Temporal sensitivity of protein kinase a activation in late-phase long term potentiation

Abstract

Protein kinases play critical roles in learning and memory and in long term potentiation (LTP), a form of synaptic plasticity. The induction of late-phase LTP (L-LTP) in the CA1 region of the hippocampus requires several kinases, including CaMKII and PKA, which are activated by calcium-dependent signaling processes and other intracellular signaling pathways. The requirement for PKA is limited to L-LTP induced using spaced stimuli, but not massed stimuli. To investigate this temporal sensitivity of PKA, a computational biochemical model of L-LTP induction in CA1 pyramidal neurons was developed. The model describes the interactions of calcium and cAMP signaling pathways and is based on published biochemical measurements of two key synaptic signaling molecules, PKA and CaMKII. The model is stimulated using four 100 Hz tetani separated by 3 sec (massed) or 300 sec (spaced), identical to experimental L-LTP induction protocols. Simulations show that spaced stimulation activates more PKA than massed stimulation, and makes a key experimental prediction, that L-LTP is PKA-dependent for intervals larger than 60 sec. Experimental measurements of L-LTP demonstrate that intervals of 80 sec, but not 40 sec, produce PKA-dependent L-LTP, thereby confirming the model prediction. Examination of CaMKII reveals that its temporal sensitivity is opposite that of PKA, suggesting that PKA is required after spaced stimulation to compensate for a decrease in CaMKII. In addition to explaining the temporal sensitivity of PKA, these simulations suggest that the use of several kinases for memory storage allows each to respond optimally to different temporal patterns.

Figure 1. Model of L-LTP induction in hippocampal CA1 pyramidal neurons.

(A) Signaling pathways in model activated by pre-synaptic stimulation. Calcium (from glutamate activation of NMDA receptors) binds to Calmodulin (CaM). Calcium-calmodulin can bind to and activate protein phosphatase 2B (PP2B), adenylyl cyclase types 1 and 8 (AC), phosphodiesterase type 1B (PDE1B), or CaMKII. Gs-coupled receptors, such as dopamine D1 and β-adrenergic receptors, synergistically activate adenylyl cyclase, which produces cAMP, which binds to PKA. Two types of phosphodiesterase (PDE1B and PDE4) degrade cAMP leading to PKA de-activation. Active PKA phosphorylates inhibitor-1 (I1), which then binds to and inhibits protein phosphatase 1 (PP1). CaMKII can autophosphorylate itself, and is dephosphorylated by PP1. (B) Stimulation protocols used for induction of L-LTP in the model. A single Ca²⁺ pulse rises instantaneously to an amplitude of 0.7 µM and decays with a time constant of 0.14 sec. Summation of Ca²⁺ pulses in the train gives a maximum elevation of Ca²⁺ of ∼10 µM (top panel). A spaced pattern consisted of four 100 Hz trains (each 1 sec duration and fixed total number of pulses (400)) with an inter-train interval of 300 sec. The massed pattern provides the same four 100 Hz trains, but with an inter-train interval of 3 sec. Time intervals shown are not to scale. (C) Simulated time course of Ca²⁺ and dopamine input for a single 1 sec train of synaptic input at 100 Hz with the magnitude of 10 µM and 1 µM respectively.

Figure 2. Temporal sensitivity of PKA and CaMKII activation during L-LTP induction.

(A) Activation of PKA by different temporal patterns of stimulation. A1 shows 1400 sec of simulation time and A2 shows the first 200 sec to better show the four trains of massed stimulation. Though the peak is 1.4 times greater for massed trains, the cumulative activity of PKA activity is ∼60% greater for spaced trains (2321 nM-sec versus 1455 nM-sec). (B) The increase in phosphoCaMKII activity (sum of Ca₄-calmodulin trapped and autonomous forms) is smaller for spaced than massed stimulations. The positive feedback loop is visible in the spaced case, in which increments of phosphorylated CaMKII increase with subsequent trains. (C) Cumulative PKA activity (measured as area under the curve) shows an exponential increase (τ = 8.5 sec) as inter-train interval is increased. The peak phosphoCaMKII decreases exponentially with temporal interval, exhibiting a frequency sensitivity opposite to that of PKA. The time constant of the decrease, τ, is 20.8 sec. The sum of the normalized kinase activity (divided by two for graphical purposes) is constant for inter-train intervals greater than 3 sec, suggesting that the increase in PKA is compensating for a phosphoCaMKII deficit with larger for inter-train intervals.

Figure 3. Experiments to verify that the critical inter-train interval for the PKA-dependence of L- LTP is ∼60 sec.

(A) LTP induced using a 80 sec inter-train interval (n = 7) is blocked by KT5720 (p<0.05), which demonstrates PKA dependence. (B) In contrast, when using a 40 sec inter-train interval (bottom; n = 7), LTP in KT5720 slices is not different from vehicle control (p>0.05), demonstrating PKA independence. In both panels, fEPSPs for KT5720 treated slices are shown with red circles, and vehicle controls with blue circles. (C) Summary of temporal sensitivity of PKA dependence of L-LTP. Data for 3 sec, 20 sec and 300 sec are from .

Figure 4. Simulations were repeated with the dopamine receptor blocked, to evaluate the contribution of dopamine to L-LTP.

Cumulative PKA activity is reduced significantly with both massed and spaced stimulation intervals. The PKA activity with spaced stimulation and dopamine blocked is comparable to PKA activity with massed stimulation and dopamine unblocked. The reduction in PKA activity for the massed stimulation case is not functionally significant because PKA is not needed for L-LTP produced by massed stimulation.

Figure 5. Mechanism underlying sensitivity of PKA activation to different temporal patterns of stimulation.

In both (A) and (B) the left column (A1, B1) shows 1400 sec of simulation time and the right column (A2, B2) shows the first 180 sec to better show the four tetani of massed stimulation. (A) With massed trains, activation of adenylyl cyclase begins to saturate with the first train, leading to sublinear summation of adenylyl cyclase activity in response to subsequent trains; (B) cAMP concentration exhibits sub-linear summation with massed stimuli; thus less cAMP is produced with massed than with spaced trains. (C) Depletion of adenylyl cyclase and calmodulin contribute to the non-linear summation. Fractions of unbound adenylyl cyclase 1 and calmodulin are much lower for the second stimulus train for massed stimulation (C1), but not for spaced stimulation (C2). Dashed horizontal line allows comparison of unbound calmodulin between first and second train. Dotted horizontal line allows comparison of unbound adenylyl cyclase 1 between first and second train.

Figure 6. The increase in PKA activity due to spaced stimulation propagates to downstream targets inhibitor-1 and protein phosphatase 1.

(A) The amount of phosphorylated inhibitor-1 is ∼50% higher for spaced than massed stimulation. The initial decrease in phosphorylated inhibitor-1 (below baseline) is due to activation of protein phosphatase 2B. (B) The sharp peaks of protein phosphatase 1 during stimulation are caused by the initial decrease of phosphorylated inhibitor-1. These transient increases are followed by longer periods of decrease in free protein phosphatase 1 below basal level, caused by an increase of phosphorylated inhibitor-1. This simulation shows the concentration of protein phosphatase 1 is reduced ∼50% more by the spaced stimulation protocol than the massed protocol (in the legend a negative value of area under the curve denotes a decrease in activity).