Phospholipases and Oxidases

Christian Hölscher Abstract

Memory formation is dependent on a series of biochemical cascades that alter synaptic transmission and neuronal activity. Phospholipases are key enzymes in these cascades that produce second messengers, which interact with a host of target systems, such as transmitter uptake systems, transmitter release, and intracellular calcium stores. One of the main second messengers is arachidonic acid, which also acts as a substrate for lipoxygenases and cyclooxygenases. These enzymes metabolise arachidonic acid to second messengers such as prostaglandins and leukotrienes. All of the transmitters have been shown to be of importance in the induction of synaptic plasticity and in learning and memory formation in rodents. In learning tasks such as a passive avoidance task in day-old chicks, inhibitors of phospholipases prevented the consolidation of memory from 1 h after training onwards, while inhibitors of cyclooxygenases blocked memory consolidation from 2 h onwards. These results show that messengers synthetised by phospholipases and oxidases are most likely part of a serial messenger cascade that underlies memory formation. Such a cascade could enable the system to filter information and enable forgetting before complete consolidation of long-term memory.

Introduction

As has been described in detail in the previous chapters, memory formation is a process that largely depends on neuronal metabolic mechanisms. Neurotransmitters are passed on between neurons and activate specific receptors on the postsynaptic site, and the information has to be transmitted beyond the neuronal membrane. Some receptors are linked to ion channels that promote influx of Ca2+ (a second messenger; see the chapter on Ca2+channels), other receptors are linked to G-proteins that are located on the other side of the membrane inside the cell. These in turn activate enzymes that release second messengers or modulate ion channels. An important family of enzymes that generates second messenger is the group of phospholipases. The activity of the phospholipases induces the release of a range of second messengers. These messengers then can be metabolised further by downstream enzymes, such as the cyclooxygenases. All of these messengers interact with a multitude of target systems: ion channels, receptors, intracellular calcium stores, transmitter release systems, cytoskeleton modifying systems, immediate early gene activation, and more. This chapter will describe some of the known types of phospholipases and their role in the complex network of cellular activities that underlies memory formation.

From Messengers to Molecules: Memories Are Made of These, edited by Gernot Riedel and Bettina Platt. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishers.

Figure 1. A) Shown is the structure of phosphatidylcholine, a phospholipid. Note the saturated fatty acid residue at the C1 position (top) and the unsaturated fatty acid residue at the C2 position (left). The lower right is a choline residue linked to the glycerol-3-phosphate frame. Triacylglycerates do not contain such groups, instead they have 3 fatty acid residues. B) Phospholipases are defined by their enzymatic actions. Shown is the molecule phosphoinositol bisphosphate, which contains inositol-1,4,5-triphosphate as a residue (lower right molecule). This residue is an important second messenger that modulates intracellular Ca2+ concentrations (see text for details). The locations where the phospholipases splice the molecule are shown. For details see Karlson.57

Figure 1. A) Shown is the structure of phosphatidylcholine, a phospholipid. Note the saturated fatty acid residue at the C1 position (top) and the unsaturated fatty acid residue at the C2 position (left). The lower right is a choline residue linked to the glycerol-3-phosphate frame. Triacylglycerates do not contain such groups, instead they have 3 fatty acid residues. B) Phospholipases are defined by their enzymatic actions. Shown is the molecule phosphoinositol bisphosphate, which contains inositol-1,4,5-triphosphate as a residue (lower right molecule). This residue is an important second messenger that modulates intracellular Ca2+ concentrations (see text for details). The locations where the phospholipases splice the molecule are shown. For details see Karlson.57

Phospholipases

What Are Phospholipases?

Phospholipases are enzymes that degrade phospholipids. Phospholipids are glycerolphosphate groups with two fatty acid residues and a functional group (see Fig. 1A). They are lipophilic and part of cell membranes. Depending on the pathway of catabolism one differentiates between several types of phospholipases. Phospholipase type A1 (PLA1) degrades phospholipids by cutting off a fatty acid residue at a defined site (see Fig. 1B for details on phospholipase metabolism). Two molecules are produced that both act as second messengers. A different group is called phospholipase type A2 (PLA2) which separates the substrate at a different site (see Fig. 1B). Furthermore, phospholipase type B, C, and D (PLB-PLD) are known. PLC metabolises phosphoinositol bisphosphate (PIP2) to diacylglycerates (DAG) and inositoltriphosphate (IP3). Both DAG and IP3 are important second messengers. PLD metabolises phospholipids at a different site than PLC (see Fig. 1B)(for further details on phospholipases see refs. 16,36,57,99). The best described phospholipases are PLA2 and PLC and they appear to play the most important roles in neuronal metabolism.

It is important to state that phospholipases are found in all cell types. They are a part of the basic biochemical machinery that is required for cell metabolism. However, neuronal isoforms of phospholipases are known that have particular properties and play specific roles in neuronal communication. These isoforms and their neuron-specific roles will be described in this chapter.

