Physiological temperature during brain slicing enhances the quality of acute slice preparations

Shiwei Huang,Marylka Y Uusisaari

doi:10.3389/fncel.2013.00048

Abstract

We demonstrate that brain dissection and slicing using solutions warmed to near-physiological temperature (~ +34°C), greatly enhance slice quality without affecting intrinsic electrophysiological properties of the neurons. Improved slice quality is seen not only when using young (<1 month), but also mature (>2.5 month) mice. This allows easy in vitro patch-clamp experimentation using adult deep cerebellar nuclear slices, which until now have been considered very difficult. As proof of the concept, we compare intrinsic properties of cerebellar nuclear neurons in juvenile (<1 month) and adult (up to 7 months) mice, and confirm that no significant developmental changes occur after the fourth postnatal week. The enhanced quality of brain slices from old animals facilitates experimentation on age-related disorders as well as optogenetic studies requiring long transfection periods.

Highlights

In cellular neuroscience research, acute brain slice preparation is the work-horse method for investigating microcircuits, single neurons, and sub-neuronal processes (Andersen, 1981), despite the necessity of other approaches when behavioral effects or large-scale circuit dynamics are studied
Typically, when slices from mature (>2 months) animals are prepared by the conventional “ice-cold” method, i.e., by cooling the brain to nearly 0◦C before and during slicing (Andersen, 1981), without trans-cardiac pre-perfusion (Moyer and Brown, 2002), the deep cerebellar nuclei (DCN) slice surface appears uneven and damaged (Figure 1, top left)
When slices were cut in exactly the same manner, but with cutting solutions warmed to near-physiological temperatures (+34 – 35◦C), slice appearance improved significantly (Figure 1, right panel)

Summary

Introduction

Acute brain slice preparation is the work-horse method for investigating microcircuits, single neurons, and sub-neuronal processes (Andersen, 1981), despite the necessity of other approaches (e.g., using living animals) when behavioral effects or large-scale circuit dynamics are studied. Various essential studies have been rendered difficult or even impossible because of this limitation It has become a grudgingly accepted fact that all in vitro work from the deep cerebellar nuclei (DCN) is limited to using young animals (Linnemann et al, 2006; Bagnall et al, 2009) and no in vitro data from animals older than 28–30 postnatal (P) days have been published (Uusisaari and Knöpfel, 2010; Person and Raman, 2011). Studies aiming at answering questions related to aging or neuro-degenerative diseases cannot be investigated in this key structure of the olivo-cerebellar circuitry. Another example is the expression of virally transfected proteins in optogenetic studies. Post-transfection times of weeks are often required to reach sufficient expression levels (Shimano et al, 2012); it is essential to be routinely able to perform slice experiments using brains of animals older than at least 6 weeks

Methods

Results

Conclusion