TY - JOUR
T1 - Differential modulation of cortical synaptic activity by calcineurin (phosphatase 2B) versus phosphatases 1 and 2A
AU - Thomas, Gail D.
AU - O'Rourke, Brian
AU - Sikkink, Robert
AU - Rusnak, Frank
AU - Marban, Eduardo
AU - Victor, Ronald G.
N1 - Funding Information:
neurons ( = i .5-I I-b.) is similar to the low frequency stimulation t:~,ed to elicit LTD [36]. Application of caly- culin A a!‘ter LTD induction in the rat hippocampus r-e- verses this synaptic depression [22]. I-!owcva-. in the pre- sent study L*;llyculin A had no effccr on thp: fqwn~y OS‘ spontaneous synaptic activity in cortical neurons. suggest- ing that rclicf of endogcnous LTD cannot cxpluin the: increased currem frequency mediated by calcineurin inhibition. The effect of calyculin A in the present study to increase EPSC and mEPSC amplitudes without affecting current frequency provides compelling evidence to suggest that the main function of phosphatases 1 and 2A is to regulate the postsynaptic responsiveness of the rat cortical neurons. Although calyculin A is specific in terms of its ability to inhibit phosphatascs ! and 2A [ 1.31,i t is not vcr> specific in terms of its site of action: its ability to partition readily into and to cross cell membranes precludes discrimination between pre-and postsynaptic actions. Wc therefore repeated the experiments described above using microcystin LR. which is membrane-irnpcri~~c~~i~t,t o inhibit phosphatases I and 2A only in the postsynaptic cell being studied [21]. We found that inclusion of microcystin LR in the internal solution yielded essentially the same results as the calyculin experiments: EPSC and mEPSC amplitudes were increased while frequencies were unaffected. Thus these data confirm and extend the conclusions of the calyculin experiments by providing additional evidence that the main site of action of phosphatases 1 and 2A in cultured rat cortical neurons is postsynaptic. These data are in agreement with studies showing that inhibition of phosphatases I and 2A potentiated peak NMDA [3q] and kainatc [38] currents in hippocumpal neurons. but contrast with reports of enhanced neurotrailsmitter release from rat dorsal root gmglion cells [I I]. for&rain synap~o-soms [33], and frog neuromuscular junctions [I]. However, the present findings support biochemical and recent immunocytochemical evidence favoring a postsynaptic role for phosphatase I: phosphatase 1 is found in high concentrations in postsynaptic densities [6,30] and in dendritic spines [26]. In conclusion, this study provides evidence for selective pre-and postsynaptic modulation of glutamatergic neurotransmission by calcineurin (phosphatase 2B) versus phosphatases 1 and 2A in rat cortical neurons. The results extend our previous observation that the Ca’ ‘/calmodulin-dependent phosphatase calcineurin functions presynaptically to restrain neurotransmission and demonstrate that the effects of calcineurin are not mediated by changes in phosphatase 1 and/or 2A activities. The specific presynaptic mechanism by which calcineurin modulates glutamate release remains to be determined. In contrast, the actions of phosphatases 1 and 2A appear to be predominantly postsynaptic, affecting the amplitude of glutamate receptor responses. Although calcineurin and phosphatases 1 and 2A appear to be components of different intracelluhr Thib work was supported by Nations1 institutes tit‘ Health Grants HL-44010 (R.G.V.). GM36865 (F.R.), HL36957 (E.M.), and Training Grant T32 HLO7.360 (G.D.T.), an Established Investigatorship from the American Heart Association (R.G.V.). and a fellowship grant from the Muscular Dystrophy Association (G.D.T. 1.
PY - 1997/2/21
Y1 - 1997/2/21
N2 - Reversible protein phosphorylation is thought to play an important regulatory role in synaptic neurotransmission. We recently have shown in cultured rat cortical neurons that inhibition of the Ca2+/calmodulin-dependent phosphatase calcineurin (phosphatase 2B) increases the frequency, but not the amplitude, of postsynaptic glutamatergic currents, implicating a presynaptic site of action for calcineurin. The specific presynaptic phosphoprotein substrates for calcineurin are unknown, however, calcineurin has been implicated in the control of the Ca2+-independent phosphatases, phosphatases 1 and 2A. To determine whether calcineurin's effects on synaptic transmission are direct or are mediated by changes in phosphatase 1 and/or 2A activities, we used whole-cell voltage clamp to record spontaneous and miniature excitatory postsynaptic currents in the presence of calyculin A (1 μM in bath solution), a membrane permeant inhibitor of phosphatases 1 and 2A which has no effect on calcineurin. Calyculin increased postsynaptic current amplitude without changing current frequency. In these same neurons, subsequent inhibition of calcineurin with cyclosporine A or FK506 had no further effect on current amplitude, but increased current frequency. The increased current amplitude seen with calyculin involved a postsynaptic mechanism, since the effect was reproduced by microcystin (10 μM in pipette solution), which is a membrane-impermeant inhibitor of phosphatases 1 and 2A. Thus, in rat cortical neurons, glutamatergic neurotransmission appears to be frequency-modulated through a presynaptic mechanism by calcineurin, and amplitude-modulated through a postsynaptic mechanism by phosphatases 1 and 2A.
AB - Reversible protein phosphorylation is thought to play an important regulatory role in synaptic neurotransmission. We recently have shown in cultured rat cortical neurons that inhibition of the Ca2+/calmodulin-dependent phosphatase calcineurin (phosphatase 2B) increases the frequency, but not the amplitude, of postsynaptic glutamatergic currents, implicating a presynaptic site of action for calcineurin. The specific presynaptic phosphoprotein substrates for calcineurin are unknown, however, calcineurin has been implicated in the control of the Ca2+-independent phosphatases, phosphatases 1 and 2A. To determine whether calcineurin's effects on synaptic transmission are direct or are mediated by changes in phosphatase 1 and/or 2A activities, we used whole-cell voltage clamp to record spontaneous and miniature excitatory postsynaptic currents in the presence of calyculin A (1 μM in bath solution), a membrane permeant inhibitor of phosphatases 1 and 2A which has no effect on calcineurin. Calyculin increased postsynaptic current amplitude without changing current frequency. In these same neurons, subsequent inhibition of calcineurin with cyclosporine A or FK506 had no further effect on current amplitude, but increased current frequency. The increased current amplitude seen with calyculin involved a postsynaptic mechanism, since the effect was reproduced by microcystin (10 μM in pipette solution), which is a membrane-impermeant inhibitor of phosphatases 1 and 2A. Thus, in rat cortical neurons, glutamatergic neurotransmission appears to be frequency-modulated through a presynaptic mechanism by calcineurin, and amplitude-modulated through a postsynaptic mechanism by phosphatases 1 and 2A.
UR - https://www.scopus.com/pages/publications/0031028491
UR - https://www.scopus.com/pages/publications/0031028491#tab=citedBy
U2 - 10.1016/S0006-8993(96)01305-4
DO - 10.1016/S0006-8993(96)01305-4
M3 - Article
C2 - 9070633
AN - SCOPUS:0031028491
SN - 0006-8993
VL - 749
SP - 101
EP - 108
JO - Brain Research
JF - Brain Research
IS - 1
ER -