsin function (
      • Bazan N.G.
      Supply of n-3 polyunsaturated fatty acids and their significance in the central nervous system.
      ,
      • Bush R.A.
      • Malnoë A.
      • Remé C.E.
      • Williams T.P.
      Dietary deficiency of N-3 fatty acids alters rhodopsin content and function in the rat retina.
      ,
      • Brown M.F.
      Modulation of rhodopsin function by properties of the membrane bilayer.
      ). Moreover, retinal DHA, like brain DHA, is very resistant to n-3 fatty acid dietary deprivation.
      Because of this constant systemic flow of DHA via a “long loop,” it is highly relevant that retinitis pigmentosa (RP) is associated with alterations in lipoprotein metabolism (
      • Bazan N.G.
      • Gordon W.C.
      • B Rodriguez de Turco E.
      The uptake, metabolism and conservation of docosahexaenoic acid (22:6-3) in brain and retina: alterations in liver and retina 22:6 metabolism during inherited progressive retinal degeneration.
      ). The most frequently reported phenotype is low plasma and red blood cell levels of DHA (
      • Converse C.A.
      • Hammer H.M.
      • Packard C.J.
      • Shephard J.
      Plasma lipid abnormalities in retinitis pigmentosa and related conditions.
      ,
      • Anderson R.E.
      • Maude M.B.
      • Lewis R.S.
      • Newsome D.A.
      • Fishman G.A.
      Abnormal plasma levels of polyunsaturated fatty acid in autosomal dominant retinitis pigmentosa.
      ,
      • Gong J.
      • Rosner B.
      • Rees D.G.
      • Berson E.L.
      • Weigel-Di-Franco C.A.
      • Schaefer E.J.
      Plasma docosahexaenoic acid levels in various genetic forms of retinitis pigmentosa.
      ,
      • Hoffman D.R.
      • Birch D.G.
      Omega 3 fatty acid status in patients with retinitis pigmentosa.
      ). In Usher's syndrome, an autosomal recessive RP that is associated with hearing loss, lower plasma levels of 22:6- and 20:4-phospholipids are also found (

      Bazan, N. G., and E. B. Rodriguez de Turco. 1996. Alterations in plasma lipoproteins and DHA transport in progressive rod-cone degeneration (prcd). Retinal Degeneration and Regeneration, Proceedings of an International Symposium in Kanazawa, Japan. 1995.

      ). Interestingly, changes show a direct correlation to the severity of disease, and are more accentuated in patients with Usher's type I than in those with the less severe type II form (
      • Maude M.B.
      • Anderson E.O.
      • Anderson R.E.
      Polyunsaturated fatty acids are lower in blood lipids of Usher's type I but not Usher's type II.
      ).
      Low plasma levels of 22:6n-3 in RP patients have also been interpreted as a mechanism to protect the retina from oxidative damage resulting from photochemical function of rhodopsin (
      • Anderson R.E.
      • Maude M.B.
      • Alvarez R.A.
      • Acland G.
      • Aguirre G.D.
      A hypothesis to explain the reduced blood levels of docosahexaenoic acid in inherited retinal degenerations caused by mutations in genes encoding retina-specific proteins.
      ). Stress signals originating from retinas undergoing degeneration (e.g., RP) may shut off the communication between the retina and the liver (
      • Scott B.L.
      • Bazan N.G.
      Membrane docosahexanoate is supplied to the developing brain and retina by the liver.
      ), reducing the systemic liver supply of 22:6n-3. In dogs with progressive rod-cone degeneration (prcd) (
      • Aguirre G.D.
      • Acland G.M.
      • Maude M.B.
      • Anderson R.E.
      Diets enriched in docosahexaenoic acid fail to correct progressive rod-cone degeneration (prcd) phenotype.
      ) as well as in other retinal-degeneration models (
      • Anderson R.E.
      • Maude M.B.
      • Alvarez R.A.
      • Acland G.
      • Aguirre G.D.
      A hypothesis to explain the reduced blood levels of docosahexaenoic acid in inherited retinal degenerations caused by mutations in genes encoding retina-specific proteins.
      ), low levels of DHA in photoreceptor phospholipids occur. Moreover, the synthesis of DHA from 20:5n-3 in RPE cultures from prcd is not affected by the retinal degeneration (
      • Chen H.
      • Ray J.
      • Scarpino V.
      • Acland G.M.
      • Aguiree G.D.
      • Anderson R.E.
      Synthesis and release of docosahexaenoic acid by the RPE cells of prcd-affected dogs.
      ), and dietary supplementation with DHA failed to prevent the loss of photoreceptor DHA and the prcd phenotype (
      • Aguirre G.D.
      • Acland G.M.
      • Maude M.B.
      • Anderson R.E.
      Diets enriched in docosahexaenoic acid fail to correct progressive rod-cone degeneration (prcd) phenotype.
      ). Moreover, cultured hepatocytes from prcd dogs display higher accumulation of 22:6-phospholipids in the liver and impaired hepatocyte secretion of DHA-containing VLDLs (

      Bazan, N. G., and E. B. Rodriguez de Turco. 1996. Alterations in plasma lipoproteins and DHA transport in progressive rod-cone degeneration (prcd). Retinal Degeneration and Regeneration, Proceedings of an International Symposium in Kanazawa, Japan. 1995.

      ), supporting the hypothesis that in retinal degenerations a systemic DHA metabolic defect occurs that leads to reduced liver DHA supply to the retina. Whether DHA dietary supplementation protects the retina of RP patients is not known.

