Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Summary

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Abstract

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

Introduction

Dynamic alterations in the number and structure of synapses are hallmarks of development, aging, and numerous neurodegenerative disorders^1-3. The ability of neurons to receive and integrate synaptic information depends upon dendritic morphology and dynamic alterations in synaptic connections. Indeed, a positive correlation exists between dendritic spine and synapse number, which both impact cognitive function⁴. Thus, it is not surprising that decrements in dendritic spine number have been associated with cognitive dysfunction in a number of neurological disorders^5-7, prompting great interest in dendritic spine quantification. Neverthel....

Protocol

NOTE: Experiments were conducted in accordance with the ethical standards approved by the Committee on Animal Research at the Veterans Affairs Palo Alto Health Care System.

1. Golgi-Cox Staining

1. Brain extraction and staining
   1. On the 1st day, deeply anesthetize mice with 100 mg/kg ketamine and 10 mg/kg xylazine before euthanizing via exsanguination.
   2. Carefully remove the calvarium and dissect out the brain.
      1. First remove the skin on the top of the skull, placing a curved scissor on the top of the cerebellum and gently cut through the calvarium parallel to the central sulcus with the tip of the sc....

Results

The extent of arborization arising from extant and newly born dentate granule cells was analyzed in wild type mice using either Golgi-Cox or DCX staining (Figure 1). Dendritic segments of DCX-positive cells were found to be 13-36 microns long. The normal distribution of dendritic length was tested using Kolmogorov-Smirnov test (D = 0.1217, p <0.01, Liliefors p <0.001; Figures 4 and 5).

In the analysis of length of segments per order of a.......

Discussion

Here, two methods were described to quantify the extent of dendritic arborization in mature and newly-born neurons using conventional staining methods in conjunction with RTI and EDFI. The acquisition of high resolution images of neurons provides an extremely useful method for testing the deleterious effects of neurodegenerative disorders and, in turn, provides a means to assess therapeutic strategies that target hippocampal neurons.

While the RTI method was utilized to capture in-depth data p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Modified Golgi-cox staining solution	Weill Cornell Medical College	NA	store at 4°C till use
1x Developing Solution (Stock 10x)	Weill Cornell Medical College	NA	store at 4°C till use
30% Sucrose,	Sigma	CAS # 57-50-1	make fresh  in ddH2O
0.3% Gelatin	Sigma	CAS # 9000-70-8	NA
Graded Ethanol Solutions (20%, 30%, 40%, 50%, 80%. 90%, 95%. 100%)	Sigma	CAS 603-003-00-5	NA
Xylene	Sigma	CAS # 1330-20-7	NA
DPX Medium	EMS	#13510	NA
Superfrost (+) white	Electron Microscopy Sciences	71869-10	NA
Coverslip 22x50mm (VWR #48393-059)	VWR	#4811-703	NA
DCX Antibody	Santa Cruz Biotechnology	sc-8066	4 C
DAB	Sigma	CAS Number 91-95-2	-20
OCT	Tissue-tek	4583	NA
Tris	Sigma	CAS Number 77-86-1	NA
ABC Lite	Vector	PK4000	NA
Microscope	Nikon	Eclipse 80i
Digital Camera	Nikon	DS-Ri1
12 bit Camera	QImaging	01 MBF2000RF-CLR-12
Neurolucida System	MBF Bioscience	V.10
Image Composite Editor	Microsoft	1.4.4.0
NIS Elements	Nikon	F 3.0
Image Pro Plus	Mediacy	Versin 7.00