Activation of presynaptic cAMP-dependent protein kinase is required for induction of cerebellar long-term potentiation

Abstract

Cerebellar long-term potentiation (LTP) is a persistent increase in the strength of the granule cell-Purkinje neuron synapse that occurs after brief stimulation of granule cell axons at 2-8 Hz. Previous work has indicated that cerebellar LTP induction requires presynaptic Ca influx, stimulation of Ca-sensitive adenylyl cyclase, and activation of PKA. The evidence implicating PKA has come from bath application of drugs during LTP induction, an approach that does not distinguish between PKA activation in the presynaptic or postsynaptic cell. Although bath application of PKA inhibitor drugs (KT5720, Rp-8CPT-cAMP-S) blocked LTP induction in granule cell-Purkinje neuron pairs in culture, selective application to granule cell or Purkinje neuron somata via patch pipettes did not. We hypothesized that presynaptic PKA activation is required for LTP induction but that drugs applied to the granule cell soma cannot diffuse to the terminal within this timescale. To test this hypothesis, we transfected cerebellar cultures with an expression vector encoding a peptide inhibitor of PKA [Rous sarcoma virus (RSV)-protein kinase A inhibitor (PKI)]. Transfection of RSV-PKI into presynaptic granule cells, but not postsynaptic Purkinje neurons or glial cells, blocked LTP induction produced by either synaptic stimulation or an exogenous cAMP analog. An expression vector encoding a control peptide with no PKA inhibitory activity was ineffective. These results show that induction of cerebellar LTP requires a presynaptic signaling cascade, including Ca influx, stimulation of Ca-sensitive adenylyl cyclase, and activation of PKA, and argue against a requirement for postsynaptic Ca signals or their sequelae.

Fig. 1.

High concentrations of postsynaptic Ca chelator fail to block cerebellar LTP induction in granule cell–Purkinje neuron pairs. Purkinje neurons were loaded with a Cs-based patch pipette saline that contained either 10 or 35 mm BAPTA. LTP was induced by 4 Hz stimulation for 100 pulses, as indicated by thethick horizontal bar at t = 0 min.Inset illustrates current traces representing the average of 10 consecutive responses (including failures) recorded from a granule cell–Purkinje neuron pair loaded with 10 mmBAPTA. Traces are from the time points indicated on the graph.

Fig. 2.

PKA inhibitor drugs block cerebellar LTP induction in granule cell–Purkinje neuron pairs when applied in the bath but not in patch pipettes. A, PKA inhibitors KT5720 (10 μm) and Rp-8CPT-cAMP-S (100 μm) and the PKG inhibitor KT5823 (10 μm) were applied in the bath starting at t = −17.5 min, as indicated by thethin horizontal bar. The control group in this graph is the same as the 10 mm BAPTA group from Figure1 and is reproduced here for comparison. B, PKA inhibitors Rp-cAMP-S, Rp-8CPT-cAMP-S, and KT5720 (all 1 mm) were included in the patch pipette saline of either the granule cell or Purkinje neuron. The time of initiating whole-cell recording was att = 15 min (or slightly earlier), as indicated by the thin horizontal bar. C, Rp-cAMP-S (2 mm) was included in the granule cell patch pipette saline with an extended baseline recording to allow for maximal presynaptic perfusion. Recordings were made using either our standard electrode for granule cell recording (5–6 MΩ) or a somewhat larger electrode (3 MΩ) to further maximize perfusion. The inset illustrates current traces representing the average of 10 consecutive responses (including failures) recorded from a granule cell–Purkinje neuron pair recorded with a 5–6 MΩ electrode.

Fig. 3.

The Na channel blocker QX-314 fails to block axonally evoked retrograde spike current when applied in the granule cell patch pipette. QX-314 was included in granule cell patch pipette at a concentration of either 1 or 5 mm and was delivered beginning with the initiation of whole-cell recording (thin horizontal bar). Retrograde spike current was evoked by extracellular stimulation of the granule cell terminals–axon, and the effect of QX-314 perfusion was assessed with test pulses applied at either 0.1 or 1 Hz. Inset illustrates single current traces from a cell perfused with 5 mm QX-314 and stimulated at 1 Hz, taken at the time points indicated on the graph.

Fig. 4.

Transfection of the granule cell with a PKA inhibitor construct in a granule cell–Purkinje neuron pair blocks LTP induction. Granule cells or Purkinje neurons were selectively transfected with either a PKA inhibitor peptide expression vector (RSV-PKI) or a mutant peptide expression vector that is inactive with respect to PKA (RSV-PKImut), 3–4 d before recordings. Localization was determined by cotransfection with GFP and subsequent illumination at 488 nm. A, Photomicrograph illustrating a GFP-transfected granule cell (far right) and Purkinje neuron (center) illuminated with 488 nm light. Scale bar, 10 μm. B, LTP experiments.Inset illustrates current traces representing the average of 10 consecutive responses recorded from a granule cell–Purkinje neuron pair in which the granule cell was transfected with the RSV-PKI construct.

Fig. 5.

Transfection of the granule cell with a PKA inhibitor construct in a granule cell–Purkinje neuron pair blocks synaptic potentiation induced by an exogenous cAMP analog. Recordings were made from cell pairs in which the granule cell was transfected with either the PKA inhibitor construct RSV-PKI or the control construct RSV-PKImut. The cAMP analog Sp-8CPT-cAMP-S was applied in the bath from t = 0–5 min (as indicated by thethin horizontal bar). At t = 22.5 min, 4 Hz stimulation for 100 pulses was applied to the granule cell (as indicated by the thick horizontal bar).

Fig. 6.

Transfection of a glial cell with a PKA inhibitor construct in a granule cell–glial cell pair has no effect on LTP induction. The PKA inhibitor peptide expression vector RSV-PKI was selectively transfected into either glial cells or granule cells before 4 Hz stimulation for 100 pulses. Inset illustrates current traces representing the average of 10 consecutive responses recorded from a granule cell–glial cell pair in which the granule cell was transfected with the RSV-PKI construct.