      DHA: TARGET OF FREE RADICAL-CATALYZED PEROXIDATION AND PRECURSOR OF DOCOSANOIDS

      DHA-containing phospholipids are a target for lipid peroxidation, and as a result of free radical-catalyzed peroxidation, F4-isoprostanes are formed (
      • Nourooz-Zadeh J.
      • Liu E.H.C.
      • Anggard E.E.
      • Halliwell B.
      F4-isoprostanes: a novel class of prostanoids formed during peroxidation of docosahexaenoic acid.
      ,
      • Roberts II, L.J.
      • Montine T.J.
      • Markesbery W.R.
      • Tappert A.R.
      • Hardy P.
      • Chemtob S.
      • Dettbarn W.D.
      • Morrow J.D.
      Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid.
      ). F2-isoprostanes are also derived from free-radical-catalyzed peroxidation, although from AA instead (
      • Morrow J.D.
      • Hill K.E.
      • Burk R.F.
      • Tarek M.
      • Nammour T.M.
      • Badr K.F.
      • Robert J.L.
      A series of prostaglandin F2-like compounds are produced in vivo in human by a non-cyclooxygenase, free radical-catalyzed mechanism.
      ). F4-isoprostanes are found esterified in phospholipids, and it has been reported that their content is increased in the brains of patients with Alzheimer's disease (
      • Nourooz-Zadeh J.
      • Liu E.H.C.
      • Yhlen B.
      • Anggard E.E.
      • Halliwell B.
      F4-isoprostanes as specific marker of docosahexaenoic acid peroxidation in Alzheimer's disease.
      ).
      Docosanoids, enzyme-derived DHA metabolites, were identified initially in the retina (
      • Bazan N.G.
      • Birkle D.L.
      • Reddy T.S.
      Docosahexaenoic acid (22:6, n 3) is metabolized to lipoxygenase reaction products in the retina.
      ), and have been proposed to elicit neuroprotective actions (
      • Bazan N.G.
      Supply of n-3 polyunsaturated fatty acids and their significance in the central nervous system.
      ). Very recently it was ascertained by tandem liquid chromatography-photodiode array-electrospray ionization-mass spectrometric-mass spectrometry (LC-PDA-ESI-MS-MS)-based lipidomic analysis that the synthesis of DHA-oxygenation messengers occurs during brain ischemia-reperfusion (
      • Marcheselli V.L.
      • Hong S.
      • Lukiw W.J.
      • Tian X.H.
      • Gronert K.
      • Musto A.
      • Hardy M.
      • Gimenez J.M.
      • Chiang N.
      • Serhan C.N.
      • Bazan N.G.
      Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression.
      ). Two DHA-oxygenation pathways operate: one that gives rise to 10,17S-docosatriene, and the second that results in the synthesis of resolvin-type messengers (17R-DHA). Resolvins were elegantly identified outside the nervous system as a response to aspirin treatment (
      • Serhan C.N.
      • Hong S.
      • Gronert K.
      • Colgan S.P.
      • Devchand P.R.
      • Mirick G.
      Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals.
      ). The brain resolvins are also made upon treatment with aspirin (
      • Marcheselli V.L.
      • Hong S.
      • Lukiw W.J.
      • Tian X.H.
      • Gronert K.
      • Musto A.
      • Hardy M.
      • Gimenez J.M.
      • Chiang N.
      • Serhan C.N.
      • Bazan N.G.
      Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression.
      ). Both DHA-oxygenation pathways generate messengers that are counter-proinflammatory signals (
      • Serhan C.N.
      • Hong S.
      • Gronert K.
      • Colgan S.P.
      • Devchand P.R.
      • Mirick G.
      Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals.
      ,
      • Serhan C.N.
      • Clish C.B.
      • Brannon J.
      • Colgan S.P.
      • Chiang N.
      • Gronert K.
      Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2: nonsteroidal antiinflammatory drugs and transcellular processing.
      ,
      • Hong S.
      • Gronert K.
      • Devchand P.R.
      • Moussignac R.L.
      • Serhan C.N.
      Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells.
      ). The docosanoid 10,17S-docosatriene is a potent inhibitor of brain ischemia-reperfusion-induced polymorphonuclear leukocyte (PMN) infiltration, as well as of nuclear factor-κB (NF-κB) and COX-2 expression (
      • Marcheselli V.L.
      • Hong S.
      • Lukiw W.J.
      • Tian X.H.
      • Gronert K.
      • Musto A.
      • Hardy M.
      • Gimenez J.M.
      • Chiang N.
      • Serhan C.N.
      • Bazan N.G.
      Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression.
      ). Moreover, marked attenuation of the stroke volume was observed when 10,17S-docosatriene was infused into the third ventricle during ischemia-reperfusion (
      • Marcheselli V.L.
      • Hong S.
      • Lukiw W.J.
      • Tian X.H.
      • Gronert K.
      • Musto A.
      • Hardy M.
      • Gimenez J.M.
      • Chiang N.
      • Serhan C.N.
      • Bazan N.G.
      Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression.
      ). Discovery of these DHA-oxygenation messengers sheds new light on how the brain modulates its response to inflammatory injury, and raises the exciting potential for designing new drugs to treat neurologic disorders that have a neuroinflammatory component, such as stroke, traumatic brain injury, or spinal cord injury. In particular, the very high biological activity of the docosanoid 10,17S-docosatriene marks it as potential effector of neuroprotection.

