1.16.1. Primary events
Since its discovery by Ito and colleagues (Ito & Kano, 1982; Ito et al., 1982) there has been a wealth o f investigations to elucidate the molecular mechanisms o f pf-PC LTD induction. The majority of the early studies use the induction protocols originally developed by Ito - conjunctive stimulation of p f stimulation preceding cf stimulation by 0-20 ms, repeated every 1 second for 5 minutes. Based on these studies, it is suggested that there are three key events which act as external triggers for the induction of pf-LTD. Three key events are: (1) a glutamate release from the parallel fibres that activate AMPA receptors (those that eventually undergo modifications) and mGluj receptors, (2) release of NO and the subsequent activation of the soluble
guanylyl cyclase in the Purkinje cell cytoplasm, (3) activation of the climbing fibre, leading to the powerful Purkinje cell depolarisation and Ca2+ influx through voltage gated Ca2+ channels (see Ito, 2001 for review).
The sources and exact manner of glutamate and NO for triggering the above cellular events inside PCs are, however, not as clear as stated above. For example, the glutamate released from the climbing fibre may also activate mGlui receptors (Dzubay and Otis, 2002). NO is thought to be produced in parallel fibre terminals. The production o f NO is a Ca2+-dependent process, as the enzyme involved in the
production of the molecule, the neuronal NO synthase, requires Ca2+. The required Ca2+ rise may be associated with an activation of the presynaptic NMDA receptors (Casado et al., 2002), though this is still debated. But there is also a suggestion that climbing fibres could also provide NO (Southam & Garthwaite, 1991).
1.16.2. Secondary events: crucial molecular events that follow the primary events
The pf-PC LTD is expressed when the AMPA receptors mediating parallel fibre EPSPs are appropriately modified. It is suggested that depression of pf-PC synapses is achieved by a decrease in the AMPA receptor density in the postsynaptic site
(Matsuda et al., 2000). This process results from the phosphorylation of the AMPA receptor, specifically the ser-880 residue of the GluR2 subunit, and involves PKC activation (Chung et al., 2003). Whether or not PKC directly phosphorylates the receptor is not clear, as there are other kinases whose activation is involved in pf-PC LTD. For example the induction o f pf-LTD by a PKC activator requires protein tyrosine kinase function (Boxall et al., 1996). And another kinase, the alpha type calcium-calmodulin dependent kinase II (aCamKII) is also required for the pf-PC LTD induction (Hansel et al., 06). The phosphorylation of AMPA receptors reduces their affinity to the postsynaptic specialisation that contains postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (zo-1) (PDZ domain). The AMPA receptors thus released from the grip of anchoring proteins are internalised in a clathryn-dependent manner (Wang & Linden, 2000). Phosphatases oppose the phosphorylation of AMPA receptors by kinases and, in the PC, phosphatases such as PP1, 2A/B act to increase the efficacy of pf-PC synapses (Belmeguenai & Hansel, 2005). Thus, the induction of pf-PC LTD
requires the inhibition of phosphatases; consistent with this, pf-PC LTD induction is blocked by an intracellular infusion of an activated PP2B.
How do the initial, external events described earlier culminate in the phosphorylation of the AMPA receptors? mGlui receptor activation leads to the activation o f one or more G proteins that include Gq and Gn (Hartmann et al., 2004). The a-subunits of these G proteins activate phospholipase Cp (Jiang et al., 1994). When activated, this enzyme breaks down phosphatidylinositol-4,5-biphosphate (PIP2), which is a
membrane constituent, into the two signalling molecules, IP3 and diacylglycerol
(DAG). PLCp4 is the most widely expressed isoform, but PLCp3 is also present in a subset of Purkinje cells that do not express PLCp4 (Sama et a l , 2006). IP3, in
conjunction with Ca2+, activates IP3 receptors on the endoplasmic reticulum and
causes Ca release from there. DAG, on the other hand, activates protein kinase C (PKC) leading to the phosphorylation of AMPA receptors as described above. DAG is further broken down into 2-arachidonylglycerol (2AG) by the action of DAG lipase (Bisogno et al., 2003). 2-AG is an endocannabinoid that is thought to activate CBi receptors located presynaptically (Kreitzer & Regehr, 2002). The activation of CBi
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receptors is also required for the pf-PC LTD induction (Safo & Regehr, 2005), although at the moment it is not known how activation of presynaptic CB1 receptors leads to the expression of pf-PC LTD, which is postsynaptic.
The role of NO in the pf-PC LTD induction appears to be to target soluble guanylyl cyclase within the Purkinje cell; this enzyme catalyses the conversion of GTP into cyclic GMP (cGMP) - an activator of protein kinase G. One of the substrates of activated protein kinase G is G-substrate which, when phosphorylated by PKG,
inhibits protein phosphatases, especially PP1 and PP2A (Endo et al., 1999). Thus by opposing the action of phosphatases, the NO signalling pathway augments LTD induction.
Ca2+ influences many of the steps described above. The release o f Ca2+ from internal stores is one. The translocation of PKCa to the membrane, where, upon activation by
' j i
DAG, it phosphorylates the AMPA receptors, requires a rise in [Ca ] and the activity of many other enzymes, including PLCp, is enhanced by Ca2+. As mentioned earlier, the Ca2+ rise is strongly related to the coincidence detection i.e. a mechanism that reports a convergence of simultaneous parallel and climbing fibre activations. It is likely that this coincidence signal, in the form of high [Ca2+], is detected at multiple stages of signalling involved in the pf-PC LTD.