Phospholipases are mostly located near the membranes close to their substrates. However, a cytosolic Ca2+-dependent isoform of PLA2 exists. Similar to y-protein kinase C (PKCy), PKA2 binds to the cell membrane when activated by Ca2+. This isoform is neuron-specific and appears to play an important role in neuronal communication and in the induction of synaptic plasticity (long-term potentiation of synaptic transmission, LTP).70,113

Phospholipases produce two molecules when degrading phospholipids. One is a functional group (such as IP3, or arachidonic acid), the other is diacylglycerate (DAG). All of those molecules act as a second messengers. DAG can activate PKC (see Nogues et al in this book, or ref. 15 for review). The released DAG can be further degraded by a DAG lipase to release more fatty acids.29,73

The type of fatty acids that is released by phospholipase activity depends on the kind of phospholipids that are present in the membranes. In non-neuronal cells (eg. in adipose cells), the types of fatty acids are quite diverse and can be saturated, unsaturated, or poly-unsaturated. In neurons, however, the concentration of the poly-unsaturated fatty acid arachidonic acid (ArA) is relatively high in cell membranes, and the percentage of ArA that is released after stimulation of neuronal activity and transmission is very high compared to other fatty acids, such as oleic acid or linoleic acid.9,15,19

The second messenger ArA has a number of important biological properties. It evokes Ca2+ release from intracellular stores,62,91 increases glutamate release,69 modulates ion channels,107 and inhibits uptake of glutamate in neurons and astrocytes.107,114

How Are Phospholipases Activated?

Phospholipases are activated by several mechanisms. Ca2+ -sensitive PLA types are activated after neuronal activity which opens voltage-dependent Ca2+ channels, or the Ca2+ channel that is associated with the ^-methyl-^-aspartate sensitive glutamate receptor (NMDA receptor). A second mechanism is the direct activation via metabotropic receptors that are linked to a G-protein. G-proteins act as interfaces between metabotropic receptors and intracellular target molecules, such as second messenger generating systems4 or membrane-bound channels.92 One family of metabotropic receptors that plays important roles in neuronal communication is the metabotropic glutamate receptor family (mGluR)10,54 (see also Riedel et al in this book).

As has been described in the section 'Glutamate receptors', there are several subtypes of mGluRs, divided into three main groups. Group I (mGluR1 and 5) is coupled to a PLC via G proteins and modulate the synthesis of inositol-1,4,5-triphos can also be linked to PLD,24,81 but most likely only transiently during development.62

Other metabotropic receptors that are linked to PLC are the acetylcholine receptors. The release of ArA via PLC can be triggered by carbachol, an acetylcholine agonist.101 In primary cortical cultures from mice lacking the muscarinergic type 1 acetylcholine receptor, agonist-stimulated phosphoinositide hydrolysis was reduced by more than 60% compared to cultures from wild type mice.39 Release of ArA can also be induced by activation of a type 2 serotonin receptor.31

It is of interest to note in this context that beta-amyloid fragments, a class of polypeptides that accumulates in the brains of Alzheimer's disease, inhibited the cytosolic PLC in the presence of increased Ca2+ concentrations.102 In another study, it was found that acetylcholine receptors are uncoupled from PLC by beta-amyloid fragments and the production of inositol phosphates was compromised.60

Phospholipase activity is under strict control. Piomelli and Greengard83studied PLA2 activity in in vitro enzyme assays and found that it is modulated by casein kinase II, CaMkinase II, and protein kinase A.

Arachidonic Acid (ArA), a Second Messenger

ArA is one of the main messengers produced by phospholipase activity.73 Unlike other second messengers, the eicosanoids such as ArA and metabolites of ArA are able to leave the cell in which they are generated and act as first messengers on neighbouring cells. Therefore, a role as an intercellular messenger is plausible.84

The conditions for release and the neurophysiological effects of ArA have been investigated in numerous experiments. Evidence for a role of ArA in neuronal transmission in particular will be discussed here.

Release of ArA

As mentioned above, ArA is released by metabolising phospholipids by PLA2 or PLC activity.4 In striatal neurons, ArA was released by coactivation of the a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid sensitive glutamate receptor (AMPA receptor, an ionotropic glutamate receptor subtype) and mGluRs and voltage gated Ca2+ channels.10,95 In primary cultures of striatal neurons, this release was sensitive to quinacrine, an inhibitor of PLA2, that also blocked LTP in this preparation. An inhibitor of diacylglycerol (DAG) lipase, another source for ArA release,29,73 had no effect on LTP formation at early time points. 9

In the hippocampal slice of the rat, NMDA receptor activation caused release of ArA and oleic acid. This release was inhibited by inactivation of PLA2, or by NMDA receptor blockade with MK-801 (dizocilpine).82

Other studies showed that release of ArA following activation of the NMDA receptor is due to the activation of a Ca2+-dependent PLA2. For example, the NMDA- stimulated ArA release in primary cultures of rat hippocampal neurons was inhibited by the NMDA receptor channel blockers Mg2+, 5-amino phosphonovalerate (AP5), or the PLA2 and lipoxygenase inhibitor Nordihydroguaiaretic acid (NDGA).92,103

These results suggest that there is a chain of events that starts with the activation of NMDA receptors, an increase of intracellular Ca2+ levels, and the activation of Ca2+-dependent phospholipases that induce ArA release.