      PAF-MEDIATED CELL SIGNAL TRANSDUCTION IN THE NERVOUS SYSTEM

      Excitatory synaptic neurotransmission promotes activation of phospholipases that in turn cleave synaptic membrane phospholipid reservoirs, releasing biologically active moieties: the lipid messengers. One such reservoir is the phospholipids that typically contain an alkyl-acyl chain in the C1 position and either arachidonate (20:4) or docosahexaenoate (22:6) in the C2 position of the glycerol backbone, and that have a phosphorylcholine ester. These membrane phospholipids are the PCs, and are the target of phospholipases A2, which cleave the fatty acid linked to C2. The products of this reaction are lyso-PAF and a free PUFA, either arachidonic or DHA. Subsequently, lyso-PAF is acetylated and its product, PAF, is a potent phospholipid messenger (remodeling pathway; Fig. 2). Therefore, PAF, like prostaglandins and other lipid messengers, is not maintained in membranes as a preformed structure; rather, it is rapidly synthesized upon specific stimulation.
      Figure thumbnail gr2
      Fig. 2Pathways of PAF synthesis and degradation. In the center of this figure is the remodeling pathway or “PAF cycle” from the membrane phospholipid PAF precursor, alkyl-acyl-glycerophosphorylcholine (GPC, on the left) to the the biologically active PAF (at right). The remodeling route includes the production of lyso-PAF, which is generated from the PAF precursor alkyl-acyl-GPC either directly by the action of phospholipase A2 or by the transfer of the sn-2 acyl moiety to a “donor” lyso-plasmalogen (top), which is itself mobilized from membrane plasmalogen by phospholipase action. The de novo route of PAF synthesis (upper right) involves the direct transfer of a choline moiety to alkyl-acetylglycerol. Note that PAF acetylhydrolase (bottom) inactivates all PAF molecules, regardless of their biosynthetic route, and additionally inactivates oxidatively damaged phospholipids (shorter peroxidated acyl group at C2) that possess biological activity at the PAF receptor.
      PAF is a physiologic mediator that modulates glutamate release and acts as a retrograde messenger of LTP (
      • Kato K.
      • Clark G.D.
      • Bazan N.G.
      • Zorumski C.F.
      Platelet activating factor as a potential retrograde messenger in Ca1 hippocampal long-term potentiation.
      ,
      • Arai A.
      • Lynch G.
      Antagonists of the platelet-activating factor receptor block long-term potentiation in hippocampal slices.
      ,
      • Clark G.D.
      • Happel L.T.
      • Zorumski C.F.
      • Bazan N.G.
      Enhancement of hippocampal excitatory synaptic transmission by platelet-activating factor.
      ,
      • Wieraszko A.
      • Li G.
      • Kornecki E.
      • Hogan M.V.
      • Ehrlich Y.H.
      Long-term potential in the hippocampus induced by platelet-activating factor.
      ). PAF modulates neuronal calcium ionization through the PAF receptor (
      • Bito H.
      • Nakamura M.
      • Honda Z.
      • Izumi T.
      • Iwatsubo T.
      • Seyama Y.
      • Ogura A.
      • Kudo Y.
      • Shimizu T.
      Platelet-activating factor (PAF) receptor in rat brain: PAF mobilizes intracellular Ca2+ in hippocampal neurons.
      ) and is also generated by NMDA receptor activation. PAF functions are thought to be modulated by synthesis and degradation of the lipid messenger by tightly regulated enzymes. In fact, brain contains several PAF acetylhydrolases (PAF-AH) (
      • Arai H.
      • Koizumi H.
      • Aoki J.
      • Inoue K.
      Platelet-activating factor acetylhydrolase (PAF-AH).
      ), which rapidly remove the acetate moiety from C2 (see below), thus rendering PAF biologically inactive. The existence of these “off-signal” enzymes highlights the fact that PAF is a potent messenger (
      • Bazan N.G.
      Inflammation: a signal terminator.
      ).
      PAF and the free PUFAs released during PAF synthesis accumulate in brain upon stimulation (
      • Bazan N.G.
      • Allan G.
      Platelet-activating factor and other bioactive lipids.
      ). In the retina and in neurons in culture, neurotransmitters trigger Ca2+-independent de novo PAF synthesis through cytidine 5′-diphosphocholine (
      • Bussolino P.
      • Gremo F.
      • Tetta C.
      • Pescarmona G.
      • Camussi G.
      Production of platelet-activating factor by chick retina.
      ,
      • Bussolino F.
      • Torelli S.
      • Stefanini E.
      • Gremo F.
      Platelet-activating factor production occurs through stimulation of cholinergic and dopaminergic receptors in the chick retina.
      ). Since PAF stimulates ATP release from PC12 cells, it has been implicated in vesicular release of neurotransmitters (
      • Kornecki E.
      • Ehrlich Y.H.
      Neuroregulatory and neuropathological actions of the ether-phospholipid platelet-activating factor.
      ), perhaps through a mechanism analogous to that engaged in vesicular release of vasoactive substances from platelets and eosinophils (
      • Prescott S.M.
      • Zimmerman G.A.
      • Stafforini D.M.
      • McIntyre T.M.
      Platelet-activating factor and related lipid mediators.
      ).
      PAF is rapidly degraded by deacetylation to the biologically inactive lipid, lyso-PAF. Several PAF-AH are expressed in the central nervous system (
      • Arai A.
      • Lynch G.
      Antagonists of the platelet-activating factor receptor block long-term potentiation in hippocampal slices.
      ). Use of the stable PAF analog, methyl carbamyl-PAF (mc-PAF), allows the study of PAF action while avoiding enzyme-mediated inactivation. Bath-applied mc-PAF specifically augments hippocampal excitatory synaptic transmission using synaptic pairs of neurons in culture (
      • Clark G.D.
      • Happel L.T.
      • Zorumski C.F.
      • Bazan N.G.
      Enhancement of hippocampal excitatory synaptic transmission by platelet-activating factor.
      ). This study also demonstrated that PAF action is receptor mediated and presynaptically localized, and supported the notion that PAF is a second messenger in the central nervous system. Additional mc-PAF enhanced the amplitude of evoked postsynaptic currents, while diminishing the size of presynaptic action potentials. Interestingly, the mc-PAF effect on excitatory synaptic transmission is very selective, since it does not increase general presynaptic function (
      • Clark G.D.
      • Happel L.T.
      • Zorumski C.F.
