Applied Methods in Spinal Cord Neurophysiology

Whole-cell patch-clamp experiments

 


 

The patch-clamp method was first described  by Bert Sakmann and Erwin Neher in the 1970s, and the Nobel Prize was awarded in 1991 for their invention. Otherwise the idea is quite simple: a glass pipette is used to make a tight contact with the cell surface. In whole-cell patch clamp experiments the membrane is usually ruptured by a gentle suction and the solution used for filling the glass electrode becomes continuous with the cytoplasm. In this configuration the membrane potential or the ionic currents flowing through the cell membrane can be measured by ultrasensitive amplifiers. This technique can be used for the investigation of isolated and cultured cells, but experiments on cells in tissue slices can also be carried out.

 

Click on the image for a short animation!

Back to top

 

Our experiments are conducted on young, 3-4 weeks-old Wistar rats of either sex. (Neurons in slices prepared from young rats have a better survival rate, while the visual identification of cells becomes almost impossible in rats over 5 weeks due to the myelinization.) Animals are anesthetized with urethane. Following a longitudinal incision along the length of the vertebrae, the muscles and connective tissue are removed and lumbosacral laminectomy is performed. The spinal cord is placed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF). All the ventral roots are cut off and the pia-arachnoid membrane is removed. The lumbar spinal cord is mounted on a vibratome and transverse slices are cut. The thickness varies between 300 and 800 µm, depending on the purpose of the experiment. Entering dorsal roots are also reserved if possible. Spinal cord slices are incubated for at least half an hour prior recordings at temperature of 33-35C.

 

Back to top

 

Single slices are transferred into the recording chamber and are perfused continuously with oxygenated aCSF. Our microscope is equipped with water immersion objective, DIC (differential interference contrast) prisms and digital camera, which make possible the visualization of neurons in near-infrared light without any kind of staining.

In blind patch experiments lower magnification is used when the target area is selected, for example the substantia gelatinosa appears as a translucent band across the spinal dorsal horn. Constant voltage steps are applied continuously through the glass electrode during searching for cells, and changes in current response appear when the tip of the electrode gets in contact with the cell membrane.In this method cells of the region of interest are patched randomly, and the investigator is not biased by the appearance (size, shape, etc.) of the cell

In visually-guided experiments higher magnification is used which makes possible the visual identification of individual neurons. The appearance of the cells provides important information about their viability: healthy neurons can be distinguished from dead ones. So no time is wasted for cells in bad condition.

   

Back to top

 

After the membrane has been ruptured two types of measurements, the voltage-clamp and the current-clamp methods can be carried out. In the voltage-clamp mode the membrane potential is held at a required level determined by the purpose of the experiment. In this case the current necessary for maintaining the holding potential is measured. In current-clamp experiments the changes in membrane potential are recorded while currents varying in magnitude and direction are injected into the cell.

     

Back to top

 

Excitatory postsynaptic potentials and currents are evoked by dorsal root stimulation. Aδ- and C-fiber-evoked eEPSPs/eEPSCs can be distinguished on the basis of the conduction velocity of afferent fibers (C-fiber: 2-13 m/s and Aδ-fiber <0.8m/) and stimulus treshold (C-fiber: 150-550µA, Aδ-fiber: 10-60µA). The Aδ-fiber and C-fiber responses can be considered as monosynaptic in origin when the latency remaines constant and there is no failure during stimulation at 20 Hz for the Aδ-fiber eEPSPs/EPSCs, and 2 Hz for  the C-fiber eEPSPs/EPSCs.

Back to top


 

Our patch-clamp setup

Microscope

Olympus BX51WI   >>>

(equipped with water-immersion objective,

DIC prisms, epifluorescent illumination and filters)

Patch-clamp Amplifiers

Axoclamp-2A

Axopatch-200B   >>>

 

Camera

F-View II digital camera   >>>

Data Acquisition System

Axon Digidata 1320

Axon MiniDigi

Micromanipulator

Sutter MP-225   >>>

Stimulator and stimulus isolator

A.M.P.I. Master-8   >>>

A.M.P.I. Iso-Flex   >>>

Electrode puller

Sutter P-97   >>>

 

Others

Hitachi oscilloscope

PC computer

     

 Back to top