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Department of Molecular Physiology

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Pain
Cultured dorsal root ganglion neurons

Pain cells (nociceptors) are difficult to examine. Their most interesting parts – the sensory endings – are very thin fibers, embedded in a tough layer of skin tissue. Conventional methods for physiological experimentation like calcium imaging or electrophysiology can hardly be applied to these fibers. Consequently, there is little direct physiological information about what happens when painful stimuli hit the skin. One approach to this problem is to isolate the somata of pain cells out of the dorsal root ganglia, to keep them in primary culture, and to study transduction proteins in these cultured neurons. The basic assumption is that the cultured pain cells express the same proteins that mediate pain transduction the sensory endings in vivo.

The dorsal root ganglia (DRG) contain the cell bodies of nociceptors. A bifurcated axon emanates from each cell body. At its central end this axon forms a synapse within the spinal cord, the peripheral, sensory ending lies in the skin or in other pain-sensitive tissues. Primary cultures from dorsal root ganglia grow well on a surface covered with laminin, an extracelluar matrix protein. Non-neuronal cells grow a dense carpet over the entire surface and form a support on which pain cells (large, round cells) can live for several weeks. Pain cells often grow neurites which connect several cells, a process which is promoted by neuronal growth factors.

Which transduction proteins can be studied in primary cultures of pain cells? A good way to detect the proteins is to use specific antibodies, labeled with a fluorescent dye. The image on the right shows a pain cell culture stained with an antibody that was raised against the heat-sensitive ion channel TRPV1. This channel mediates the perception of noxious heat; it is openend by tempereatures above 40 oC. Neurons expressing TRPV1 (“transient receptor potential vanilloid receptor type 1”) are labeled red on the image, the blue stain (DAPI) shows cell nuclei.

TRPV1 channels can not only be opened by heat, but also by capsaicin, the pungent component of chilli peppers. Capsaicin opens TRPV1 channels in sensory endings of heat-sensitive nociceptors and, thereby, produces a fake “hot” sensation. This effect can be studied in pain cell cultures using the patch-clamp technique. A microelectrode is used to record the membrane potential of a pain cell. The resting, unstimulated cell displays membrane potentials in the range of -60 to -80 mV. Applying 1 µM capsaicin depolarizes the neuron and causes it to fire action potentials. Thus, the cultured DRG neuron responds to a painful stimulus by generating an electrical signal.



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