      • Bazan N.G.
      Enhancement of hippocampal excitatory synaptic transmission by platelet-activating factor.
      ). Also, c-PAF elicts these actions independently of postsynaptic glutamate receptors. Moreover, mc-PAF augmented the frequency of spontaneous miniature excitatory synaptic events, sparing their amplitude and their time course.
      Retrograde messengers of LTP are candidates to up-regulate memory. Retrograde messengers released from the postsynaptic neuron are thought to enhance excitatory neurotransmitter release from the presynaptic neuron; this is believed to increase synaptic efficiency of LTP. The signaling that links the postsynaptic neuron to presynaptic modulation of neurotransmitter release is in accord with the Hebbian concept of synaptic potentiation. Several mediators have been postulated to be retrograde messengers, including arachidonic acid, nitric oxide, carbon monoxide, and PAF. In the case of arachidonic acid, even though this fatty acid is released and increases excitatory synaptic transmission when coupled with presynaptic stimulation, it requires relatively high concentrations (
      • Williams J.H.
      • Errington M.L.
      • Lynch M.A.
      • Bliss T.V.P.
      Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus.
      ), and in CA1 neurons, AA effects are blocked by DL-2-amino-phosphopentanoic acid (APV) (
      • O'Dell T.J.
      • Hawkins R.D.
      • Kandel E.R.
      • Arancio O.
      Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger.
      ,
      • Kato K.
      • Uruno K.
      • Saito K.
      • Kato H.
      Both arachidonic acid and 1-oleoyl-2-acetyl glycerol in low magnesium solution induce long-term potentiation in hippocampal CA1 neurons in vitro.
      ), consequently limiting arachidonic acid's potential as a retrograde messenger.
      PAF, on the other hand, fulfills several aspects of a retrograde messenger in LTP: it is synthesized in brain during stimulation as well as in neurons in culture, it activates hippocampal excitatory synapses by increasing presynaptic glutamate release, and it augments hippocampal CA1 LTP.
      Infusion of PAF into specific brain regions promotes memory facilitation in rats. In experiments using the step-down inhibitory avoidance or spatial-habitation task, PAF infused 10 min before training or immediately after training enhanced retention scores. However, PAF infused 60 min post-training had no effect on retention (
      • Izquierdo I.
      • Fin C.
      • Schmitz P.K.
      • Da Silva R.C.
      • Jerusalinsky D.
      • Quillfeldt J.A.
      • Ferreira M.B.G.
      • Medina J.H.
      • Bazan N.G.
      Memory enhancement by intrahippocampal, intraamygdala, or intraentorhinal infusion of platelet-activating factor measured in an inhibitory avoidance task.
      ). In PAF-receptor-deficient mice, marked attenuation of LTP was observed. Infusion of PAF into brain regions enhanced memory formation in a time-dependent, neuroanatomically specific fashion. A presynaptic PAF-receptor antagonist had a clear amnesic effect in several memory-task tests.
      Post-training intrahippocampal infusion of intracellular forms of the PAF receptor did not modify memory for an inhibitory avoidance task (
      • Izquierdo I.
      • Fin C.
      • Schmitz P.K.
      • Da Silva R.C.
      • Jerusalinsky D.
      • Quillfeldt J.A.
      • Ferreira M.B.G.
      • Medina J.H.
      • Bazan N.G.
      Memory enhancement by intrahippocampal, intraamygdala, or intraentorhinal infusion of platelet-activating factor measured in an inhibitory avoidance task.
      ,
      • Jerusalinsky D.
      • Fin C.
      • Quillfelot J.A.
      • Beatriz C.F.
      • Schmitz P.K.
      • DaSilva R.C.
      • Walz R.
      • Bazan N.G.
      • Medina J.H.
      • Izquierdo I.
      Effects of antagonists of platelet-activating factor receptors on memory of inhibitory avoidance in rats.
      ). However, post-training injection of PAF into either amygdala, hippocampus, or entorhinal cortex enhances memory in an inhibitory avoidance task (
      • Izquierdo I.
      • Fin C.
      • Schmitz P.K.
      • Da Silva R.C.
      • Jerusalinsky D.
      • Quillfeldt J.A.
      • Ferreira M.B.G.
      • Medina J.H.
      • Bazan N.G.
      Memory enhancement by intrahippocampal, intraamygdala, or intraentorhinal infusion of platelet-activating factor measured in an inhibitory avoidance task.
      ), whereas injection of a PAF-receptor antagonist that selectively targets the presynaptic PAF receptor impairs memory in this task. Moreover, post-training intrahippocampal and intradorsal striatal injections of either PAF or PAF-receptor antagonists modulate memory in hidden and visible platform water-maze tasks, respectively (
      • Packard M.G.
      • Teather L.
      • Bazan N.G.
      Effects of intrastriatal injections of platelet-activating factor and the PAF antagonist BN 52021 on memory.
      ,
      • Bazan N.G.
      • Packard M.G.
      • Teather L.
      • Allan G.
      Bioactive lipids in excitatory neurotransmission and neuronal plasticity.
      ,
      • Teather L.A.
      • Packard M.G.
      • Bazan N.G.
      Effects of posttraining intrahippocampal injections of platelet-activating factor and PAF antagonists on memory.
      ).
      The NMDA-receptor antagonist MK-801 markedly attenuated the memory-enhancing effects of intradorsal striatal infusion of PAF on the visible platform water-maze task. On the other hand, in a hidden platform water-maze task, intrahippocampal infusion of PAF blocks the memory-perturbing actions of MK-801. The mechanism allowing interactions between the phospholipid messenger and the NMDA receptor in the mnemonic functions of hippocampus and striatum may underlie the participation of the intracellular form of the PAF receptor or of signaling cascades evolving from the cell-surface PAF receptor to transcription factors and genes (
      • Teather L.A.
      • Packard M.G.
      • Bazan N.G.
      Differential interaction of platelet-activating factor and NMDA receptor function in hippocampal and dorsal striatal memory processes.
      ).
