Cerebellar Regional Dissection for Molecular Analysis

Summary

Different cerebellar regions have been implicated to play a role in distinct behavioral outputs, yet the underlying molecular mechanisms remain unknown. This work describes a method to reproducibly and quickly dissect cerebellar cortex of the hemispheres, anterior and posterior regions of the vermis, and the deep cerebellar nuclei in order to probe for molecular differences by isolating RNA and testing for differences in gene expression. 

Abstract

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies indicate that distinct cerebellar regions contribute to these different functions. Molecular studies examining regional cerebellar differences are lagging as they are mostly done on whole cerebellar extracts thereby masking any distinctions across specific cerebellar regions. Here we describe a technique to reproducibly and quickly dissect four different cerebellar regions: the deep cerebellar nuclei (DCN), anterior and posterior vermal cerebellar cortex, and the cerebellar cortex of the hemispheres. Dissecting out these distinct regions allows for the exploration of molecular mechanisms that may underlie their unique contributions to balance, movement, affect and cognition. This technique may also be used to explore differences in pathological susceptibility of these specific regions across various mouse disease models.

Introduction

The cerebellum contains over half of the neurons in the brain and has historically been referred to as a motor control and balance center in the brain¹. More recently, studies have demonstrated that the cerebellum plays a key role in various other functions including cognition, reward processing, and affect^2,3,4,5.

The cerebellum has well-described anatomy: the cortex region is composed of granule, Purkinje, and molecular layers. Granule cells form the granule cell layer and send input via ....

Protocol

1. Setup

1. Gather necessary equipment including decapitation scissors, blunt forceps, dissection scissors, vascular scissors, microspatula, sagittal mouse brain matrix, razor blades, 200 µL pipet tips, glass petri dish, glass slide, and ice bucket. Lay out all equipment on an absorbent pad.
2. Place petri dish, glass plate, and brain matrix on ice.
3. Using a razor blade at a perpendicular angle, trim about 5 mm off the tip of one 200 µL pipet tip. This makes the size of the opening at the end of the tip about 1 mm wide. This should be sufficient to punch out the DCN. But this can also be adjusted as needed ....

Results

For these experiments, four eleven-week-old female wild type C57/Black6 mice were used. One mouse was used to conduct a full cerebellar dissection which is referred to as 'bulk cerebellum' and allowed for the comparison of RNA levels in dissected regions to a full dissection. The other three mice were used to conduct the cerebellar dissection described in this protocol. Using three mice makes it possible to ensure that the trends detected in the levels of RNA are reproducible acro.......

Discussion

The method described here makes it possible to assess the underlying gene expression and molecular mechanisms within four distinct cerebellar regions – the deep cerebellar nuclei (DCN), the anterior cerebellar cortex of the vermis (CCaV), the posterior cerebellar cortex of the vermis (CCpV), and the cerebellar cortex of the hemispheres (CCH). The ability to assess these regions separately will expand our knowledge of the heterogeneity of specific cerebellar regions and possibly shed light on their contribution to v.......

Materials

Name	Company	Catalog Number	Comments
1.5 Microcentrifuge tubes	ThermoScietific	3456
100% Isopropyl Alcohol	VWR Life sciences	1106C361
200 ul Pipet tips	GeneMate	P-1237-200
Adult Mouse Brain Matrix Sagittal	Kent Scientific Corporation	RBMA-200S
Blunt forceps
Chloroform	Macron	220905
Decapitation Scissors
Dissecting Scissors
Ethyl Alcohol	Pharmco	111000200
Glass Slide (for electrophoresis)	BIORAD
Homogenizer	Kimble	6HAZ6
Ice Bucket
Insulin Syringe (.5ml)	BD	329461
iScript Adv cDNA kit for RT-qPCR	BIORAD	1725037
Micro Spatula
Needle Nose forceps
Petri Dish	Pyrex
Primetime Primer for Aldolase C	IDT	Mm.PT.58>43415246
Primetime Primer for Kcng4	IDT	Mm.PT.56a.9448518
Primetime Primer for Parvalbumin	IDT	Mm.PT.58.7596729
Primetime Primer Rps18	IDT	Mm.PT.58.12109666
Single Edge Rzor Blades	Personna GEM
Sterile, sigle-use pestles	FisherScientific	12141364
TRIzol Reagent	Ambion by Life technologies	15596018
Vascular Scissors