Early establishment of chloride homeostasis in CRH neurons is altered by prenatal stress leading to fetal HPA axis dysregulation

Abstract

Corticotropin-releasing hormone (CRH) neurons play an important role in the regulation of neuroendocrine responses to stress. The excitability of CRH neurons is regulated by inhibitory GABAergic inputs. However, it is unclear when GABAergic regulation of CRH neurons is established during fetal brain development. Furthermore, the exact progression of the developmental shift of GABA action from depolarization to hyperpolarization remains unelucidated. Considering the importance of CRH neuron function in subsequent hypothalamic-pituitary-adrenal (HPA) axis regulation during this critical phase of development, we investigated the ontogeny of GABAergic inputs to CRH neurons and consequent development of chloride homeostasis. Both CRH neuron soma in the paraventricular nucleus (PVN) and axons projecting to the median eminence could be identified at embryonic day 15 (E15). Using acute slices containing the PVN of CRF-VenusΔNeo mice, gramicidin perforated-patch clamp-recordings of CRH neurons at E15, postnatal day 0 (P0), and P7 were performed to evaluate the developmental shift of GABA action. The equilibrium potential of GABA (E_GABA) was similar between E15 and P0 and showed a further hyperpolarizing shift between P0 and P7 that was comparable to E_GABA values in adult CRH neurons. GABA primarily acted as an inhibitory signal at E15 and KCC2 expression was detected in CRH neurons at this age. Activation of the HPA axis has been proposed as the primary mechanism through which prenatal maternal stress shapes fetal development and subsequent long-term disease risk. We therefore examined the impact of maternal food restriction stress on the development of chloride homeostasis in CRH neurons. We observed a depolarization shift of E_GABA in CRH neurons of pups exposed to maternal food restriction stress. These results suggest that Cl^- homeostasis in early developmental CRH neurons attains mature intracellular Cl^- levels, GABA acts primarily as inhibitory, and CRH neurons mature and function early compared with neurons in other brain regions, such as the cortex and hippocampus. Maternal food restriction stress alters chloride homeostasis in CRH neurons of pups, reducing their inhibitory control by GABA. This may contribute to increased CRH neuron activity and cause activation of the HPA axis in pups.

Keywords: CRH neuron; GABA; HPA axis; KCC2; chloride homeostasis; prenatal stress.

FIGURE 1

Developmental change of E_GABA in CRH neurons. Gramicidin-perforated patch-clamp recordings of CRH neurons from E15–E17, P0–P2, and P7–P9 mice. (A) A representative 100 μM GABA-evoked current trace at –70 mV holding potential (upper panel). The duration of GABA application is indicated with horizontal bars. Two 1-sec voltage ramps from –90 to –10 mV were applied before and during 5-sec puff application of GABA; sample I-V curves before (black) and after GABA application (red) (lower panel). E_GABA was estimated from the voltage axis intercept (detailed further in “Materials and methods”). (B) E_GABA of CRH neurons at E15–E17, P0–P2, and P7–P9 (*p < 0.05 by Kruskal–Wallis test; E15–E17: n = 8, P0–P2: n = 11, P7–P9: n = 11; closed circle indicates single cells). The lines in each box depict the lower quartile, median, and upper quartile values. The whiskers extending from each end of a box depict the minimum and maximum ranges. (C) sIPSCs recorded from E15 CRH neurons after the neuronal membrane was ruptured during perforated-patch clamp-recording. Scale bar as shown in the figure inset.

FIGURE 2

GABAergic inhibitory regulation of CRH neurons is established during early development. (A) Representative current-clamp recordings from P0–P2 CRH neurons in gramicidin-perforated patch mode showing the effect of 100 μM GABA on membrane potential. Five-second application of GABA hyperpolarized (upper panel) or depolarized (lower panel) membrane potential. (B) Application of GABA blocked spontaneous action potentials in P0–P2 CRH neurons (n = 2). (C) Summary of the proportions of each response type (depolarization or hyperpolarization) recorded upon GABA application in P0–P2 CRH neurons (n = 14). (D) The RMP of CRH neurons at P0–P2 (n = 13). Error bars represent SEM. Scale bar as shown in the figure inset.

FIGURE 3

KCC2 is expressed in the somata of CRH neurons in PVN of E15–E17 mice. (A) Representative images of PVN from WT E15 mice immunostained for CRH (green) and KCC2 (red). III, third ventricle. White dotted line indicates the PVN with cell bodies of CRH neurons. (B) Representative images of ME from WT E15 mice immunostained for CRH (green) and KCC2 (red). III, third ventricle. White arrowheads indicate CRH neuron terminals in the ME. Scale bars: 50 μm.

FIGURE 4

Maternal food restriction stress causes a depolarizing shift in E_GABA of CRH neurons in pups. (A) Body weights of control and FR mothers. The lines in each box depict the lower quartile, median, and upper quartile values. The whiskers extending from each end of a box depict the minimum and maximum ranges (*p < 0.05, **p < 0.01 by Mann–Whitney U test; n = 8 in each group). (B) Body weights of pups from control or FR mothers (ns, not significant; control: n = 12, FR: n = 12; closed circle indicates single cells). (C) Serum corticosterone level of control and FR mothers (**p < 0.01 by unpaired t-test, n = 8 in each group; closed circle indicates single cells). Error bars represent SEM. (D) Fetal corticosterone level of control and FR mothers (**p < 0.01 by unpaired t-test; control: n = 12, FR: n = 12; closed circle indicates single cells). Error bars represent SEM. (E) Representative 100 μM GABA-evoked current traces at –70 mV holding potential in CRH neurons of P0–P2 pups from FR mothers. Currents were recorded under gramicidin-perforated voltage-clamp condition. The duration of GABA application is indicated with horizontal bars. (F) Two 1-sec voltage ramps from –90 to –10 mV were applied before and during 5-sec puff application of 100 μM GABA; sample I-V curves before (black) and after GABA application (red). E_GABA was estimated from the voltage axis intercept (detailed further in “Materials and methods”). (G) Plot of E_GABA of CRH neurons from pups of control or FR mothers (**p < 0.01 by unpaired t-test; control: n = 11, FR: n = 15; closed circle indicates single cells). Error bars represent SEM.