Induction and maintenance of late-phase long-term potentiation in isolated dendrites of rat hippocampal CA1 pyramidal neurones

Abstract

Expression of N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in the CA1 region of the hippocampus can be divided into an early (1-2 h), protein synthesis-independent phase and a late (>4 h), protein synthesis-dependent phase. In this study we have addressed whether the de novo protein synthesis required for the expression of late-LTP can be sustained solely from the translation of mRNAs located in the dendrites of CA1 pyramidal neurones. Our results show that late-LTP, lasting at least 5 h, can be maintained in hippocampal slices where the dendrites located in stratum radiatum have been isolated from their cell bodies by a microsurgical cut. The magnitude of the potentiation of the slope of field EPSPs in these 'isolated' slices was similar to that recorded in 'intact' slices. Incubation of the slices with the mRNA translation inhibitor cycloheximide or the mammalian target of rapamycin (mTOR) inhibitor rapamycin blocked late-LTP in both 'intact' and 'isolated' slice preparations. In contrast, incubation of slices with the transcription inhibitor, actinomycin D, resulted in a reduction of sustained potentiation, at 4 h, in 'intact' slices while in 'isolated' slices the magnitude of potentiation was similar to that seen in untreated slices. These results indicate that late-LTP can be induced and maintained in 'isolated' dendritic preparations via translation of pre-existing mRNAs.

Figure 1. Properties of fEPSPs recorded from ‘isolated’ dendritic hippocampal slice preparations

A, upper panel, view of an ‘isolated’ hippocampal slice preparation. The separation of the cell body layer from the dendritic layer is clearly visible. The glass recording electrode is placed in the stratum radiatum immediately below the area which has been cut away from the stratum pyramidale and stratum oriens. Two stimulating electrodes are placed to either side of the recording electrode in order to activate independent inputs. The lower panel shows a representative cresyl violet-stained slice. The cell bodies of neurones located in the dentate gyrus and CA3 regions are visible. Although the somata of some CA1 neurones are still present in the slice, there is no evidence of any CA1 pyramidal cell bodies in the region, as indicated by the asterisk, where the recording electrode was placed. Scale bar 500 μm. B, plot of the amplitudes of fEPSPs recorded in ‘isolated’ slice preparations shows that they are blocked by the AMPA receptor antagonist, CNQX (30 μm). Representative fEPSPs recorded at the times indicated are shown above the plot. C, examples of fEPSPs recorded in ‘intact’ (left-hand panel) and ‘isolated’ slices (right-hand panel) in response to increasing stimulus intensities (ranges 20–100 μA for ‘intact’; 100–250 μA for ‘isolated’). Evidence of population spikes is seen only in recordings from the ‘intact’ slice. D, plot of fEPSP slope against stimulus intensity for data shown in C.

Figure 2. Late-LTP can be induced and maintained in ‘isolated’ hippocampal slice preparations

A and B, pooled data (n = 10 for both) showing the time-course of LTP induced in ‘intact’ (A) and isolated (B) hippocampal slices. In both types of slice preparation, LTP can be maintained for at least 5 h following tetanic stimulation of the Schaeffer collateral/commissural pathway (S1). Non-tetanized (S2) pathways show no significant increase in fEPSP slopes during these recordings. Examples of fEPSPs recorded as a result of the stimulation of S1 and S2 pathways in both ‘intact’ and ‘isolated’ slice preparations are shown above each graph. C, LTP in ‘isolated’ slices is blocked when tetanic stimulation is carried out in the presence of the NMDA receptor antagonist, d-AP5 (n = 3). A small amount of post-tetanic potentiation is visible in the S1 pathway. D, plot of paired-pulse facilitation ratios recorded prior to and 1 h and 4 h after the application of the tetanus for LTP recorded in ‘intact’ (filled bars) and ‘isolated’ (shaded bars) slice preparations (n = 4 for both). Although PPF ratios are higher in ‘intact’ slices, when compared to ‘isolated’ slices there is no significant difference in these ratios within each preparation at the time points indicated.

Figure 3. Late-LTP in both ‘intact’ and ‘isolated’ hippocampal slices is blocked by inhibitors of mRNA translation

A and C, pooled data obtained from ‘intact’ slices where LTP was induced in the presence of the mRNA translation inhibitors cycloheximide (A, n = 8) or rapamycin (C, n = 5). In each case these inhibitors did not affect the initial size of the potentiation obtained but prevented the establishment of stable late-LTP. Thus, 4 h after the application of the tetanic stimulation the slope values for the fEPSPs have returned to control (pretetanus) levels. B and D, pooled data obtained from ‘isolated’ hippocampal slices treated with cycloheximide (B, n = 7) or rapamycin (D, n = 9). As is the case for ‘intact’ slices, late-LTP was blocked under these recording conditions. Representative fEPSPs recorded from stimulation of the S1 pathway and taken from the time points are shown above each of the plots. For comparison, in each of the plots shown, the magnitudes and time-courses of late-LTP obtained in control slices (and shown in Fig, 2) are indicated by the light grey symbols.

Figure 4. Differential effect of mRNA transcription inhibitors on late-LTP in ‘intact’ and ‘isolated’ hippocampal slices

A, pooled data obtained from ‘intact’ slices (n = 8) where LTP was induced in the presence of the mRNA transcription inhibitor actinomycin D. The LTP obtained in the presence of actinomycin D did not show a stable profile. B, late-LTP in ‘isolated’ slice preparations (n = 6) in the presence of actinomycin D shows a similar time-course and magnitude to late-LTP obtained in ‘isolated’ slices in non-drug-treated preparations. Again, for ease of comparison, in each of the plots shown, the magnitudes and time-courses of late-LTP obtained in control slices (and shown in Fig 2) are indicated by the light grey symbols.

Figure 5. mRNA translation inhibitors reduce the incorporation of ³⁵S-methionine in hippocampal slices

Autoradiograph of a SDS-PAGE gel showing the levels of ³⁵S-methionine incorporation into untreated hippocampal slices (lane 1) and slices treated with rapamycin (1 μm, lane 2) or cycloheximide (300 μm, Lane 3). Clear reductions in these levels are seen in lanes 2 and 3.