In a different study, it was observed that the fatty acids ArA, 12-hydroxy-6,8,11,l4-eicosatetraenoic acid (12-HETE), and 12-hydroperoxyeicosatetraenoic acid (12-HPETE) were released after LTP induction in the rat in vivo. However, the concentrations of 12-HETE release declined already after 1 h to baseline,8 while ArA levels remained high. In a follow up study, after the induction of LTP in the hippocampal slice, ArA concentration in the postsynaptic membrane fraction increased, due to PLA2 activity in the first minutes after induction. 45 min and 3 h later, PLC activity had been responsible for the release.19 This shows that PLC plays a role in the LTP-induced increase of ArA, but the release is later than PLA2 dependent ArA release. In synaptoneurosomes of rat cortex neurons, 30% of arachidonic acid release was inhibited by neomycin, an inhibitor of PLC, and 60% by quinacrine, an inhibitor of PLA2. The effect was additive when both inhibitors were given.

Time Course of Release

After the induction of LTP in the hippocampal slice, ArA concentration in the postsynaptic membrane fraction increased. This increase had a defined time course. Release ofArA that was due to PLA2 activity, which took place in the first 2.5 minutes after induction, but went down shortly thereafter. The PLC and DAG lipase-linked pathway of ArA release had a different time course. ArA concentrations were increased as late as 45 min and 3 h after stimulation.19,20 This later wave of ArA release can hardly be due to the time that PLC or DAG need to release ArA. The data might show a second step needed for LTP consolidation by prolonging duration of a second messenger signal.

Targets of ArA

ArA has a number of effects in neurons, from channel modulation to reuptake inhibition. All these different activities seem to work towards changing the basal state of the neuron to a state of increased excitability.

In the rat hippocampal slice, ArA can directly mobilise intracellular Ca2+ independent of IP3,68'91 in rat hippocampal synaptosomes, ArA and metabolites ofArA67-69 were able to stimulate hydrolysis of PIP268'69 and release of glutamate.69 ArA inhibits uptake of glutamate in neurons and astrocytes in cultures of rat cortex tissue. The uptake mechanisms in neurons are 20 times more sensitive to modulation than the ones in astrocytes.114 In a patch-clamp study, ArA strongly inhibited glutamate uptake in glial cells.6

Ci's-fatty acids such as ArA can activate protein kinase C (PKC), an enzyme involved in key processes of LTP formation, as well as in memory formation (see Nogues et al in this book). As described before, PLC turns PIP2 over into IP3 and DAG. Lester et al65 and Kato et al58 showed that ArA and DAG act synergistically to activate PKC in vitro and in vivo. In another study, ArA increased B-50 phosphorylation,93 a presynaptic protein which is a PKC substrate and which is associated with transmitter release.27,93 ArA is rapidly cleared from the cytosol, which is important for the deactivation of the messenger signal after the transmitter-releasing stimulus has stopped.73

ArA and Metabolites of ArA As Transmitters and 'Retrograde Messengers' in Synaptic Plasticity

Apart from playing a role as intracellular messengers, a further role that these messengers might play is that of a feedback signal to the presynaptic site (Fig. 2). Since changes can be observed at the presynaptic site after induction of LTP, it has been speculated that a feedback signal from the postsynaptic site must exist to relay the information that the presynaptic neuron has successfully activated the postsynaptic neuron.109

Figure 2. Proposed model ofincrease or decrease of transmitter release after the activation of phospholipases and the postsynaptic release of ArA. Presence of ArA reduces presynaptic Ca2+ influx which will in turn reduce transmitter release. If ArA is present along with DAG and Ca2+, PKC will be activated which causes increases in transmitter release. The DAG could be released by a PLC which is linked to a presynaptic mGluR. The IP3 released by the PLC increases intracellular Ca2+ levels via internal store depletion. See text for details.

Figure 2. Proposed model ofincrease or decrease of transmitter release after the activation of phospholipases and the postsynaptic release of ArA. Presence of ArA reduces presynaptic Ca2+ influx which will in turn reduce transmitter release. If ArA is present along with DAG and Ca2+, PKC will be activated which causes increases in transmitter release. The DAG could be released by a PLC which is linked to a presynaptic mGluR. The IP3 released by the PLC increases intracellular Ca2+ levels via internal store depletion. See text for details.

In sensory neurons of the sea snail Aplysia, where synaptic plasticity appears to be expressed mostly at the presynaptic site, extensive studies of the activity of ArA in neuronal communication have been conducted. Metabolites ofArA are released by sensory neurons in response to inhibitory transmitters and directly target a class of K+ channel, increasing the probability of their opening. This causes hyperpolarisation and shortening of action potentials. In vertebrate neurons, other types of K+ channels have been found to be sensitive to ArA and to other polyunsaturated fatty acids.107

In the motor end plate of Xenopus, an ArA metabolite has been found to play a role as a retrograde messenger. In the muscle, a G-protein dependent release of ArA into the membrane was observed. Injecting a non-hydrolysable GTP analogue into the muscle to activate the G-protein increased spontaneous firing rates of the innervating neurons. Since the muscle is the postsynaptic site and the motor neuron the presynaptic site, retrograde communication must have taken place. Analysis of the ArA metabolites in the neurons identified 5-HPETE as a prime candidate.40

Prostaglandins (PG) were shown to be excreted after activation of neurons that use noradrenaline (NA) or acetylcholine (ACh) as transmitters. The prostaglandin PGE seems to have a negative effect on NA release. Indomethacin, a cyclooxygenase (COX) inhibitor which prevents PG formation, increased NA release. In intestines, PG increased ACh evoked muscle contraction, and indomethacin produced a decrease of contractions in the gut. While PG played a negative feedback role in the NA system, it activated the ACh system in the peripheral nervous system in a positive feedback manner41

These ArA and ArA metabolite -related changes in synaptic transmission suggest that these messengers indeed travel between neurons and act as some kind of retrograde messenger.

