RESEARCH ARTICLE Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation

A developmental “switch ” in chloride transporters occurs in most neurons resulting in GABAA mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABAA receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH) neurons, which control puberty and repro-duction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABAA receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABAA receptor activation. Anion exchang-ers (AEs) are also potential modulators of responses to GABAA activation since they accu-mulate chloride and extrude bicarbonate. To evaluate the mechanism(s) underlying GABAA mediated depolarization, GnRH neurons were analyzed for 1) expression of chloride trans-porters and AEs in embryonic, pre-pubertal, and adult mice 2) responses to GABAA recep-tor 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 GABAA ago-nist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide elimi-nated the depolarization/calcium response to muscimol in 40 % of GnRH neurons fromWT mice. In the remaining GnRH neurons, HCO3- mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABAA mediated transmission in GnRH neurons