Profound molecular changes following hippocampal slice preparation: loss of AMPA receptor subunits and uncoupled mRNA/protein expression

Abstract

The acute hippocampal slice preparation is a convenient, in vitro model widely used to study the biological basis of synaptic plasticity. Although slices may preserve their electrophysiological properties for several hours, profound molecular changes in response to the injury caused by the slicing procedure are likely to occur. To determine the magnitude and duration of these changes we examined the post-slicing expression kinetics of three classes of genes known to be implicated in long-term synaptic plasticity: glutamate AMPA receptors (GluR), transcription factors and neurotrophins. Slicing resulted in a striking loss of GluR1 and GluR3, but not of GluR2 proteins suggesting that rapid changes in the composition of major neurotransmitter receptors may occur. Slicing caused a significant induction of the transcription factors c-fos, zif268, CCAAT enhancer binding protein (C/EBP) b and d mRNAs and of the neurotrophin brain-derived neurothophic factor (BDNF) mRNA. In contrast, there was no augmentation, and sometimes a decline, in the levels of the corresponding proteins. These data reveal that significant discrepancies exist between the slice preparation and the intact hippocampus in terms of the metabolism of molecular components known to be involved in synaptic plasticity.

Alteration of Neuronal Firing Properties afterIn VivoExperience in a FosGFP Transgenic Mouse

“…5 and data not shown). Other investigators have shown that c-fos mRNA is induced after slice preparation (Taubenfeld et al, 2002) but that this is uncoupled from translation. Thus, the amount of protein produced is low, and protein levels are not sufficient to overcome tissue autofluorescence and remain undetectable.…”

Protein synthesis and consolidation of memory-related synaptic changes

“…One possibility is suggested by evidence that the preparation of the slice causes transient increases in the activity of kinases involved in synaptic plasticity (Ho et al, 2004) followed by a return to conditions closer to those found in vivo. In parallel there is surge in the expression of transcriptional regulators (Taubenfeld et al, 2002;Zhou et al, 1995) and synaptic proteins. Thus, it is possible that at least some of the proteins necessary for consolidation are present at supra normal levels in freshly prepared slices, thereby creating an artificial condition in which inhibitors are ineffective.…”

Number and Density of AMPA Receptors in Individual Synapses in the Rat Cerebellum as Revealed by SDS-Digested Freeze-Fracture Replica Labeling

“…Otherwise, the discrepancy may be ascribable to differences in sample preparations. It has been reported that slicing can cause drastic changes in synapses (Kirov et al, 1999) and loss of GluR1 and GluR3 proteins (Taubenfeld et al, 2002) in the hippocampus. In addition, we cannot exclude the possibility that immunoreactive AMPARs may be partly nonfunctional, although our previous data suggested a close correlation between number of functional AMPAR channels and immunogold particles in immature CF-PC synapses (Tanaka et al, 2005).…”