Is the lectin IB4 a specific marker for those sensory neurones that are selectively activated by peripheral tissue damage?

Overview

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Pain is an undesirable symptom of virtually every disease state and yet, even today, effective forms of pain control may be severely debilitating and addictive. Unmyelinated primary sensory neurones are responsible for the detection of peripheral tissue damage and relay this information to the central nervous system. Over the last ten years we have collected a large body of evidence to suggest that the neurones which detect peripheral tissue damage (and consequently are responsible for the sensation of pain) selectively express a unique cell-surface carbohydrate. If the hypothesis is substantiated, this carbohydrate will be an extremely valuable tool in the study of pain circuits as well as the delivery of drugs into these neurones which could act as new and novel analgesics. The aim of this series of experiments is to determine whether unmyelinated primary sensory neurones selectively express the characteristic cell-surface carbohydrate identified by binding sites for the lectin IB4. The results of this study have a high probability of generating a technology that has far reaching applications for both the study and treatment of pain.

Approaches

Microelectrodes filled with the fluorescent marker Neurobiotin will be used to make intracellular recordings from primary sensory neurones that innervate the tail in deeply-anaesthetised rats. The tail of the animal will then be mapped in order to identify the neurone's receptive field. Where a receptive field can be identified, this will be fully characterised using a battery of stimuli including brush, pressure, pinch, pin prick, noxious chemicals, cold, warm and heat. The conduction velocity of the neurone will then measured by electrical stimulation of the centre of the cutaneous receptive field. Cells will then be stained by iontophoresis of the Neurobiotin using depolarising pulses of 3 nA (100 ms pulse width at 3 Hz) for between 2 and 20 minutes. Throughout the injection the resting membrane potential and action potential shape will be monitored to ensure that the recording remains stable. In order to permit unambiguous identification, only one cell will be stained in each ganglion with up to six ganglia being injected in any one animal. At the end of the experiment, animals will be euthansed and the lumbar dorsal root ganglia removed for histochemical analysis. Frozen serial sections will then be cut through each ganglion, Neurobiotin filled cells identified using a rhodamine-avidin conjugate and then screened for IB4 binding sites using a IB4-FITC conjugate.

 

A. Intracellular recordings from a rat primary sensory neurone. B. Confocal image of the neurone characterised showing that it does not bind IB4.

Image A. Intracellular recordings from a rat primary sensory neurone.

Image B. Confocal image of the neurone characterised above (red fluorescence) showing that it does not bind IB4 (green fluorescence).

References

  1. Thornton, P.J., Gerke, M.B. and Plenderleith, M.B. (2005) Histochemical localisation of a galactose-containing glycoconjugate expressed by sensory neurones innervating different peripheral tissues. Journal of the Peripheral Nervous System. 10: 47-57.
  2. Gerke, M.B. and Plenderleith, M.B. (2004) Ultrastructural analysis of the central terminals of primary sensory neurones labelled by transganglionic transport of Bandeiraea simplicifolia I-isolectin B4. Neuroscience. 127: 165-175.
Study level
Honours
Supervisors
QUT
Organisational unit

Science and Engineering Faculty

Research area

Medical Sciences

Contact
Please contact the supervisor.