How to Ensure Selectivity ofArA Messenger Activity

If ArA and metabolites would change synaptic activity in an indiscriminate manner, any ArA molecules that diffuse to other neurons than to the target neuron would create a chaotic situation by indiscriminately upregulating neuronal transmission. There is evidence that the target neurons require more than one signal for modulation to prevent a non-selective change in synaptic activity. ArA can modulate the release of the neurotransmitter glutamate. Ca2+-dependent glutamate transmitter release was found to be inhibited by ArA in a PKC independent fashion in cerebrocortical synaptosomes.42,44 This effect seemed to be due to the reduction of Ca2+ entry into the presynaptic site that was caused by ArA.43 Yet, if the preparation was incubated with ArA and low concentrations of phorbol esters simultaneously, PKC was activated, and glutamate release potentiated.73 The same result was obtained when incubating the preparation with ArA and a synthetic DAG analogue. The increase of glutamate release was Ca2+ dependent.115 In the presence of ArA, only very low concentrations of phorbol esters were needed to activate PKC and to increase glutamate release. The authors suggest that ArA serves as a retrograde messenger which only increases transmitter release if it coincides with a second signal such as Ca2+ entry into the presynapse or diacylglycerol formation.45,46 This mechanism would ensure that only previously active neurons are upregulated in their transmitter release, as these neurons have increased intracellular Ca2+ concentrations when the ArA signal arrives. There is further evidence for such a mechanism. A PKC substrate which is associated with transmitter release is the presynaptic protein B-50.27 Perfusion with high concentrations of ArA increased B-50 phosphorylation in a synaptoneurosome preparation. Addition of Ca2+ to the medium facilitated this, presumably because activation of the Ca2+ -sensitive PKC required only low amounts of ArA to increase its activity.93

This 'simultaneity detection system' is not unique. Similar modes of operation have been suggested for other molecules, such as

See also Bourne and Nicoll13 for a discussion on coincidence detecting systems in the nervous system. Since ArA will diffuse into neighbouring cells and synapses which are not activated at the same time as the neuron that releases ArA, a potentiation of that inactive neuron would not be sensible. Instead, the inhibiting effect could suppress neurons that are not firing at the same time as the active neurons which causes ArA release. If the neighbouring neuron is not active, or if this activity is not in synchrony with the ArA releasing neuron, it is most probably not working in cooperation with the active neuron. Suppression of such 'non-cooperating' neurons thereby keeps spontaneous firing 'noise' and interference down. If ArA reaches a synapse that recently has been active, Ca2+ levels will be high due to activation of voltage dependent channels, and diacylglycerol will be available, for instance via activation of presynaptic glutamate metabotropic receptors that are linked to a PLC.23,44,45

Contradictory results observed in the hippocampal slice preparation could be explained by this model. Perfusion of the slice preparation with the mGluR agonist 1S,3R-1-amino-cyclopentyl-1,3-dicarboxylic acid (1S-3R-ACPD) produced LTP in area CA1.11 O'Mara et al77 and Collins and Davies, 2 however, did not find a potentiation of transmission after perfusion with 1S-3R-ACPD in the CA1 area. Instead, a depression (LTD) was observed. Only when ArA was perfused along with 1S-3R-ACPD, a potentiation developed over 30 min. ArA alone also created a slight depression of field excitatory postsynaptic potentials (EPSPs).22 A similar result was published by Zhang and Dorman115 who found that KCl-induced depolarisation increased glutamate release when ArA and a diacylglycerol analogue were added. This process was Ca2+ dependent.

The Role of ArA in LTP Formation

In various experiments, ArA and metabolites of ArA were shown to play a role in the induction of LTP In in vivo studies of the dentate gyrus of the rat, NDGA blocked the synaptic component of LTP and the associated increase in release of glutamate. LTP produced a sustained increase of ArA release that was blocked by NDGA.68 After the induction of LTP in the hippocampal slice, ArA concentration in the postsynaptic membrane fraction increased. The release was PLA2 and PLC dependent.19 The specific PLA2 inhibitor bromophenacyl bromide caused a large reduction in the magnitude of LTP in the CA1 field of the hippocampal slice.70

In vivo blockade of PLA2 by quinacrine in the hippocampus of the rat inhibited oleate release and LTP formation, the effect was reversible by application of oleate.66 In the CA1 region of rat slices, ArA produced LTP and LTD, which was inhibited by NDGA28,76 or AP5.76 ArA together with 1-oleyl-2-acetyl glycerol (a DAG analogue) induced LTP in the CA1 region of guinea pig slices in low Mg2+ concentration, this was blocked by the phospholipase inhibitors neomycin and 2-nitro-4-carboxylphenyl-N,N-diphenylcarbamate (NCDC).59 ArA induced activity-dependent LTP in the hippocampus of the rat in vitro and in vivo. ArA itself was not capable of inducing synaptic plastic effects. However, weak tetanic stimulation that did not potentiate synaptic transmission by itself was required to induce LTP in the slice.8,76,108

It seems to follow from these results that ArA is of importance in LTP induction. However, the comparatively slow effect of ArA in LTP induction made Williams et al108 suggest that ArA only plays a role as a 'slower' retrograde messenger, while faster messengers such as NO precede the ArA signal (see also Fig. 3).

