Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABA(A) Mediated Excitation

A developmental “switch” in chloride transporters occurs in most neurons resulting in GABA(A) mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABA(A) receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH) neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABA(A) receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABA(A) receptor activation. Anion exchangers (AEs) are also potential modulators of responses to GABA(A) activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s) underlying GABA(A) mediated depolarization, GnRH neurons were analyzed for 1) expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2) responses to GABA(A) receptor activation in NKCC1(-/-) mice and 3) function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1(-/-) mice retained relatively normal responses to the GABA(A) agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO(3) (-) mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABA(A) mediated transmission in GnRH neurons.