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Università "Federico II," Naples, Italy
1 NIMH, NIH, Bethesda, MD
The presence of RNA in axons has been known for nearly 40 years (1). In large model axons, such as the goldfish Mauthner axon and the squid giant axon, axoplasmic RNA includes the main cytoplasmic species, that is, tRNA, rRNA, and mRNA. These findings have significantly contributed to the demonstration of an active protein synthetic system in axons (for reviews, see 2–4). In the squid, protein synthesis also occurs in the presynaptic terminals of the retinal photoreceptor neurons (5).
The above data have led us to question the cellular origin of axonal and presynaptic RNAs, often located at considerable distances from the neuronal soma. Evidence that axonal RNAs may not exclusively derive from cell bodies, but may also be synthesized locally, has been reported in several systems, including the isolated squid giant axon (6; for reviews, see 3,4). Indeed, when the giant axon was incubated with [3H]uridine, all axoplasmic RNA species were labeled (6).
To further investigate this process, we have been using the perfused squid giant axon—a cannulated axon still embedded in the stellate nerve but lacking the nerve cell bodies. The axon is placed in a sealed chamber containing artificial seawater (ASW), and perfusion is routinely continued for about 2 h, at which time EM analyses show the lack of axonal damage.
Upon addition of [3H]uridine to the chamber, [3H]RNA appears in the perfusate after a brief lag period, and continues to accumulate at a linear rate for at least 2 h. The perfusate [3H]RNA is transcribed from a nuclear DNA template, as shown by its reproducible inhibition by actinomycin D added to the external medium (20–50 µg/ml), and by electrophoretic analyses indicating tRNA and eukaryotic rRNA as its main components. As noted for axoplasmic [3H]RNA (6), these observations suggest that the perfusate [3H]RNA is synthesized by periaxonal glial cells, thus opening the way to investigations of the mechanisms modulating RNA transfer from glia to axon.
In initial studies, we assessed the effect of raising the concentration of K+ ions to 100 mM in the ASW bathing the perfused axon. Under this depolarizing condition, the rate at which [3H]RNA appeared in the perfusate increased markedly, often in more than one episode. Similar effects were obtained when the concentration of K+ ions in the axon perfusate was drastically reduced, thereby inducing depolarization of the axon but not of the glia. These results suggested that the increased rate of synthesis, delivery, or both, of [3H]RNA to the axon perfusate was mediated by the release of one or more neurotransmitters from the depolarized axon that activated receptors in the glial plasma membrane.
These receptors include two glutamate receptors (NMDA and metabotropic of class II), and a nicotinic ACh autoreceptor, identified on the basis of voltage changes induced in periaxonal glia by neurophysiological stimulation of squid or crayfish giant axons, or by addition of agonists or antagonists of these receptors (7,8). None of these receptors is present on the axonal membrane. While glutamate was initially considered the candidate neurotransmitter released by the axon, recent studies have shown that the true neurotransmitter is the dipeptide N-acetylaspartylglutamate (NAAG), which specifically binds to the metabotropic glutamate receptor, and is rapidly degraded to N-acetylaspartate and glutamate by a glial ectocarboxypeptidase (9).
In our most recent experiments, we have used several agonists of the glutamate and ACh glial receptors, singly or in association with specific antagonists, to examine their effects on the rate of appearance of [3H]RNA in the perfusate. Our data indicate that NAAG, glutamate, NMDA, or carbachol markedly increase the delivery of glial [3H]RNA to the perfusate, generally in more than one wave (Fig. 1). Activation of either glutamate receptor in the presence of the specific antagonist of the other (MK801 for the NMDA receptor, EGLU for the metabotropic II receptor) elicits comparable effects.
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We gratefully acknowledge friendly support from the MBL staff, grants from the University of Naples "Federico II" and MUIR to AG, and NIH support to BBK. We thank R. De Stefano, C. Cefaliello, and D. De Martino for the [3H]RNA analyses.
Literature Cited
This article has been cited by other articles:
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  A. Giuditta, J. Tai Chun, M. Eyman, C. Cefaliello, A. P. Bruno, and M. Crispino Local Gene Expression in Axons and Nerve Endings: The Glia-Neuron Unit Physiol Rev, April 1, 2008; 88(2): 515 - 555. [Abstract] [Full Text] [PDF]
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