Learning Experiments: Evidence for the Role of Phospholipase Activity in Memory Formation

Inhibiting the activity of phospholipases in learning experiments have shown that these enzymes are of importance for memory formation or consolidation. In a one -trial passive avoidance task of the chick, animals learn to associate the unattractive taste of methylanthranilate and the colour of a bead. The animals only need one trial to form the association. This task offers the advantage of being able to 'time' the steps of memory formation.

In one study, bilateral intracerebral injections of the PLA2 and lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) or the PLA2 inhibitor aristolochic acid (AST) were made into the intermediate medial hyperstriatum ventrale (IMHV), an area that is of importance for the formation of memories formed by learning this task. Pre-training injections of either inhibitor produced lasting amnesia for the avoidance response. The onset of amnesia

Presumed mechanism of ArA activity in synaptic plasticity

presynaptic site postsynaptic site

Figure 3. Schematic graph depicting the activity of the presumed retrograde messenger ArA. After NMDA receptor activation (in the cortex, or hippocampus), or synchronous AMPA and mGluR activation (in the striatum), ArA is released via PLA2 (NMDA receptor dependent) or PLC (mGluR dependent) activation. ArA causes intracellular Ca2+ release and increases transmitter release, while transmitter re-uptake is inhibited. Additionally, membrane fluidity is changed by the increased percentage of unsaturated fatty acids, which modulates receptors activity. This enhances synaptic plasticity.

for both inhibitors NDGA and AST was at 1.25hr post-training (see Figs 4 and 5). Injection of drugs post-training had no effect on retention.53 The results support the theory that ArA release is a necessary step in the relatively early events mediating the synaptic plasticity associated with memory formation. Other phospholipase inhibitors have produced similar results. For example, the PLA2 inhibitor bromoenol lactone impaired spatial memory formation in mice.32

In further support of the notion that the release of ArA plays an important role in memory formation, a study of young and aged rats showed that aged rats with learning impairments had lower concentrations of ArA in their brain tissue, while saturated fatty acids were increased.106

Chicks were given bilateral intracerebral injections of NDGA (5^l of a 4mM solution) or saline 30min before training and tested at the stated times post-training (n=13-18 per group; *=p<0.05). For details see Hölscher and Rose.53

Chicks were given bilateral injections of 5 ^l of a 4 mM AST solution or saline 30 min before training, testing was after 1, 1.25 hr or 24 hr subsequently. (n=15-18 per group; *=p<0.05). For details see Hölscher and Rose.53

A Different Second Messenger Released by PLA2: Platelet-Activated Factor (PAF)

As an example for another cellular messenger that is released by PLA2 and that plays important roles in memory formation is the platelet-activating factor (PAF; 1-O-alkyl-2-acyl-sn-3-phosphocholine). Some results of importance are that the induction of LTP can be blocked by a PAF receptor antagonists in area CA1 of the rat hippocampus,2 in the dentate gyrus,59 and in other areas of the brain.37 Furthermore, PAF was found to inhibit ionotropic GABA receptor activity17 and to increase glutamate transmitter release, perhaps as a retrograde synaptic messenger.63 A mouse strain lacking the PAF receptor also showed impaired LTP in some areas of the hippocampus.18 PAF furthermore couples synaptic events with gene expression by

100 80

20 0

time of testing a iter training (h)

Figure 4. Effect of the phospholipase inhibitor NDGA on retention of a one-trial passive avoidance task when injected pre-training and tested post-training. Animals were trained to avoid to peck a bead dipped in an unpleasant substance. Shown are the percentages of animals pecking or avoiding the bead that they were trained on. Avoidance indicates memory retention, while pecking suggests that the animals had forgotten the task.

stimulating a FOS/JUN/AP-1 transcriptional signalling system, as well as transcription of COX-2 (inducible prostaglandin synthase, see below).

Most interestingly in the context of this chapter is the indication that PAF enhances memory formation if infused into the hippocampus or other learning-related brain areas.56,104

Oxygenases That Are of Importance in Memory Formation

ArA serves as a substrate for cyclooxygenases (COX) and lipoxygenases, leading to products such as prostaglandins and leukotrienes (see Fig. 8), which are cellular messengers themselves.23,57,73 In the following sections, a brief description of their role will be given to cast some light on other parts of the biochemical cascades that form the basis of memory formation.

Lipoxygenases

ArA metabolism via the lipoxygenase pathway leads to formation of a family of messengers, such as leukotrienes. The first step in leukotriene biosynthesis is catalyzed by the enzyme lipoxygenase and results in the formation of hydroperoxy-6,8,11,l4-eicosatetraenoic acid (5-HPETE) in the case of 5-lipoxygenase, which may be converted enzymatically (via the action of a peroxidase) or nonenzymatically to the corresponding hydroxy acid, 5-HETE (see McMillan et al, 72 for a summary).