      PAF-receptor antagonists block LTP in rat hippocampal CA1 neurons, dentate gyrus, and medial vestibular nuclei. PAF itself increases excitatory postsynaptic responses and the frequency of spontaneous excitatory postsynaptic currents, and enhances the receptor-mediated release of the excitotoxic amino acid, glutamate, from presynaptic terminals. Excitatory synaptic transmission is modulated by a variety of signaling systems, including the inhibitory neurotransmitter GABA. In a study of the effects of PAF on GABA neurotransmission in hippocampal neurons in primary culture, extracellular PAF reduced GABA-gated chloride ion current in more than 65% of cells while enhancing it in ∼23% of cells. This heterogeneous modulation of inhibitory neurotransmission may be the result of GABA receptor subtypes differing in their subunit composition or in phosphorylation sites responding to intracellular signaling molecules that transduce the extracellular PAF signal to the GABA receptor, or it may be the result of allosteric effects on the receptor regulatory sites (
      • Chen C.
      • Bazan N.G.
      Platelet-activating factor inhibits ionotropic GABA receptor activity in cultured hippocampal neurons.
      ).
      Because PAF predominantly inhibits ionic GABA-receptor activity in hippocampal neurons, it is not surprising that PAF enhances excitatory presynaptic glutamatergic neurotransmission as well as decreases inhibitory GABAergic postsynaptic activity (
      • Chen C.
      • Bazan N.G.
      Platelet-activating factor inhibits ionotropic GABA receptor activity in cultured hippocampal neurons.
      ).
      The observation that PAF-receptor antagonists elicit neuroprotection in the gerbil brain ischemia-reperfusion model (
      • Panetta T.
      • Marcheselli V.L.
      • Braquet P.
      • Spinnewyn B.
      • Bazan N.G.
      Effects of a platelet-activating factor antagonist (BN 52021) on free fatty acids, diacylglycerols, polyphosphoinositides and blood now in the gerbil brain: inhibition of ischemia reperfusion induced cerebral injury.
      ) suggested that PAF may work at the synapse. In this model, the ischemia-induced accumulation of free PUFA was decreased by the PAF antagonists that were also neuroprotective by restoring cerebral blood flow (
      • Panetta T.
      • Marcheselli V.L.
      • Braquet P.
      • Spinnewyn B.
      • Bazan N.G.
      Effects of a platelet-activating factor antagonist (BN 52021) on free fatty acids, diacylglycerols, polyphosphoinositides and blood now in the gerbil brain: inhibition of ischemia reperfusion induced cerebral injury.
      ). The ischemia-induced PUFA release is believed to result from synaptic phospholipase(s) A2 activity (
      • Bazan N.G.
      • Packard M.G.
      • Teather L.
      • Allan G.
      Bioactive lipids in excitatory neurotransmission and neuronal plasticity.
      ). In addition, the PAF antagonist shown to be neuroprotective in the gerbil ischemia-reperfusion model also selectively displaced radiolabeled PAF binding from synaptic membranes (
      • Marcheselli V.L.
      • Rossowska M.J.
      • Domingo M.T.
      • Braquet P.
      • Bazan N.G.
      Distinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex.
      ). This synaptic membrane binding site was therefore suggested to be the modulator of glutamate neurotransmitter release (
      • Clark G.D.
      • Happel L.T.
      • Zorumski C.F.
      • Bazan N.G.
      Enhancement of hippocampal excitatory synaptic transmission by platelet-activating factor.
      ). Moreover, intracellular PAF-binding sites identified in a microsomal fraction were distinct from those at the synaptic membrane fraction insofar as responses to several antagonists (
      • Marcheselli V.L.
      • Rossowska M.J.
      • Domingo M.T.
      • Braquet P.
      • Bazan N.G.
      Distinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex.
      ). At that time it was known that PAF activates early-response genes (
      • Squinto S.P.
      • Block A.L.
      • Braquet P.
      • Bazan N.G.
      Platelet-activating factor stimulates a Fos/Jun/AP-1 transcriptional signaling system in human neuroblastoma cells.
      ,
      • Squinto S.P.
      • Braquet P.
      • Block A.L.
      • Bazan N.G.
      Platelet-activating factor activates HIV promoter in transfected SH-SY5Y neuroblastoma cells and MOLT-4 T lymphocytes.
      ,
      • Bazan N.G.
      • Squinto S.P.
      • Braquet P.
      • Panetta T.
      • Marcheselli V.L.
      Platelet-activating factor and polyunsaturated fatty acids in cerebral ischemia or convulsions: intracellular PAF-binding sites and activation of a Fos/Jun/AP-1 transcriptional signaling system.
      ), therefore the intracellular receptor was thought to be the signaling linkage to the nucleus. Cloning of the seven-transmembrane domain PAF receptor (
      • Honda Z.
      • Nakamura M.
      • Miki I.
      • Minami M.
      • Watanabe T.
      • Seyama Y.
      • Okado H.
      • Toh H.
      • Ito K.
      • Miyamoto T.
      • Shimizu T.
      Cloning by functional expression of platelet-activating factor receptor from guinea-pig lung.
      ,
      • Kunz D.
      • Gerard N.P.
      • Gerard C.
      The human leukocyte platelet-activating factor receptor. CDNA cloning, cell surface expression, and construction of a novel epitope-bearing analog.
      ,
      • Sugimoto T.
      • Tsuchimochi H.
      • McGregor C.G.
      • Mutoh H.
      • Shimizu T.
      • Kurachi Y.
      Molecular cloning and characterization of the platelet-activating factor receptor gene expressed in the human heart.
      ) and the discovery of specific PAF receptor-mediated Ca2+ influx into neurons (
      • Bito H.
      • Nakamura M.
      • Honda Z.
      • Izumi T.
      • Iwatsubo T.
      • Seyama Y.
      • Ogura A.
      • Kudo Y.
      • Shimizu T.
      Platelet-activating factor (PAF) receptor in rat brain: PAF mobilizes intracellular Ca2+ in hippocampal neurons.
      ) further illuminated neural PAF signaling. In studies of LTP in hippocampal neurons from PAF-receptor-deficient mice, both incidence and size of LTP, defined as increased excitatory postsynaptic potentials, were attenuated in PAF-receptor-knockout mice, as compared with LTP in wild-type mice. Moreover, PAF receptor-deficient mice display a marked attenuation of LTP upon stimulation of the lateral, but not the medial, perforant path (
      • Chen C.
      • Magee J.C.
      • Marcheselli V.L.
      • Hardy M.
      • Bazan N.G.
      Attenuated long-term potentiation in hippocampal dentate gyrus neurons of mice deficient in the platelet-activating factor receptor.
