Late-phase, protein synthesis-dependent long-term potentiation in hippocampal CA1 pyramidal neurones with destabilized microtubule networks

Abstract

Background and purpose: Protein synthesis-dependent late-long term potentiation (L-LTP) is an enduring form of synaptic plasticity that has been shown to rely on, at least partly, protein synthesis at synaptic and/or dendritic sites. Evidence suggests that somatic transcription of new mRNAs may provide a significant contribution to the availability of mRNAs at synaptic sites where they are made available for dendritic translation. Transport of mRNAs from somatic to dendritic sites might be expected to involve movement along a microtubule network. In this study we examined whether it was possible to maintain L-LTP in hippocampal slices with destabilized microtubule networks.

Experimental approach: Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from rat hippocampal slices and following a period of baseline recording, stimuli were given that induced LTP. LTP was monitored for 5 h in both control slices and slices treated with vincristine to depolymerize tubulin.

Key results: L-LTP was induced and maintained in vincristine-treated slices. Four hours after tetanic stimulation fEPSPs were 196+/-19% of baseline values. The magnitude of potentiation was similar to that seen in untreated slices (175+/-15%). L-LTP in vincristine-treated slices was, however, not maintained in the presence of the protein synthesis inhibitor, rapamycin. Immunohistochemistry and confocal microscopy of vincristine-treated slices verified that the microtubule network had been destabilized.

Conclusions and implications: Communication between somatic and synaptic sites through protein and/or mRNA trafficking via an intact microtubule network is not required for protein synthesis dependent L-LTP.

Figure 1

Incubation of acute hippocampal slices in vincristine destabilizes the microtubule network in CA1 pyramidal cells. (a) Immunofluorescent labelling of the CA1 region of the hippocampus with α-tubulin (green) co-stained with TOPRO-3 (blue) in a ‘control' (untreated slice). (b) Immunofluorescent labelling of the CA1 region of the hippocampus with α-tubulin co-stained with TOPRO-3 in a slice exposed to vincristine (5 μM). Note that the α-tubulin staining is more diffuse throughout the section in (b) compared to that seen in (a). The boxed areas in (a) and (b) highlight the fact that in (b) there is diffuse pooling of α-tubulin around the cell soma, whereas in (a) there is a lack of α-tubulin staining. Scale bar in (a) and (b) is 50 μm. (c) Comparison of input–output curves for vincristine-treated and control slices. No significant differences were observed over the stimulus intensities examined.

Figure 2

L-LTP can be induced and maintained in hippocampal slices with destabilized microtubule networks. (a) Upper panel, typical fEPSP waveforms (average of five consecutive samples) recorded in the S1 pathway before and 4 h after the application of the LTP-inducing tetanus. Lower panel, typical fEPSP waveforms (average of five consecutive samples) recorded in the S2 (non-tetanized) pathway at similar time points shown in the upper traces. (b) Pooled data showing the time course of L-LTP induced in vincristine-treated (n=10) and control (n=9) hippocampal slices. The extent and magnitude of L-LTP is similar for both groups. Non-tetanized (S2) pathways in both vincristine-treated and control slices show no significant increase in fEPSP slopes during these recordings. (c) Comparison of PPF ratios recorded before and 1 and 4 h after the application of the LTP-inducing tetanus in vincristine-treated and control slices. There is no significant difference in the PPF ratios at the different time points shown or between the two groups. fEPSP, field excitatory postsynaptic potential; L-LTP, late-long term potentiation; PPF, paired-pulse facilitation.

Figure 3

L-LTP in vincristine-treated slices is dependent on protein synthesis. (a) Typical fEPSP waveforms (average of five consecutive traces) recorded in the S1 pathway before the delivery of the tetanus (trace 1), around the peak of the potentiation (trace 2) and 2 h following the tetanus (trace 3). (b) Pooled data (n=5) showing the time course of potentiation of fEPSPs in vincristine-treated slices in the presence of rapamycin (1 μM). After an initial large potentiation, slope values of fEPSPs decay to near baseline levels within 3 h. The non-tetanized (S2) pathway remains stable throughout the recording period indicating that the decrease in the fEPSPs seen in the S1 pathway is not due to a deterioration in the slice preparation when exposed to the protein synthesis inhibitor. For comparison, the magnitudes and time courses of L-LTP obtained in slices treated with vincristine only are reproduced from Figure 2b. fEPSP, field excitatory postsynaptic potential; L-LTP, late-long term potentiation.