Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Summary

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Abstract

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g. obesity) increase with age.

Introduction

The brain regulates energy homeostasis through the neuroendocrine and autonomic nervous systems. The ventromedial hypothalamus (VMH), comprised of the ventromedial nucleus (VMN) and the arcuate nucleus (ARC), is important for the central regulation of energy homeostasis. Specialized glucose sensing neurons, within the VMH, link neuronal activity and peripheral glucose homeostasis¹. There are two types of glucose sensing neurons; glucose excited (GE) neurons increase while glucose inhibited (GI) neurons decrease their activity as extracellular glucose increases. VMH glucose sensing neurons are generally studied using electrophysiology or calcium/membrane pot....

Protocol

1. Animals

1. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Medicine and Dentistry of New Jersey.
2. Group house male C57BL/6 mice on a 12 hr light/12 hr dark schedule and allow ad libitum access to water and food. Sacrifice at 4-5 months old. Euthanasia of the mice was performed using the surgical plane of anesthesia and a secondary form of euthanasia (i.e. penetrating incision into the chest cavity via the diaphragm). This is consistent with the AVMA Guidelines on Euthanasia.

2. Preparation of Perfusion Solution, Coverslips, Glass Pipet....

Results

The precise dissection of the VMH away from other hypothalamic areas is important to obtain consistent results. The inclusion of other areas could dilute the VMH neuronal population, changing the % of depolarized neurons calculated. Furthermore, glucose sensing neurons have been identified in other hypothalamic regions, such as the lateral hypothalamus, which may differ functionally and mechanistically from VMH glucose sensing neurons. Figure 1 illustrates the correct anatomical locations for proper diss.......

Discussion

The key to being able to study activity of neurons from adult mice is the ability to dissociate healthy neurons. Dissociation of hypothalamic neurons from adult mice is more difficult at several key steps in the protocol compared to neurons from juvenile mice. We have overcome this problem in a number of ways. Making thick 500 µm brain slices minimizes mechanical damage to neurons compared to the usual 250-350 µm slices used for brain tissue from younger mice. However, thicker slices require greater attention t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neurobasal-A Medium (Custom)	Invitrogen	0050128DJ	custom made glucose free
Hibernate-A Medium (Custom)	BrainBits		custom made glucose free
Penicillin streptomycin (20,000 U/ml)	Invitrogen	15140	other vendors acceptable
Stericup vacuum filter units (0.22 μm)	Millipore		other vendors acceptable
25 mm Glass coverslips	Warner	#1 25mm round
18 mm Glass coverslips	Warner	#1 18mm round
GlutaMAX	Invitrogen	35050
B27 minus insulin (50x)	Invitrogen	0050129SA
Razor blade	VWR	55411
Vibratome & cooling chamber	Vibratome	Series 1000 Sectioning system
Vibratome blades	Polysciences	22370	injector or double edge blades from other vendors acceptable
Papain, suspension	Worthington	LS003124
BSA, suitable for cell culture	Sigma		other vendor acceptable
DNAse, for cell culture	Invitrogen		other vendor acceptable
cloning cylinders, 6 mm x 8 mm	Bellco Glass	2090-00608
Membrane Potential Dye (blue)	Molecular Devices	R8042
In-line heater	Warner	SF-28
Syringe pumps	WPI	sp100i	other vendor acceptable
Closed chamber	Warner	RC-43C
Polyethylene tubing	Warner	PE-90
Metamorph	Molecular Devices		alternate image analysis software acceptable
Microscope	Olympus	BX61 WI	used with 10X objective
Camera	Photometrics	Cool Snap HQ
Narrow Cy3 Filter Set	Chroma	41007a
Illumination System	Sutter Instruments	Lambda DG-4