A Ca2+-dependent lipoxygenase has been described.72 This isoform is activated by increased intracellular Ca2+levels, as are observed after activation of neurons, due to opening of voltage dependent Ca2+ channels, opening of NMDA receptor associated Ca2+ channels, or due to release of Ca2+ from intracellular stores. Activation of neurons therefore could activate or prime the lipoxygenase through Ca2+ influx, as it is the case with many other Ca2+ dependent enzymes. In the canine brain, a 12-lipoxygenase was found to be widely distributed: it was localised in hippocampus, cortex, and basal ganglia.75 Lipoxygenases and their products (e.g., 15-HETE) also have been found in the chick cerebrum and cerebellum.38

As described earlier, extensive studies of the activity of ArA metabolites in neuronal communication have been conducted. 12-lipoxygenase derivatives of ArA are released by sensory neurons in response to inhibitory transmitters and directly target a class of K+ channel, increasing the probability of their opening. This causes hyperpolarisation and shortening of action potentials. In vertebrate neurons, other types of K+ channels have been found to be

time of testing a iter training (h)

Figure 5. Effect ofthe phospholipase inhibitor aristolochic acid (AST) on retention when injected pre-training (4mM) and tested post-training.

sensitive to ArA, to lipoxygenase products, and to other polyunsaturated fatty acids.107 Piomelli et al84 isolated lipoxygenase metabolites of ArA, 5-HETE and 12-HETE, from Aplysia nervous tissue, as well as cyclooxygenase products, prostaglandins such as PGE2 and PGF2a.

In a behavioural study, the lipoxygenase product 1-oleoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylcholine (ODHPC), a phosphatidylcholine, enhanced discriminatory shock avoidance learning in rats. Also, ODHPC enhanced LTP of population spikes in the CA1 region.55 These results indicate that lipoxygenase products are involved in the induction of synaptic plasticity and memory formation.

Cyclooxygenases

What Are Cyclooxygenases?

Cyclooxygenases (COX) convert ArA into a variety of metabolites, mainly into prostacyclins, prostaglandins, or thromboxanes57 (see also Fig. 7). The enzymes oxidise two double bonds with two oxygen molecules and form a two-ring system (PGG2) in an epoxy-reaction. This metabolite is very unstable and can be converted to any of the three main groups of COX products.

COXs are found in all tissues. Enzyme concentrations vary greatly depending on tissue type.99 At least two isoforms exist. COX-1 is a constitutive enzyme, the enzyme is always present and the level of expression is regulated in a steady state mode. The enzyme is present in neurons, macrophages, fibroblasts, and endothelial cells.

COX-2 is an inducible form of cyclooxygenase. It is induced by pro-inflammatory agents including interleukin-1 P and lipopolysaccharides. Anti-inflammatory steroids such as dexam-ethasone inhibit the induction of COX-2 but do not affect levels of COX-1. Additionally, COX-2 synthesis can be induced by a number of other molecules of which cAMP, interleukin-1, leukotrienes, and ArA are noteworthy. The expression of the enzyme is furthermore modulated by PKC, phorbol esters and DAG-induced prostaglandin synthesis. COX-2 is predominantly expressed in endothelial cells, fibroblasts, and macrophages, and above all, in neurons.1,14,111 In an in vivo dialysis study in the hippocampus, an increase in the release of prostaglandins after stimulation of the NMDA receptor was observed.64 The time course of COX-2 induction is surprisingly constant, the time measured after COX had been induced by various agents was always around 2 h.26

Figure 6. Effect of inhibitors of cyclooxygenase inhibitors on retention of a one-trial passive avoidance task when injected pre-training and tested post-training.

Learning Experiments: Evidence for the Role of COX-2 Activity in Memory Formation

To test if arachidonic acid is metabolised to other messengers by COXs, the effect of inhibitors of these enzymes were tested in a one-trial passive avoidance task in the chick. The cyclooxygenase inhibitors Indomethacin, Naproxen, and Ibuprofen caused amnestic effects at all concentrations tested when injected intracerebrally (i.c.) before training. The onset of amnestic effects was always 2 h after training, independent of drug type, concentration, and injection time before training (see Fig. 6, and ref. 47). In a second study, the injection of the selective COX-2 inhibitor, SC58125 (see ref. 97) or dexamethasone before training showed amnestic effects for training on a one-trial passive avoidance task at 2 h but not 1 h after training.48

Figure 7. Scheme of metabolism of ArA via the cyclooxygenase pathway II to prostaglandins (PG) or prostacyclins (PGI2) and thromboxanes (TX) as an example of the diversity of these pathways. Seven major pathways are known, three lipoxygenase (5-, 12, and 15-lipoxygenase), two cyclooxygenase, and two P-450-cytochrome oxidase pathways.36'99

Figure 7. Scheme of metabolism of ArA via the cyclooxygenase pathway II to prostaglandins (PG) or prostacyclins (PGI2) and thromboxanes (TX) as an example of the diversity of these pathways. Seven major pathways are known, three lipoxygenase (5-, 12, and 15-lipoxygenase), two cyclooxygenase, and two P-450-cytochrome oxidase pathways.36'99

A follow up study analysed the release of COX products (prostaglandins) from brain tissue using the ELISA technique. The release of cyclooxygenase products into the extracellular fluid was measured at 1, 2, and 3 h post-training. An increase of prostaglandin production was see after 2 and 3 h, but not after 1 h. A cyclooxygenase inhibitor, ibuprofen, inhibited the training-dependent increase of cyclooxygenase products 2 h and 3 h after learning when injected pre-training, as did dexamethasone which prevents cyclooxygenase induction. The selective COX-2 inhibitor, SC58125 had the same effect.97

The delay of release 2 h after training suggests that the drugs prevent induction of COX-2, which takes around 2 h.26'74 The results indicate that COX-2 products play a role in memory consolidation in the chick when learning this task. They also suggest that COX-2 induction plays a role in memory consolidation several hours after the learning experience (see Fig. 7, and refs. 47,48).

