Pp2a

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Initial studies employed the drugs Okadaic Acid (OA) and Calyculin A (CalA), agents widely acknowledged to be specific and selective phosphatase inhibitors.54 Both drugs disrupted retention from two different time points depending on the concentration of drug administered and the time relative to training at which administration took place.17,224 At concentrations of OA that might be expected to selectively inhibit PP2A (0.5 nM per hemisphere), bilateral intracranial administration of either drug close to the time of training induced retention deficits from approximately 40-50 minutes post-training. This most probably indicates a role for PP2A in a process that normally occurs at or before this time relative to training and that is critical to a subsequent stage of memory formation.

The apparent requirement for PP2A was later shown to be confined to the left hemisphere of the chick. Administration of putative PP2A inhibitors to the right hemisphere was without effect on retention levels at any time tested, while administration to the left hemisphere mimicked the effects observed following bilateral administration (Bennett, Moutsoulas, Lawen & Ng, unpublished observations). These data are summarised schematically in Figure 3. The effects of PP2A inhibition were observed also to vary with the strength of reinforcement associated with the training experience. While left hemisphere inhibition of PP2A following strongly reinforced training resulted in significant memory loss (Fig. 3), left hemisphere inhibition following weakly reinforced training facilitated memory formation (Bennett, Moutsoulas, Lawen & Ng, unpublished observations).

Previous data from our laboratory have demonstrated that PKC inhibition has an amnestic effect following PAL which is evident by 25-30 minutes post training and which is also lateral-ized to the left hemisphere.228 Transient changes in phosphorylation of the PKC substrate, growth associated protein of ~50 kD weight (GAP-43), are also observed to occur at around this time in critical areas of the chick brain,1,226 and within a similar time frame following single-trial inhibitory-avoidance learning in the rat.28 The amnestic effect of PP2A inhibition occurred soon after the effect observed following PKC inhibition. As PP2A is known to de-phosphorylate GAP-43,168 it is possible that PP2A may be a critical element in a proposed reinforcement-associated memory switch believed to require PKC activity.143 Dephosphoryla-tion of GAP-43 may be just as essential as its phosphorylation. The memory 'switch' is believed to determine whether information is retained beyond approximately 30 minutes post-training, but evidence supporting this speculation is circumstantial so far. Other candidate substrates for PP2A include a number of kinases that are dephosphorylated by this enzyme,7 the protein called myristoylated alanine-rich C kinase substrate (MARCKS), which shows increased phosphorylation following imprinting in the chick;173 the protein RC3/neurogranin, which shows enhanced phosphorylation during LTP;30 and the protein tau, which shows altered dephos-

Figure 3. Schematic diagrams depicting the effects of inhibiting PP2A in one hemisphere of the chick following strongly-reinforced learning (SRL). Groups of chicks were trained to discriminate between a previously aversive red coloured bead and a previously pleasant blue coloured bead. They were then administered either OA (0.5 nM) or CalA (0.1 nM) into the left (LH) or right (RH) hemisphere and retention was tested at the specified time post-training. Groups of chicks that received either drug in the left hemisphere showed good retention, indicated by selective avoidance of a red coloured bead, only up until approximately 40-50 minutes post-training. All groups tested after this time showed significant retention deficits. Groups of chicks administered either drug in the right hemisphere showed good retention at all times of test.

Figure 3. Schematic diagrams depicting the effects of inhibiting PP2A in one hemisphere of the chick following strongly-reinforced learning (SRL). Groups of chicks were trained to discriminate between a previously aversive red coloured bead and a previously pleasant blue coloured bead. They were then administered either OA (0.5 nM) or CalA (0.1 nM) into the left (LH) or right (RH) hemisphere and retention was tested at the specified time post-training. Groups of chicks that received either drug in the left hemisphere showed good retention, indicated by selective avoidance of a red coloured bead, only up until approximately 40-50 minutes post-training. All groups tested after this time showed significant retention deficits. Groups of chicks administered either drug in the right hemisphere showed good retention at all times of test.

phorylation in the brains of patients with Alzheimer's disease,186 a disorder characterised by memory loss.

It remains difficult to elucidate roles for PP1 in complex systems, as all available cell-permeable inhibitors also curtail PP2A activity and most do so with greater potency. While some inhibitors, such as OA, differ in their potency towards the two enzyme classes in vitro,63 it is not generally considered appropriate to generalize from in vitro to in vivo contexts as many factors can influence drug potencies in complex in vivo systems.37 Nevertheless, previous studies have used differences in drug potencies to support arguments that either PP1 or PP2A is involved in a particular regulatory system in intact tentative evidence in support of a role for PP1 in a relatively early stage of memory formation based on such an approach.

