Anesthetics disrupt growth cone guidance cue sensing through actions on the GABA_A α2 receptor mediated by the immature chloride gradient

Background: General anesthetics (GAs) may exert harmful effects on the developing brain by disrupting neuronal circuit formation. Anesthetics that act on γ-aminobutyric acid (GABA) receptors can interfere with axonal growth cone guidance, a critical process in the assembly of neuronal circuitry. Here we investigate the mechanism by which isoflurane prevents sensing of the repulsive guidance cue, Semaphorin 3A (Sema3A). Methods: Growth cone sensing was assayed by measuring growth cone collapse in dissociated neocortical cultures exposed to recombinant Sema3A in the presence or absence of isoflurane and/or a panel of reagents with specific actions on components of the GABA receptor and chloride ion systems. Results: Isoflurane exposure prevents Sema3A induced growth cone collapse. A GABA_A α2 specific agonist replicates this effect (36.83 ± 3.417% vs 70.82 ± 2.941%, in the Sema3A induced control group, p < 0.0001), but an α1-specific agonist does not. Both a Na-K-Cl cotransporter 1 antagonism (bumetanide, BUM) and a chloride ionophore (IONO) prevent isoflurane from disrupting growth cone sensing of Sema3A. (65.67 ± 3.775% in Iso + BUM group vs 67.45 ± 3.624% in Sema3A induced control group, 65.34 ± 1.678% in Iso + IONO group vs 68.71 ± 2.071% in Sema3A induced control group, no significant difference) (n = 96 growth cones per group). Conclusion: Our data suggest that the effects of isoflurane on growth cone sensing are mediated by the α2 subunit of the GABA_A receptor and also that they are dependent on the developmental chloride gradient, in which Cl⁻ exhibits a depolarizing effect. These findings provide a rationale for why immature neurons are particularly susceptible to anesthetic toxicity.