These studies of the role of COX in memory formation in chicks have been corroborated by similar studies in rats, suggesting that the molecular mechanisms are similar in different species. For example, it was shown that the effects of the Cox inhibitor ibuprofen impaired spatial learning in rats as well as the development of LTP in vivo.78 In another study it was shown that conditioning of animals in a lever-pressing task was dependent on COX activity. Interesting enough, this investigation concluded that the main effect of the cannabinoid (CB1) receptor agonist tetrahydrocannabinol on behaviour is mediated through COX activity as it was blocked by COX antagonists such as diclofenac or indomethacin.11

In transgenic mice that overexpressed COX-2 in neurons, memory impairments were observed.1 This finding suggests that an uncontrolled release or an unphysiologically high level of COX products interferes with mechanisms necessary for normal memory formation.

Chicks were given bilateral intracerebral injections of the cyclooxygenase inhibitors indomethacin, naproxen, ibuprofen, or saline 30min before training and were tested at the stated times subsequently (n=13-18 per group; *=p<0.05, **=p<0.01). For details see ref. 47.

Cooperation of ArA and Metabolites of ArA As Messengers in Neuronal Systems

The question arises why several neuronal messenger systems are in operation that appear to serve similar functions. One argument in favour of parallel messenger systems is that the system is very stable through redundancy. In fact, biological systems tend to make use of redundancy when processes are concerned that are essential for life. A different possibility is that only a subset of neurons use a particular type of messenger while others use a different type to avoid cross talk. This appears to be the case for the neurotransmitter nitric oxide synthase (NOS), which is produced in the different areas of the hippocampus of the rat at different quantities50. A different line of arguments comes from a theoretical approach. Neuronal signalling networks were constructed with experimentally obtained constants and analysed by computational methods to understand their role in complex biological processes. These networks exhibited emergent properties such as integration of signals across multiple time scales, generation of distinct outputs depending on input strength and duration, and self-sustaining feedback loops. Feedback can result in bistable behaviour with discrete steady-state activities, well-defined input thresholds for transition between states and prolonged signal output, and signal modulation in response to transient stimuli. These properties of signaling networks raise the possibility that information for "learned behaviour" of biological systems may be stored within intracellular biochemical reactions that comprise signalling pathways.7 Hence, a multitude of biochemical inputs can be more than just the sum of its parts and can produce surprising effects and produce novel qualities.

Evidence for the parallel use of different signal systems has been collected in many investigations in several species. In the marine mollusc Aplysia, Piomelli et al83 identified the lipoxygenase pathway products 5-HETE and 12-HETE as well as COX products such as the prostaglandins PGE2 and PGF2a as neurotransmitters. In the hippocampal slice of the rat, 12-HPETE and 12-HETE release was increased after LTP induction.68,9 In postganglionic neurons, both messengers NO and ArA modulate calcium currents. In one study, the effect of the NO generating drug nitroprusside was abolished by the NOS inhibitor NG-nitro-L-arginine methyl ester (NAME), while the effect of ArA was unchanged by NAME,61 showing that both systems are independent from each other. Finally, in the sensory pain-pathway, NO86 and COX products3 act as messengers, they are co-located with dopamine, Histamine and neuropeptides.

These results show us that several signaling systems act simultaneously in neuronal communication. So what does this tell us about the different function of these transmitter system? Perhaps we have to look at other parameters. If we go back to the one-trial passive avoidance task (PAT) of the chick we can have a look at the timing of memory formation and consolidation.

The Timing of Memory Formation

Discrete Time Windows of Messenger Activity

Studies with the NOS inhibitors nitro-L-arginine (L-NARG) and 7-nitro indazole49'51'52 produced amnesia for the one-trial passive avoidance task in the chick. The interesting observation here was that injection of the nitric oxide synthase inhibitors pre-training resulted in amnesia for the task after 15 minutes of training.

As shown earlier, inhibitors of phospholipases are effective from 1 h onwards after training,53 the time point when release of arachidonic acid into the extracellular fluid was the highest.20

Injection of cyclooxygenase inhibitors before training produced amnestic effects from 2 h onwards after training. 7 The learning-related increase of release of prostaglandins observed in the saline group followed the same time course, i.e., a large increase of release after 2 h post-training compared to 1 h post-training values.48 The delay of 2 h can be explained by the time the induction process of COX-2 takes, as measured in different cell types. As mentioned before, the time course takes about 2 h.26

It appears that nitric oxide, arachidonic acid, and arachidonic acid metabolites act together as messengers, lined-up in a linear cascade. Nitric oxide is an uncharged molecule that is released quickly and that diffuses across cell membranes without much resistance.33,100 In contrast, lipids are released rather slowly and tend to 'stick' to membranes for a longer time.36,99 Therefore, they are better suited as longer-lasting messengers. Arachidonic acid is a molecule that is fairly unstable due to its double bonds, it is not only metabolised by oxidases but by oxygen radicals or other free radicals.36 Hence, ArA is not a good messenger for longer time durations. Oxidase products, however, have a longer life span but take longer to be synthetised.26 These properties could explain the observed time windows of memory formation. Hence, induction of key enzymes in neurons appear to be of importance for memory consolidation and synaptic plastic processes. This has been shown before in experiments that analysed the time-course of development of LTP in the hippocampus. Several enzymes such as Ca2+/ calmodulin-dependent kinase II, and y-PKC were found to be induced in the course of LTP consolidation. 05

Figure 8 summarises the three different time courses of amnesia development after drug injection. One has to postulate that each messenger has a peak of production and is present only in low concentration before and after this peak. Otherwise, a compensation of effects due to loss of one messenger by other messengers should occur, and no amnesia would develop. This might well be the case in other areas of the chick brain or in brains of other species.

