The t286 point mutant mouse (T286A) lacks the autophosphorylation site on calcium/calmodulin-dependent protein kinase II (CaMKII). Experiments performed on T286A null mutants prevent experience-dependent plasticity in vivo and long term potentiation (LTP) in vitro (Hardingham et al. 2003). In this study, we aimed to discover whether CaMKII autophosphorylation is required for pre- or postsynaptic changes in synaptic efficacy or indeed both.
Coronal cortical slices were prepared from 4- to 6-week-old wild-type and T286A mice (humanely killed by cervical dislocation). Layer 2/3 neurones in barrel cortex were recorded in current-clamp and stimulated extracellularly in layer 4 of an adjacent barrel. Stimulation was set to a minimal level where single inputs are believed to be activated. Under these circumstances, 1/CV2 plots can be used to probe the locus of any change in mean amplitude (Faber & Korn, 1991). Conditioning was achieved by pairing 200 post-synaptic action potentials with presynaptic stimulations at an interval of pre 10 ms before post and at 2 Hz.
In T286A mice, increases in mean amplitude were transient and consistently produced 1/CV2 plots consistent with predominantly presynaptic changes (n = 11). In wild-types, increases in mean amplitude were stable and produced 1/CV2 plots consistent with predominantly postynaptic changes (n = 12). 1/CV2 plots were statistically different between T286A and wild-type mice (two sample t test, P < 0.05). On occasions amplitude frequency histograms had statistically reliable peaks both in control periods of recording and after pairing, possibly referring to the quantal size of excitation. At 5-10 min post-pairing in the control animals, the average distance between peaks often increased (n = 9), whereas they remained the same in T286A null mutants (n = 6).
Further experiments using APV (50 µM) in the perfusing solution blocked LTP, but produced short-term potentiation (STP) consistent with that seen in T286A mice (n = 10). APV had no effect on the STP seen in the T286A mice (n = 10) whilst pharmacological blockade of CaMKII with AIP (autocamtide-2-related inhibitory peptide) also blocked potentiation (n = 5). Data also suggest long term depression (LTD) is unaffected in the T286A mutants (n = 11).
The conclusions of this study are that STP is presynaptic and does not depend on CaMKII autophosphorylation, while LTP also involves a postsynaptic component and is dependent on CaMKII autophosphorylation. LTD is also independent of CaMKII autophosphorylation and can be either pre- or postsynaptic.