      ). In another study, PAF-deficient mice had unaltered LTP; however, there were substantial differences between the experimental conditions of the two studies (
      • Kobayashi K.
      • Ishii S.
      • Kume K.
      • Takahasi T.
      • Shimizu T.
      • Manabe T.
      Platelet-activating factor receptor is not required for long-term potentiation in the hippocampal CA1 region.
      ). Furthermore, PAF-receptor antagonists reduced LTP in wild-type mice, but not in PAF-receptor knockout mice. These results further support the hypothesis that PAF is involved in hippocampal synaptic plasticity (
      • Chen C.
      • Magee J.C.
      • Marcheselli V.L.
      • Hardy M.
      • Bazan N.G.
      Attenuated long-term potentiation in hippocampal dentate gyrus neurons of mice deficient in the platelet-activating factor receptor.
      ). Most recently, the intracellular PAF receptors were further characterized. One form is confined to the endosomes (
      • Ihida K.
      • Predescu D.
      • Czekay R-P.
      • Palade G.E.
      Platelet activating factor receptor (PAF-R) is found in a large endosomal compartment in human umbilical vein endothelial cells.
      ) and the other to the nuclear membrane (
      • Marrache A.M.
      • Gobeil Jr., F.
      • Bernier S.G.
      • Stankova J.
      • Rola-Pleszczynski M.
      • Choufani S.
      • Bkaily G.
      • Bourdeau A.
      • Sirois M.G.
      • Vazquez-Tello A.
      • Fan L.
      • Joyal J-S.
      • Filep J.G.
      • Varma D.R.
      • Ribeiro-da-Silva A.
      • Chemtob S.
      Proinflammatory gene induction by platelet-activating factor mediated via its cognate nuclear receptor.
      ). Both intracellular forms of the receptor may be part of the intracellular microsomal form described earlier (
      • Marcheselli V.L.
      • Rossowska M.J.
      • Domingo M.T.
      • Braquet P.
      • Bazan N.G.
      Distinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex.
      ). At any rate, we know that neurons, astrocytes, and microglia, as well as endothelial cells, express the PAF seven-transmembrane domain receptor. It remains to be defined whether the intracellular PAF receptor(s) have a different molecular structure or whether they are in fact an intracellular state of the cell-surface PAF seven transmembrane domain receptor. Does the cell-surface PAF receptor internalize? Is the cell-surface PAF receptor destined to insertion in the membrane already active, and if so, can PAF itself internalize to access either of these PAF-receptor forms located inside the cell? Finally, regarding PAF's transcriptional actions, at least two mechanisms are possible: the cell surface receptor triggers the signaling cascade, or the intracellular form establishes interactions with specific kinases/phosphatases or transcription factors with or without specific scaffolding proteins.
      During ischemia, seizures, and in other pathologic conditions involving oxidative stress, the rates of PAF synthesis and degradation no longer maintain a modulated PAF pool size; consequently, PAF concentration increases and it becomes a pro inflammatory messenger and a mediator of neurotoxicity. As such, PAF activates COX-2 expression (
      • Bazan N.G.
      • Fletcher B.S.
      • Herschman H.R.
      • Mukherjee P.K.
      Platelet-activating factor and retinoic acid synergistically activate the inducible prostaglandin synthase gene.
      ) as well as that of several early-response genes that encode transcription factors (
      • Squinto S.P.
      • Block A.L.
      • Braquet P.
      • Bazan N.G.
      Platelet-activating factor stimulates a Fos/Jun/AP-1 transcriptional signaling system in human neuroblastoma cells.
      ,
      • Squinto S.P.
      • Braquet P.
      • Block A.L.
      • Bazan N.G.
      Platelet-activating factor activates HIV promoter in transfected SH-SY5Y neuroblastoma cells and MOLT-4 T lymphocytes.
      ). PAF activates apoptosis and PMN and their adhesion to the microvasculature (
      • Prescott S.M.
      • Zimmerman G.A.
      • Stafforini D.M.
      • McIntyre T.M.
      Platelet-activating factor and related lipid mediators.
      ). This event has critical consequences for cell survival. Leukocyte infiltration mediates neural injury in head trauma, stroke, spinal cord injury, and other diseases. PAF also enhances the synthesis and release of IL-6, IL-8, IL-10, tumor necrosis factor-α, and of other mediators of the inflammatory response. PAF activates phospholipases through its receptor, resulting in additional PAF synthesis, along with that of prostaglandins and leukotrienes. Overall, PAF is a potent neuronal injury messenger. PAF also plays a prominent role in astrocytes and in microglial cells. Many of these actions have been studied in nonneural cells and are assumed to occur in the nervous system as well. Therefore, excessive PAF promotes neuronal damage, and PAF-receptor antagonists elicit neuroprotection in various models of neural injury (
      • Panetta T.
      • Marcheselli V.L.
      • Braquet P.
      • Spinnewyn B.
      • Bazan N.G.
      Effects of a platelet-activating factor antagonist (BN 52021) on free fatty acids, diacylglycerols, polyphosphoinositides and blood now in the gerbil brain: inhibition of ischemia reperfusion induced cerebral injury.
      ,
      • Prehn J.H.M.
      • Krieglstein J.
      Platelet-activating factor antagonists reduce excitotoxic damage in cultured neurons from embryonic chick telencephalon and protect the rat hippocampus and neocortex from ischemic injury in vivo.
      ,
      • Gilboe D.D.
      • Kintner D.
      • Fitzpatrick J.H.
      • Emoto S.E.
      • Esanu A.
      • Braquet P.G.
      • Bazan N.G.
      Recovery of postischemic brain metabolism and function following treatment with a free radical scavenger and platelet-activating factor antagonists.
      ,
      • Lindsberg P.J.
      • Yue T-L.
      • Frerichs K.U.
      • Hallenbeck J.M.
      • Feuerstein G.
      Evidence for platelet-activating factor as a novel mediator of experimental stroke in rabbits.
      ,
      • Oberpichler H.
      • Sauer D.
      • Rossberg C.
      • Mennel H.D.
      • Krieglstein J.
      PAF antagonist ginkgolide-B reduces post-ischemic neuronal damage in rat brain hippocampus.
      ,
      • Kochanek P.M.
      • Dutka A.J.
      • Kumaroo K.K.
      • Hallenbeck J.M.
      Platelet-activating factor receptor blockade enhances recovery after multi-focal brain ischemia.
      ).

