Neuronal lesioning with axonally transported toxins

doi:10.1016/S0165-0270(00)00297-1

Journal of Neuroscience Methods

Volume 103, Issue 1, 15 November 2000, Pages 73-82

https://doi.org/10.1016/S0165-0270(00)00297-1 Get rights and content

Abstract

Axonally transported toxins can be used to make selective lesions of the nervous system. Collectively, these techniques are termed ‘molecular neurosurgery’ because they exploit the surface molecular identity of neurons to selectively destroy specific types of neurons. Suicide transport, is anatomically selective but not type-selective. The most widely used suicide transport agents are the toxic lectins (ricin, volkensin) and the immunotoxin, OX7-saporin. The toxic lectins and saporin are ribosome inactivating proteins that irreversibly inhibit protein synthesis. The toxic lectins have binding subunits but saporin requires a targeting vector to gain entrance into cells. Immunolesioning uses monoclonal anti-neuronal antibodies to deliver saporin selectively into neurons that express a particular target surface antigen. Neuropeptide–saporin conjugates selectively destroy neurons expressing the appropriate peptide receptors. Notable experimental uses of these agents include analysis of the function of the cholinergic basal forebrain (192-saporin) and pain research (anti-DBH-saporin, substance P-saporin). It is likely that more immunolesioning and neuropeptide-toxin conjugates will be developed in the near future.

Section snippets

Suicide transport

This term, coined by D.J. Reis, refers to the uptake and axonal transport of toxin that produces a neural lesion afferent to the site of toxin injection. In the peripheral nervous system, the toxic lectins ricin, abrin, modeccin and volkensin, have been used to ablate neurons projecting through a particular nerve (Wiley et al., 1982, Wiley and Stirpe, 1987). This approach has been used for several experimental purposes in the peripheral nervous system:

1.
To determine the cellular localization of

Immunolesioning

This term, coined by D.A. Lappi, refers to the use of anti-neuronal monoclonal antibodies to selectively target toxin to neurons expressing the appropriate surface antigen. Immunotoxins have been studied as cell type-specific killers for a number of years. The original impetus was to develop therapeutic agents for treatment of cancer or for immunosuppression (Uhr, 1984, Frankel, 1988). In order to achieve specific targeting, the monoclonal antibodies must be armed with a ribosome inactivating

Neuropeptide-toxin conjugates

Neuropeptide conjugates were first used a number of years ago in experiments on the hypothalamic–pituitary endocrine system (Samson et al., 1992a, Samson et al., 1992b, Samson et al., 1993, Samson et al., 1995, Blackburn et al., 1993, Blackburn et al., 1995a, Blackburn et al., 1995b). Recently, Mantyh and colleagues showed that substance P is selectively internalized by neurons expressing the neurokinin-1 receptor (NK-1R) (Mantyh et al., 1995). Based on this observation, we made substance

Suicide transport

In the peripheral nervous system, ricin remains a useful agent.

1.
Dilute stock ricin solution in sterile saline with 0.1% Fast Green dye. For a typical rat peripheral nerve, a concentration of 1 μg/μl is a reasonable starting point.
2.
Expose and mobilize a short segment of the nerve in an anesthetized animal.
3.
Pressure microinject 1 μl of ricin solution using a glass micropipette with the tip broken back or a fine syringe needle and a Hamilton microsyringe. Insert the pipette or needle into the nerve

Caveats – lessons learned the hard way

A number of considerations are important when using axonally transported toxins:

1.
These toxins are proteins that must be handled appropriately including minimizing freezing and thawing, keeping aliquots sterile and cold and not foaming when mixing. Standard preservatives (i.e. azide) are incompatible with the toxins.
2.
Each experiment should be preceded by pilot experiments to determine the dose and route of administration that will produce the desired lesion.
3.
Anatomical studies are always needed to

Summary

The collection of techniques described now makes possible a variety of powerful experiments generally aimed at determining the function of a particular group of neurons by analyzing the effect of selectively destroying the neurons in question. The choice of targets is continuing to increase ever more rapidly now that the usefulness of this approach has become apparent. The challenge will be to integrate this information with the other lines of inquiry in coming to conclusions about the function