Fig 1.
GnRH neurons are positive for NKCC1 and negative for KCC2 throughout development.
A1-2) Robust NKCC1 staining (brown) was detected at E14.5 in the nasal area (N) including cells in the olfactory epithelium and cells and fibers (OA, black arrows) at the nasal forebrain junction (J), with many fibers seen entering the developing olfactory bulb (OB). A2 is higher magnification of boxed area in A1. A3) GnRH neurons (green, arrows) in nasal areas were NKCC1 positive (red), showing light labeling over their cell soma (white arrows). Labeling of GnRH processes was obscured by dense labeling of olfactory axon bundles (black arrow). B) NKCC1 (red) is co-localized in GnRH neurons (green) in pre-pubertal (B1) and adult (B2) mice. NKCC1 labeling was more robust in GnRH neurons and surrounding preoptic tissue at pre-pubertal ages compared to adult. However, in the adult, some GnRH neurons remained clearly positive for NKCC1 in the cell soma and proximal processes (B2). C) NKCC1 (red) was present in the median eminence (ME), but did not co-localized with GnRH fibers (green). D) Chromagen double-labeling amplification of NKCC1 signal with NiDAB (blue-black) in sections from WT (D1) and NKCC1-/- (D2) mice demonstrates ubiquitous NKCC1 staining throughout the pre-optic area (POA) and staining of GnRH neurons (red), both cell soma and processes in WT mice (D1). E1-2) KCC2 staining (brown) was robust in the developing OB, ventral forebrain and areas around the cephalic flexure (CF). E2) Higher magnification of boxed area in E1 showing no KCC2 labeling in nasal areas (N) or at the nasal forebrain junction (J). E3) GnRH cells (green, arrows) at forebrain junction (J) are negative for KCC2 (red), while cells in the ventral forebrain (B) robustly stain for KCC2. F) GnRH cells (green) are KCC2 (red) negative in both pre-pubertal (F1) and adult (F2) mice, although robust KCC2 labeling is found throughout the brain. G) Although present in the median eminence (ME), KCC2 (red) did not co-localize with GnRH fibers (green). F) Double labeling was performed for GnRH neurons with anti-GFP and/or anti-cGnRH. Arrows indicate GnRH cells and large arrow indicate cells magnified on the right. Scale bars: A1 and B2 = 200 μM, E, F, and G = 100 μM; A3, B3, C and D = 10 μM. B = brain; T = tongue. N ≥ 3 at all stages.
Fig 2.
Anion exchanger 2 (AE2) is expressed in GnRH neurons throughout development.
GnRH cells (green) in prenatal (A, B) and pre-pubertal mice (C1-2) expressed AE2 (red). C3-4) Chromagen intensified double-labeling for AE2 (NiDAB, black) and GFP (ABC-AP, purple) confirmed that GnRH neurons express AE2 in pre-pubertal (C3) and adult (C4) mice. D) No AE2 (red) expression was detected on GnRH fibers (green) in the median eminence, whereas astrocytes (GFAP, green) in neighboring regions were AE2 positive (E). Scale bars: B, C and E = 10 μM; D = 100 μM.
Fig 3.
GnRH neurons retain depolarization to MUS in the absence of NKCC1.
A) As in vivo, GnRH cells (green) maintained in explants express NKCC1 (red), and AE2 (red). GnRH neurons were negative for KCC2, although a few non-GnRH neurons expressed KCC2 (lower panel, GnRH neuron fiber near KCC2 positive cell). B1) Example of GnRH neurons used for calcium imaging: bright field (BF), after loading with the calcium indicator (CaGreen), and after fixing and staining (GnRH). (B2) Examples of calcium imaging traces from NKCC1+/+ and NKCC1-/- GnRH neurons showed spontaneous activity in both genotypes (“Spontaneous Ca2+ transients”, B3). All GnRH neurons in both NKCC1+/+ (+/+) and NKCC1-/- (-/-) explants had calcium responses to MUS (“Ca2+ responses to MUS”, B3). Error bars = S.E.M. O.D. = optical density. Scale bars, A and B1 = 10 μM.
Fig 4.
GABAA activation elicits similar response in GnRH neurons from NKCC1-/- explants but is reduced by BUME.
A) Example of a gramicidin perforated patch clamp recording used to measure response to MUS. Trace from NKCC1-/- GnRH cell shows MUS depolarization is present before and after TTX. B) Peak amplitudes of MUS responses in GnRH neurons are similar in NKCC1+/+ (+/+, black dots) and NKCC1-/- (-/-, red dots) (B1) In contrast, BUME treatment significantly reduces the response to MUS in GnRH neurons in WT mice (B2). C) Reversal potential for GABA in GnRH neurons was evaluated by acute application of MUS (100 μM, arrowhead). All recordings were performed with CNQX/AP5 (10 μM each) and TTX (1 μM). Examples of recordings from GnRH neurons in untreated (WT) and bumetanide treated (+BUME) groups, and the voltage protocol used (C1). Reversal potentials were more negative when NKCC1 was inhibited by BUME (20 μM, red dots) than in control (WT, black dots) cells (*p<0.05, error bars = SEM) (C2).
Fig 5.
Inhibition of NKCC1 and removal of HCO3- attenuates response to MUS.
Recording periods consisted of: 1) baseline recording in ACSF, 2) application of CNQX/AP5 (10 μM each) followed by MUS (10 μM), and 3) second exposure to MUS (10 μM), followed by KCl (20mM). Groups compared were: A) Control B) BUME (NKCC1 inhibited), and C) HCO3- free ACSF with BUME. Due to general fluorescent rundown in ACSF, all responses were normalized to KCl stimulus at the end, and amplitude comparisons were made. D) Amplitude of the 1st calcium responses to MUS. E) Percentage of cells retaining a calcium response to the second application of MUS. O.D. = optical density.