      PAF DEGRADATION IN THE CENTRAL NERVOUS SYSTEM

      Multiple PAF-degrading enzymes in the central nervous system further highlight the importance of maintaining a well-controlled PAF pool size. These include tissue PAF-AH (
      • Arai H.
      • Koizumi H.
      • Aoki J.
      • Inoue K.
      Platelet-activating factor acetylhydrolase (PAF-AH).
      ) as well as plasma-type PAF-AHs (
      • Prescott S.M.
      • Zimmerman G.A.
      • Stafforini D.M.
      • McIntyre T.M.
      Platelet-activating factor and related lipid mediators.
      ). One plasma-type PAF-AH of molecular mass 45 kDa is a form of sPLA2. There is also an intracellular form, PAF-AHIb, isolated from brain. PAF-AHIb is a heterotrimer with two catalytic subunits (α1, 29 kDa and α2, 30 kDa) and one regulatory β subunit (45 kDa); the latter gene is analogous to the LIS1 gene, which causes lissencephaly in Miller-Dieker syndrome (
      • Bazan N.G.
      Inflammation: a signal terminator.
      ). Because lissencephaly results from defects in neuronal cell migration, PAF-AHIb may modulate neuronal migration during nervous system development. Moreover, PAF has been found to promote growth-cone collapse, implicating further the phospholipid messenger in developmental neurobiology (
      • Clark G.D.
      • Happel L.T.
      • Zorumski C.F.
      • Bazan N.G.
      Enhancement of hippocampal excitatory synaptic transmission by platelet-activating factor.
      ).
      PAF-AH cleaves the acetate moiety of PAF and comprises an “off” signal when PAF is no longer needed to perform a function or when the concentrations of PAF become neurotoxic. We tested this hypothesis using recombinant plasma-type PAF-AH (rPAF-AH) in primary neuronal cultures, and found that rPAF-AH in fact elicits neuroprotection (
      • Ogden F.
      • DeCoster M.A.
      • Bazan N.G.
      Recombinant plasma-type platelet-activating factor acetylhydrolase attenuates NMDA-induced hippocampal neuronal apoptosis.
      ). The human form of PAF-AH is effective in inhibiting inflammation in nonneural cells (
      • Tjoelker L.W.
      • Wilder C.
      • Eberhardt C.
      • Stafforini D.M.
      • Dietsch G.
      • Schimpf B.
      • Hooper S.
      • Trong H.L.
      • Cousens L.S.
      • Zimmerman G.A.
      • Yamada Y.
      • McIntyre T.M.
      • Prescott S.M.
      • Gray P.W.
      Anti-inflammatory properties of a platelet-activating factor acetylhydrolase.
      ). rPAF-AH elicits concentration-dependent neuroprotection against NMDA-induced apoptosis in hippocampal neurons in primary culture (
      • Ogden F.
      • DeCoster M.A.
      • Bazan N.G.
      Recombinant plasma-type platelet-activating factor acetylhydrolase attenuates NMDA-induced hippocampal neuronal apoptosis.
      ). The criteria used in this study included electron microscopy to monitor nuclear condensation, histone release, TUNEL staining, and DNA laddering. Therefore, rPAF-AH may be an alternative to PAF antagonists in regulating pathologic accumulation of PAF in several conditions involving excitotoxicity, including epileptic brain damage, head injury, stroke, glaucoma, and neurodegenerative diseases. The development of these concepts for therapeutic interventions may be helpful because rPAF-AH is relatively resistant to proteolytic degradation, contributing to sustained bioavailability after administration. Also rPAF-AH is able to hydrolyze oxidized phospholipids, which are also recognized by the PAF receptor (
      • Prescott S.M.
      • Zimmerman G.A.
      • Stafforini D.M.
      • McIntyre T.M.
      Platelet-activating factor and related lipid mediators.
      ). PAF-like oxidized phospholipids accumulate in several pathologic conditions that involve oxidative stress.

