Anesthetics interfere with axon guidance in developing mouse neocortical neurons in vitro via a γ-aminobutyric acid type A receptor mechanism.
BACKGROUND
The finding that exposure to general anesthetics (GAs) in childhood may increase rates of learning disabilities has raised a concern that anesthetics may interfere with brain development. The generation of neuronal circuits, a complex process in which axons follow guidance cues to dendritic targets, is an unexplored potential target for this type of toxicity.


METHODS
GA exposures were conducted in developing neocortical neurons in culture and in early postnatal neocortical slices overlaid with fluorescently labeled neurons. Axon targeting, growth cone collapse, and axon branching were measured using quantitative fluorescence microscopy.


RESULTS
Isoflurane exposure causes errors in Semaphorin-3A-dependent axon targeting (n = 77 axons) and a disruption of the response of axonal growth cones to Semaphorin-3A (n = 2,358 growth cones). This effect occurs at clinically relevant anesthetic doses of numerous GAs with allosteric activity at γ-aminobutyric acid type A receptors, and it was reproduced with a selective agonist. Isoflurane also inhibits growth cone collapse induced by Netrin-1, but does not interfere branch induction by Netrin-1. Insensitivity to guidance cues caused by isoflurane is seen acutely in growth cones in dissociated culture, and errors in axon targeting in brain slice culture occur at the earliest point at which correct targeting is observed in controls.


CONCLUSIONS
These results demonstrate a generalized inhibitory effect of GAs on repulsive growth cone guidance in the developing neocortex that may occur via a γ-aminobutyric acid type A receptor mechanism. The finding that GAs interfere with axon guidance, and thus potentially with circuit formation, represents a novel form of anesthesia neurotoxicity in brain development.
Department of Anesthesiology, Columbia University, New York, New York 10032, USA. cdm2134@columbia.edu
C David
CD
Mintz
Kendall M S
KM
Barrett
Sarah C
SC
Smith
Deanna L
DL
Benson
Neil L
NL
Harrison
D000704:Analysis of Variance; D000768:Anesthesia, General / Q000379:methods; D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D000818:Animals; D001369:Axons / Q000187:drug effects*; D001921:Brain / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D020439:Growth Cones / Q000187:drug effects; D066298:In Vitro Techniques; D007530:Isoflurane / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence / Q000379:methods; D009414:Nerve Growth Factors / Q000187:drug effects; D055495:Neurogenesis / Q000187:drug effects; D009474:Neurons / Q000187:drug effects*; D018079:Receptors, GABA / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects
D018685:Anesthetics, Inhalation; D009414:Nerve Growth Factors; D018079:Receptors, GABA; D007530:Isoflurane