In our early studies with OA and CalA, both drugs were found to produce two temporally distinct effects on memory formation.17,224 The retention functions were statistically identical in each case and the two times of onset were relatively independent of time of administration or drug concentration within set ranges. The most parsimonious explanation for these results is that different cellular constituents, underlying different memory stages, might be inhibited by high and low drug concentrations. The later of the two effects, discussed in the preceding section, was most probably due to inhibition of PP2A as it was induced by very low drug concentrations (up to 0.5 nM OA) as well as the higher drug concentrations (100 nM OA) used in our later studies. It seems reasonable to attribute the alternative effect obtained in these experiments, with onset of retention deficits prior to 20 minutes post-training, to inhibition of PP1, since this effect was induced only by concentrations of OA sufficient to inhibit both PP1 and PP2A in vitro.

Of some interest is the later finding that the effect of inhibiting PP1, assuming this to be the case here, was confined to the right hemisphere of the chick, with no evidence of a role for PP1

Figure 4. Schematic diagrams depicting the effects of inhibiting PP1 in one hemisphere of the chick following strongly-reinforced learning (SRL) or weekly reinforced learning (WRL). Trained groups of chicks were administered OA (100 nM) or CalA (100nM) into the left (LH) or right (RH) hemisphere and retention was tested at the specified time post-training. Groups of chicks that received either drug in the left hemisphere showed good retention, indicated by selective avoidance of a red coloured bead, only up until approximately 40-50 minutes post-training. All groups tested after this time showed significant retention deficits, most probably attributable to the effects of the drugs on PP2A (see Fig. 3). Groups of chicks administered either drug in the right hemisphere showed good retention only up until approximately 10-20 minutes post-training. Groups tested between 20 and 120 minutes post-training showed variable retention levels, with a gradual recovery being apparent. By 240 minutes post-training, retention levels in right hemisphere, drug-treated, chick groups were identical to those obtained from untreated control groups.

Figure 4. Schematic diagrams depicting the effects of inhibiting PP1 in one hemisphere of the chick following strongly-reinforced learning (SRL) or weekly reinforced learning (WRL). Trained groups of chicks were administered OA (100 nM) or CalA (100nM) into the left (LH) or right (RH) hemisphere and retention was tested at the specified time post-training. Groups of chicks that received either drug in the left hemisphere showed good retention, indicated by selective avoidance of a red coloured bead, only up until approximately 40-50 minutes post-training. All groups tested after this time showed significant retention deficits, most probably attributable to the effects of the drugs on PP2A (see Fig. 3). Groups of chicks administered either drug in the right hemisphere showed good retention only up until approximately 10-20 minutes post-training. Groups tested between 20 and 120 minutes post-training showed variable retention levels, with a gradual recovery being apparent. By 240 minutes post-training, retention levels in right hemisphere, drug-treated, chick groups were identical to those obtained from untreated control groups.

in left hemisphere memory processing being evident (Bennett, Moutsoulas, Lawen & Ng, unpublished observations). The left hemisphere retention deficits depicted in Figure 4 can be fully accounted for by the effects of the drugs on PP2A, as they were also obtained following administration of much lower drug concentrations (Fig. 3). More surprisingly, the effect on retention levels of PP1 inhibition in the right hemisphere was found to be both transient (Fig. 4) and independent of the level of reinforcement associated with the task (data not shown). Retention levels were reduced from before 20 minutes post-training through to after 60 minutes post weakly- or strongly-reinforced training, with drug-treated groups of chicks being statistically indistinguishable from control groups by 2-4 hours post-training. This pattern of results is unprecedented and, hence, difficult to explain. It was not observed when bilateral drug administration took place, but this is probably due to the effects of OA and CalA on PP2A in the left hemisphere, as described above.

It is possible that inhibition of PP1 in the right hemisphere may selectively affect retrieval of information from specific memory stages, rather than affecting memory formation per se. It is possible also that inhibition of PP1 may selectively affect only one or more of several informa-

Figure 5. Schematic diagrams showing the effects of inhibiting PP2B in one hemisphere of the chick following strongly-reinforced learning (SRL). Chicks trained using the PAL task were administered either CyA (20 nM) or FK506 (20 nM) and tested for retention at a specified time post-training. Groups of chicks that received either drug in the left hemisphere (LH) showed good retention, indicated by selective avoidance of a red coloured bead, up until approximately 70-80 minutes post-training, an effect also obtained following administration of PPIase inhibitors (data not shown). Groups of chicks administered the drugs in the right hemisphere (RH) showed a pattern of retention deficits similar to those obtained following inhibition of PP1, with good retention for approximately 5-10 minutes, followed by a period of retention loss which had resolved completely by 180-240 minutes post-training.

