Mechanical Manipulation of Neurons to Control Axonal Development

Summary

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Abstract

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 μdyne range^1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method³. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation^4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction⁷; as well as detachment and mechanical measurements on any type of cultured cell⁸.

Protocol

1. Making glass needles.

1. An adjustable micro-needle puller is used to fabricate needles with a tapered tip about 4 mm in length and that are closed solid beams. As opposed to a long flexible tip, this short 4 mm length limits vibrations of the needle tip during experiments. At the proximal region of the 4 mm fiber, the needle tapers rapidly from the diameter of the glass tubing to 15 μm within 1 mm, while the distal-most 1 mm of the fiber is 2.5 μm in diameter. We use R-6 glass capillary tube, OD 0.9 mm, ID 0.6 mm 8" length and a BB-CH puller. If the investigator is interested in measuring other force ranges, needles with different spring properties (i.e.....

Discussion

Techniques to apply and measure cellular forces have a long history⁹. Our method was originally motivated by the work of Dennis Bray, who used glass needles similar to ours to 'tow' neurons at a constant rate using a motorized hydraulic device¹⁰. There are many alternative means of applying forces to cells which include: stepper motors¹¹, magnetic beads¹², microfabricated beams¹³ and fluid flows¹⁴. The latter are similar to our approach in that the cellula.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
R-6 cap. Tube	Drummond Scientific	9-000-3111	R-6 glass OD 0.9mm, ID 0.6 mm, 8"
BB-CH puller	Mecanex S.A., Geneva, Switzerland	BB-CH puller	Use Mode 4 Alt by CP=100, PP=10, SP1=1000, SP2=1000
0.001" Chromel wire	Omega Engineering, Inc.	SPCH-001-50	unsheathed, themocouple wire, 50ft spool now called Chromega
0.003" Constatan wire	Omega Engineering, Inc.	SPCI-003-50	unsheathed, themocouple wire, 50 ft spool
fine forceps	Fine Science Tools	91150-20	Dumont Inox #5
universal microscope boom stand	Nikon Instruments	76135 or 90430	most brands or types of boom stand will work for this use
mechanical micromanipulator	Narishige International	M-152	three-axis direct-drive coarse micromanipulator
hydraulic micromanipulator	Narishige International	MO-203	now available as MMO-203, three movable axis type
needle holder	Leica Microsystems	11520145	set of 3
single instrument holder	Leica Microsystems	11520142
double instrument holder	Leica Microsystems	11520143
mechanical micromanipulator	Leica Microsystems	39430001	post mount,1 prob holder, RH Model 430001
joystick mech. micromanipulator	Leica Microsystems	11520137
Leica DM IRB	Leica Microsystems		inverted microscope
Vibraplane isolation table	Kinetic Systems	1200 series	ours is model 1201-02-12
Ringcubator	Home made (see reference 19)		reference 19, requires updated controller listed below
programable temperature controller	Instrumart.com	Fuji Electric PXR3	replaces the retired PXV3 temperature controller
Nikon Diaphot TMD	Nikon Instruments		inverted microscope, circa 1980
Nikon SMZ-10 binocular dissecting	Nikon Instruments		other dissecting microscopes will work