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. 1997 Jul 15;17(14):5357-65.
doi: 10.1523/JNEUROSCI.17-14-05357.1997.

Postsynaptic inhibitors of calcium/calmodulin-dependent protein kinase type II block induction but not maintenance of pairing-induced long-term potentiation

N Otmakhov  1 , L C Griffith, J E Lisman
Affiliations

Postsynaptic inhibitors of calcium/calmodulin-dependent protein kinase type II block induction but not maintenance of pairing-induced long-term potentiation

N Otmakhov et al. J Neurosci. .

Abstract

The role of postsynaptic kinases in the induction and maintenance of long-term potentiation (LTP) was studied in the CA1 region of the rat hippocampal slice. A peptide inhibitor for the catalytic domain of calcium/calmodulin-dependent protein kinase type II (CaM-kinase) was applied through a perfused patch pipette. The inhibitor completely blocked both the short-term potentiation and LTP induced by a pairing protocol. This indicates that the kinase or kinases affected by the peptide are downstream from depolarization in the LTP cascade. The ability to block LTP required that measures be taken to interfere with degradation of the peptide kinase inhibitor by endogenous proteases; either addition of protease inhibitors or modifications of the peptide itself greatly enhanced the effectiveness of the peptide. Protease inhibitors by themselves or control peptide did not block LTP induction. To study the effect of kinase inhibitor on LTP maintenance, we induced LTP in one pathway. Subsequent introduction of the kinase inhibitor blocked the induction of LTP in a second pathway, but it did not affect maintenance of LTP in the first. The implications for the role of kinases in LTP maintenance are discussed.

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Figures

Fig. 1.
Fig. 1.
Diffusion of dye into dendrites after the introduction of dye into the patch pipette. A, Picture of the dendritic region of a pyramidal cell after it was filled with fluorescent dye (carboxyfluorescein, 25 μm) in the patch pipette (pp; soma diagrammed atleft); an intrapipette capillary was used (ipc). Relative positioning of two stimulation electrodes (s1 and s2) is indicated.B, Time course of the dye fluorescence measured at three different distances from the soma of the neuron (see numbered regions in A). C, Magnitude of synaptic responses of the cell as a function of time during the experiment. Thirty minutes after the induction of LTP in one pathway (open circles), the dye was perfused into the patch pipette (see A, B). This perfusion did not significantly affect either the LTP or the control (filled circle) pathways (see alsoD). D, Ratio of the amplitudes of LTP versus the control synaptic responses. Each point is the ratio of the average of 20 responses. E, Series resistance for this recording. F, Averages of 20 consecutive synaptic responses before (c1) and 140 min after (c2) LTP induction.
Fig. 2.
Fig. 2.
CaMKII(AC3) inhibitor peptide does not affect LTP if applied postsynaptically without protease inhibitors. Open symbols indicate the pathway where the pairing procedure was applied at time 0 (arrow). Filled symbolsindicate the control pathway. LTP was induced after 20 min of whole-cell recording. When protease inhibitors were not included with 2 mm of CaMKII(AC3) inhibitor peptide (A), the level of LTP was not affected in comparison to the level of LTP induced with the control pipette solution (B).
Fig. 3.
Fig. 3.
Postsynaptic application of CaMKII(AC3) inhibitor peptide completely blocked induction of LTP, provided that protease inhibitors were used. Open symbols indicate the pathway where the pairing procedure was applied at time 0 (arrow). Filled symbols indicate the control pathway. LTP was induced after 20 min of whole-cell recording.A and B show individual experiments, andC–F show pooled data. In all experiments a protease inhibitor cocktail was included in the pipette solution.A, C, CaMKII(AC3) (2 mm) inhibitor peptide completely blocked both LTP and STP; when 1 mm of the kinase inhibitor was applied (B,D), LTP still could be induced in many cases, but its magnitude was decreased significantly, as compared with controls in which the pipette solution contained protease inhibitors but no kinase inhibitor (E) or 1 or 2 mm of the inactive CaMKII(AC3) control peptide (F).Insets in A, B, Averages of 20 consecutive traces of EPSCs taken in the periods indicated in the corresponding panels. Calibration: 150 pA, 100 msec forA; 500 pA, 100 msec for B.
Fig. 5.
Fig. 5.
Modified CaMKII(AC3) inhibitor (resistant to protease degradation) completely blocked induction of LTP even when protease inhibitors were not included in the patch pipette solution.A, Protected peptide (see Materials and Methods) completely blocked LTP induction 20 min after the start of whole-cell recording. B, Large LTP was produced when unprotected peptide was applied. Gray symbols are for the pathway in which LTP was induced by pairing at the arrow.Filled symbols are for the control pathway.
Fig. 4.
Fig. 4.
Postsynaptic application of CaMKII inhibitor peptides after LTP induction did not affect the maintenance of LTP. Shown are a representative experiment (A) and pooled data (B) of seven similar experiments when CaMKII(AC3) inhibitor peptide (2 mm) was perfused during the period marked by the horizontal bar.C, Pooled data of six experiments when CaMKII(273–302) inhibitor peptide (2 mm) was perfused. A–C, Protease inhibitors were included in the patch pipette during the entire experiment. D, Pooled data of four control experiments. Open symbols are for the pathway in which LTP was induced by the pairing protocol (at arrow);filled symbols are for the control pathway.Insets in A, Averages of 20 consecutive traces of EPSCs taken in the periods indicated in A. Calibration, 500 pA, 100 msec.
Fig. 6.
Fig. 6.
Ten minutes of intracellular application of protected CaMKII(AC3) inhibitor peptide completely blocked induction but did not affect early maintenance of LTP. CaMKII(AC3) peptide inhibitor was modified to resist protease degradation. Protease inhibitors also were included in the patch pipette. Six minutes after the start of whole-cell recording, LTP was induced in one pathway (open or gray symbols). Two minutes later, kinase inhibitor or inactive control CaMKII(AC3) peptide was applied (horizontal bars). Ten minutes later, the ability of the kinase inhibitor to inhibit LTP induction in the second pathway (filled symbols) was tested.A, Protected kinase inhibitor. B, Control peptide. C, Superposition of the data from the first pathways in A and B. D, Superposition of the data from the second pathways in Aand B. Arrows indicate the time when the LTP induction protocol was applied.
Fig. 7.
Fig. 7.
Intracellular application of the protected CaMKII(AC3) peptide inhibitor did not affect LTP maintenance.A1, A representative experiment shows that no decline in the potentiated (open symbols) and control (filled symbols) synaptic responses is observed over time when the kinase inhibitor is added after LTP induction (arrow). Series resistance (A2), input resistance (A3), and extracellular field potentials (A4) were stable throughout the experiment.Insets in A2, Left, Averages of 20 consecutive traces of EPSCs taken in the periods indicated in A1; calibration, 600 pA, 100 msec.Right, Averages of 20 consecutive traces of current transients used for monitoring series (Rs) and input resistance (Ri) during whole-cell recording, taken in the periods indicated in A2; calibration, 300 pA, 60 msec. Insets in A4, Averages of 20 consecutive traces of extracellular recorded EPSPs (fEPSP), taken in the periods indicated inA4; calibration, 200 μV, 60 msec. B, Summary data for active kinase inhibitor (B1), control peptide (B2), and superposition of B1 andB2 in B3. Gray oropen symbols represent the pathway in which LTP was induced by pairing at the arrow. Filled symbols represent the control pathway. Protease inhibitors were included in all experiments. Horizontal bars indicate periods when inhibitors were applied.
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