Two Behavioral Tests Allow a Better Correlation Between Cognitive Function and Expression of Synaptic Proteins.

Marta Balietti,Daniele Marcotulli,Fiorenzo Conti,Arianna Pugliese,Luca Bragina,Giorgia Fattorini

doi:10.3389/fnagi.2018.00091

Abstract

The molecular substrate of age-associated cognitive decline (AACD) is still elusive. Evidence indicates that AACD is related to synaptic impairment in hippocampus, but different hippocampal regions play different roles, with the dorsal hippocampus (DH) associated to spatial learning, and the ventral hippocampus (VH) crucial for emotionality. If changes in hippocampal function contributes to AACD, this contribution may be reflected in alterations of synaptic protein levels. A commonly used approach to investigate this issue is western blotting. When this technique is applied to the entire hippocampus and the cognitive impairment is evaluated by a single task, changes in expression of a protein might undergo a “dilution effect”, as they may occur only in a given hippocampal region. We show that two behavioral tests yield more accurate results than one test in evaluating the function of the whole rat hippocampus by studying the expression of synaptotagmin 1 (SYT1), a vesicular protein whose expression in aged hippocampus is reportedly inconsistent. Analysis of SYT1 levels in the whole hippocampus of rats selected by the Morris water maze (MWM) test only failed to highlight a difference, whereas analysis of SYT1 levels in the whole hippocampus of rats categorized by both the MWM and the step-through passive avoidance (STPA) tests demonstrated a significant increase of SYT1 level in impaired rats. These findings, besides showing that SYT1 increases in impaired aged rats, suggest that using the whole hippocampus in blotting studies may prevent false negative results only if animals are categorized with tests exploring both DH and VH.

Highlights

The molecular substrate of age-associated cognitive decline (AACD) is still elusive
Analysis of synaptotagmin 1 (SYT1) levels in the whole hippocampus of rats selected by the Morris water maze (MWM) test only failed to highlight a difference, whereas analysis of SYT1 levels in the whole hippocampus of rats categorized by both the MWM and the step-through passive avoidance (STPA) tests demonstrated a significant increase of SYT1 level in impaired rats
Mounting evidence indicates that age-associated cognitive decline (AACD) can be related to synaptic impairment in hippocampus (e.g., Vanguilder and Freeman, 2011; Vanguilder et al, 2011), and investigations on synaptic structure and function and on synaptic proteins have flourished in the recent past (e.g., Burke and Barnes, 2006; Bishop et al, 2010; Bano et al, 2011)

Summary

THE TWO BEHAVIORAL TESTS APPROACH

We reasoned that the hippocampal regions analyzed by western blotting, the amount of tissue examined and the ability of the method to detect alterations in proteins levels, can be increased by employing a larger number of behavioral tests. We tested the hypothesis that two behavioral tasks provide more accurate results than one test alone in evaluating the function of the whole rat hippocampus by studying the expression of the vesicular protein synaptotagmin 1 (SYT1). Chen et al (2007), who categorized their animals with the MWM test and used only dorsal hippocampal tissue for western blotting analysis, reported that cognitive impairment correlated with increased SYT1 levels, whereas Nicolle et al (1999), who categorized the animals by the same test but examined the whole hippocampus found no difference in SYT1 levels. Using the entire hippocampus to analyze proteins levels requires classifying animals by at least two tests, exploring the dorsal and ventral portion of the hippocampus, if false negative results are to be avoided

HOW TO CHOOSE?

Findings

CONCLUSION