Figure 5. Schematic diagrams showing the effects of inhibiting PP2B in one hemisphere of the chick following strongly-reinforced learning (SRL). Chicks trained using the PAL task were administered either CyA (20 nM) or FK506 (20 nM) and tested for retention at a specified time post-training. Groups of chicks that received either drug in the left hemisphere (LH) showed good retention, indicated by selective avoidance of a red coloured bead, up until approximately 70-80 minutes post-training, an effect also obtained following administration of PPIase inhibitors (data not shown). Groups of chicks administered the drugs in the right hemisphere (RH) showed a pattern of retention deficits similar to those obtained following inhibition of PP1, with good retention for approximately 5-10 minutes, followed by a period of retention loss which had resolved completely by 180-240 minutes post-training.

tion traces that normally form in parallel, only one of which is expressed at any given time. Several additional experiments (Bennett, Moutsoulas, Lawen & Ng, unpublished observations) have provided preliminary support for the second of these conclusions but this has yet to be fully investigated. Were this to be the case, it would support compelling data from other learning paradigms which have recently challenged the traditional view that existing memory stages are sequentially dependent.46,50,61,91,201

PP2B

In our first series of studies using the PP2B inhibitor Cyclosporin A (CyA), we demonstrated that memory loss was observed following bilateral drug administration from approximately 70-80 minutes post-training.16 We later showed (depicted in Fig. 5) that this effect was lateralized to the left hemisphere (Bennett, Moutsoulas, Lawen & Ng, unpublished observations), and that a temporally identical amnestic effect was obtained following administration of an alternative PP2B inhibitor, FK506.14 Unexpectedly, similar data were obtained following administration of a number of pharmacological analogues which share with CyA and FK506 the ability to inhibit distinct classes of immunophilins, but which do not inhibit PP2B activ-ity.15 Immunophilins (also called peptidyl prolyl cis/trans isomerases or PPIases) catalyse protein folding reactions,59 are enriched in brain tissue, and have important neuroprotective func-tions.176 It was concluded, therefore, that our evidence was consistent with a role for these enzymes, rather than for PP2B, in a later stage of memory formation. Investigation of whether PP2B is also required for memory formation at or after this time awaits the development of more specific inhibitors.

In a subsequent series of studies we proposed that, if PP2B plays a role in memory via activation of PP1, as is suggested by leading theoretical models, then the temporal and spatial parameters of this involvement might be expected to be similar. We investigated this proposal by conducting further experiments with CyA and FK506, in which drug concentrations and times and location of drug administration were systematically varied. Consistent with our proposal, various treatments thought to act by inhibition of PP2B produced results almost identical to those produced by treatments thought to act by inhibition of PP1.14 The requirement for

PP2B was shown to be localized to the right hemisphere; inhibition of either PP1 or PP2B in this hemisphere produced memory loss with an early time of onset; 10-20 minutes post-training for PP1 inhibition and 5-10 minutes post-training for PP2B inhibition. The retention loss with both classes of inhibitors was found to be transient, regardless of the level of reinforcement associated with the training experience, and both retention functions were very similar, with recovery of memory taking place by approximately 2-3 hours (Fig. 5).

This consistency is made more compelling by the fact that the pattern of retention deficits seen in both cases is so unusual. Few early drug effects on memory have been found to occur following right hemisphere drug administration, although this may be partially because many retention tests are conducted after the time at which memory recovered in our studies, so that transient effects might be overlooked. Recovery of memory following a prolonged period of retention loss is also rare following strongly-reinforced training and, as yet, has not been reported elsewhere following weakly-reinforced training. It seems reasonable to conclude, therefore, that at least one role for PP2B in memory formation in the chick may involve activation of PP1.

The exact nature of this role remains undetermined although, given the model of synaptic plasticity described previously, it is of interest that inhibition of CaMK-II in the right hemisphere of the chick also results in memory loss soon after training.223 Unlike PP1 and PP2B inhibition, CaMK-II inhibition also produces memory loss in the left hemisphere and all retention deficits induced by CaMK-II appear to be permanent. It may be instructive that CaMK-II appears to have more than one, differentially localised, role in memory for a single-trial, step-down inhibitory avoidance task in rodents,215 and that only some effects of CaMK-II inhibition in this species are permanent, while others can be attenuated by appropriate experimental protocols.9 Additional work within the chick model is required to establish whether similar effects may be observed within this species.

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