      LIPIDOMIC NEUROBIOLOGY ON THE HORIZON

      The significance of PUFAs and phospholipids in the nervous system, particularly their specific involvement in cell signaling, will be greatly expanded by newer experimental approaches. Some of the new avenues of exploration in the nervous system are also applicable to other tissues. Furthermore, newer experimental approaches are already here, and will soon be expanded. Following the genomics era, we are now in “proteomics times,” when not only is the proteome being defined, but metabolomics are emerging. Among the powerful new tools available are lipidomic analyses. Lipidomics is beginning to allow us to precisely define lipid organization, metabolism, and signal transduction in a given cell or part of a cell (e.g., dendrites). This is mainly due to the power, ease, and versatility of what has evolved from the development of electrospray ionization mass spectrometry (
      • Han X.
      • Gross R.W.
      Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics.
      ). The detailed composition of lipid classes and molecular species can now be approached in a more accurate and efficient manner. Moreover, the detailed identification of changes in the lipidome during the development, function, aging, and dysfunctions of the nervous system will be tackled.
      An example of an evolving area of the neurosciences is the understanding of the fundamental inner workings of the dendrites, which contain complex intracellular membranes rich in polyunsaturated phospholipids. Dendrites undergo profound changes during neuronal function, including the membrane vesicular transport of neurotransmitter receptors, ion channels, and other proteins destined to the dendritic spine, where critical postsynaptic elements of neurotransmission are located. Definition of the dendritic lipidome will also define the participation of lipid signaling in dendritic development and in the establishment of synaptic contacts as well as overall dendritic plasticity.
      Although AA is widely implicated in signaling in brain, there are several gaps in our understanding of the release of this fatty acid from membrane reservoirs. Reports that AA is released primarily by G-protein-mediated PLA2 activation remain to be confirmed (
      • Axelrod J.
      • Burch R.M.
      • Jelsema C.L.
      Receptor-mediated activation of phospholipase A2 via GTP-binding proteins: arachidonic acid and its metabolites as second messengers.
      ,
      • Burch R.M.
      G protein regulation of phospholipase A2.
      ). In addition, modulation of PLA2 by Ca2+ and protein kinase needs to be better defined. It is clear that NMDA-receptor activation promotes the release of AA (
      • Dumuis A.
      • Sebben M.
      • Haynes L.
      • Pin J.P.
      • Bockaert J.
      NMDA receptors activate the arachidonic acid cascade system in striatal neurons.
      ), and that a variety of eicosanoids are then generated. The modulatory events that channel AA toward specific eicosanoids are not understood. The endocannabinoid family of lipid messengers will remain an active focus of interest because of the growing evidence of their actions in synaptic function, learning, memory, and other forms of behavior (
      • Piomelli D.
      The molecular logic of endocannabinoid signaling.
      ,
      • Carlson G.
      • Wang Y.
      • Alger B.E.
      Endocannabinoids facilitate the induction of LTP in the hippocampus.
      ).
      The essential fatty acid of the linolenic acid family, DHA, is most highly concentrated in brain and retina. The significance of this polyunsaturated fatty acid, above and beyond its being a target for nonenzymatic peroxidation under various pathologic conditions, will be a focus of continued research. Clearly, DHA-containing molecular species of phospholipids confer a unique environment for ion channels, receptors, transporters, and protein-protein interactions critical in signaling. How these events are regulated, including the synthesis and remodeling of these highly unsaturated phospholipids, is not clearly understood. The enzyme-mediated synthesis of docosanoids raises the possibility of exploring the specific generation of novel DHA-oxygenation messengers that are neuroprotective (
      • Marcheselli V.L.
      • Hong S.
      • Lukiw W.J.
      • Tian X.H.
      • Gronert K.
      • Musto A.
      • Hardy M.
      • Gimenez J.M.
      • Chiang N.
      • Serhan C.N.
      • Bazan N.G.
      Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression.
      ). How these docosanoid messengers are synthesized, through which receptors they elicit their actions, how they are affected by pharmacologic agents, and how the docosanoids themselves might become a base for new drug design are questions for the near future. The use of tandem LC-PDA-ESI-MS-MS-based lipidomic analysis, in combination with other experimental approaches, will greatly contribute to our understanding of the significance of DHA in health and in pathologic conditions.
      The knowledge evolving from lipidomic neurobiology will also be potentiated by multidisciplinary approaches (e.g., multiphoton confocal analysis). Structural neurobiology will also come into play, because the lipidome will provide new insights into the precise stereochemical structure of lipids of excitable membranes, as well as of intracellular membranes. There is also growing evidence of the exquisite signaling interplay among neurons, astrocytes, oligodendrocytes, and microglia. Prostaglandins are among the lipid messengers explored to date as modulators of astrocyte release of glutamate (
      • Bezzi P.
      • Carmignoto G.
      • Pasti L.
      • Vesce S.
      • Rossi D.
      • Rizzini B.L.
      • Pozzan T.
      • Volterra A.
      Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.
      ). Microglia also actively make prostaglandins in response to injury (
      • Minghetti L.
      • Greco A.
      • Cardone F.
      • Puopolo M.
      • Ladogana A.
      • Almonti S.
      Increased brain synthesis of prostaglandin E2 and F2-isoprostane in human and experimental transmissible spongiform encephalopathies.
      ,
      • Minghetti L.
      • Levi G.
      Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide.
      ,
      • Fiebich B.L.
      • Schleicher S.
      • Spleiss O.
      • Czygan M.
      • Hull M.
      Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C.
      ), although prostaglandin E2 may be engaged in neuroprotective mechanisms (
      • Kim E.J.
      • Kwon K.J.
      • Park J.Y.
      • Lee S.H.
      • Moon C.H.
      • Baik E.J.
      Neuroprotective effects of prostaglandin E2 or cAMP against microglial and neuronal free radical mediated toxicity associated with inflammation.
      ). Moreover, the renewed interest in defining the significance of nonneuronal cells in the nervous system (
      • Barres B.A.
      What is a glial cell?.
      ,
      • Hansson E.
      • Rönnbäck L.
      Glial neuronal signaling in the central nervous system.
      ,
      • Villegas S.N.
      • Poletta F.A.
      • Carr N.G.
      Glia: a reassessment based on novel data on the developing and mature central nervous system.
      ) will be greatly enhanced by lipidomic approaches. Overall, the near future will witness breakthroughs using lipidomic neurobiology to more precisely define synaptic lipid signaling that will contribute to a renewed vista of the function of the nervous system as well as of the neurobiology of diseases.

      Acknowledgments

      The authors' studies were supported by US Public Health Service Grants R01EY05121 from the National Eye Institute, R01NS23002 from the National Institute of Neurologic Disorders and Stroke, and P20RR16816 from the COBRE Program of the National Center for Research Resources, National Institutes of Health, Bethesda, MD.

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