Defined Steps in Memory Formation

Defined time windows for activity of drugs in learning-tasks have been observed before. Rosenzweig et al90 as well as Gibbs34 discriminate between different steps of memory formation in the chick. They differentiate between three phases: short term memory (0-15 min), intermediate memory (15-55 min), and long-term memory (>55 min). Different drugs can interrupt one or several phases of this cascade, and produce amnestic effects at the end of each phase, which has not been interrupted. This interruption is independent of the concentration of drugs, or the time point of injection before training (see also ref. 90). Rose and collaborators found another time point that appears to be intrinsic in memory consolidation in the chick. The second wave of glycoprotein synthesis that was first described by Pohle et al85 in the rat and was later found

—o—

nitric oxide

ArA (or -metabolite)

— o----

COX metabolite

O 5 min 30 min lh 1.25h 1,75h 2 h 3 h

time

Figure 8. Scheme of possible production levels of neuronal messengers over time. This scheme is based on the data presented in this chapter. A first wave of NO is proposed since inhibition of NO synthase produced amnestic effects after 30 min. After NO production starts to decrease, ArA is released in greater quantity with a rapid increase after about 1 h and decrease after about 1.75 h. ArA is most likely metabolised in this time period, and the release of newly formed ArA is inhibited, since an amnestic effect becomes unmasked after 1.75 h when injecting COX inhibitors. The third wave, which consists of cyclooxygenase metabolites, starts after 1.75 h and continues for an unknown time period. For details see Hölscher and Rose;53 Hölscher.47'48

in the chick.15,96 This second wave of expression is found 6 h to 8 h post-training. Interestingly enough, an increase of spontaneous neuronal bursting in that time period had been observed35 (see refs. 88,89 for a review).

Defined phases in the metabolic cascade of memory formation in mammals are not unknown either. In memory formation of rats79 or humans,5 a division between short-term, long-term, and working memory has been suggested. McGaugh71 noted as early as 1968 that there are defined time windows for sensitivity of drugs injected in rats tested in passive avoidance training tasks. Pohle et al85 observed two defined time windows of [3H]fucose incorporation in the area CA1 and CA3 after training of rats in a discrimination task. Since fucose is incorporated into glycoproteins, a second phase of memory-related glycoprotein synthesis might be expressed in hippocampal neurons. Regan87 reported that intraventricular infusion of antibodies to neuronal cell adhesion molecules (N-CAM) disrupted consolidation of a passive avoidance response in the rat when administered between 6 and 8 h post-training.

A Potential Role for Defined Time Windows of Messenger Systems in Memory Formation

Why do distinct time windows for metabolic processes in memory formation exist? Each step in the biochemical cascade continues only for a limited amount of time, and after that, the activity of the receptor/enzyme is not essential for establishing the memory trace any more. Then, inhibiting the activation of the specific step in the chain of events cannot prevent memory consolidation. But what role would these defined time windows play in memory formation?

Clearly, it is of importance to filter information before it is stored in long-term memory. To be able to filter memory input, several steps of consolidation are required. Initial activation by NO could be a form of short-term memory, or a priming step, for further consolidation that needs input of a different quality or quantity to be maintained past this stage. The PLA2 linked ArA release might be the next step in this mechanism, which has to be followed by the 3rd step, PLC activation, to ensure intermediate memory formation. A fourth step could be the metabolism ofArA by COXs or lipoxygenases to messengers with longer half-lives that would ensure a long lasting signal. Memory consolidation could be terminated at any of these steps. If the conditions for long-term memory formation are not met, this signal could be interrupted and the memory trace would be retained only for a limited amount of time. Clearly, memory can be retained for different lengths of time. Not all information has to be kept, or should be kept, in storage for the whole lifetime. If the activation of the NMDA receptor and the subsequent activation of protein kinases, and the synthesis of glycoproteins, always resulted in long-term memory formation, the phenomenon of forgetting would be hard to explain. In a weak-stimulus passive avoidance task of the day old chick as employed by Bourne et al12 or by Crowe et al25, amnesia is observed after about 5 hours, before a second wave of glycoprotein synthesis occurs,12,96 or the main part of post-training neuronal bursting is observed.35 A termination of memory formation at this step is possible. In other words, even if the synthesis of glycoproteins, the production of immediate early genes, the upregulation of receptor sensitivity via protein kinase activity already has happened, the mechanism can still be stopped, and memory formation halts. All these different biochemical steps appear to act independently from each other, but all are required in order to establish a long-term change in synaptic efficacy. Only then, blocking the synthesis of a retrograde messenger as late as 2 h post-training could evoke amnesia. The results presented in this chapter therefore not only illuminate the process of learning and memory formation but also potential mechanisms of forgetting.

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