Acute in vivo genetic rescue demonstrates that phosphorylation of RIM1alpha serine 413 is not required for mossy fiber long-term potentiation

Abstract

While presynaptic, protein kinase A (PKA)-dependent, long-term plasticity has been described in numerous brain regions, the target(s) of PKA and the molecular mechanisms leading to sustained changes in neurotransmitter release remain elusive. Here, we acutely reconstitute mossy fiber long-term potentiation (mfLTP) de novo in the mature brains of mutant mice that normally lack this form of plasticity. These results demonstrate that RIM1alpha, a presynaptic scaffold protein and a potential PKA target, can support mfLTP independent of a role in brain development. Using this approach, we study two mutations of RIM1alpha (S413A and V415P) and conclude that PKA-phosphorylation-dependent signaling by RIM1alpha serine 413 is not required for mfLTP, consistent with conclusions reached from the study of RIM1alpha S413A knockin mice. Together, these results provide insights into the mechanism of mossy fiber LTP and demonstrate a useful acute approach to genetically manipulate mossy fiber synapses in the mature brain.

Figure 1.

Postnatal viral-mediated expression of RIM1α rescues mossy fiber LTP in RIM1α^−/− mice, without altering short-term plasticity. A, Schematic representation of the experimental strategy. B, Representative example of an acute hippocampal slice expressing EGFP, illuminated by both epifluorescent and white light. Scale bar is 500 μm. C, Summary of mfLTP experiments in hippocampal slices. Example traces are an average of three responses obtained over 1 min from periods immediately before (solid trace) and 60 min after (dashed trace) tetanus. D, Mean level of mfLTP measured from 50 to 60 min after tetanus. Sample sizes for C, D were: EGFP in RIM1α^−/− mice 12 slices/5 mice; RIM1α in RIM1α^−/− mice 11 slices/7 mice; EGFP in RIM1α^+/+ mice 6 slices/3 mice. E, RIM1α^−/− slices expressing RIM1α exhibit similar paired-pulse facilitation to those expressing control EGFP virus. F, RIM1α^−/− slices expressing RIM1α show marked frequency facilitation and sensitivity to DCG-IV, similar to RIM1α^−/− slices expressing EGFP alone. Sample sizes for E, F were: EGFP 11 slices/3 mice; RIM1α 9 slices/3 mice. Horizontal bar indicates stimulation frequency and period of drug application. DG, Dentate gyrus; MFT, mossy fiber tract.

Figure 2.

Postnatal viral-mediated expression of S413A mutant RIM1α rescues mfLTP in RIM1α^−/− mice. Traces are from periods immediately before (solid trace) and 60 min after (dashed trace) tetanus. Sample sizes were: EGFP, 12 slices/5 mice; S413A, 12 slices/6 mice.

Figure 3.

Postnatal viral-mediated expression of the enhanced 14-3-3 binding mutant V415P of RIM1α rescues mfLTP in RIM1α^−/− mice. Traces are from periods immediately before (solid trace) and 60 min after (dashed trace) tetanus. Sample sizes were: EGFP, 12 slices/5 mice; V415P, 12 slices/7 mice.