GABA regulates synaptic integration of newly generated neurons in the adult brain

Abstract

Adult neurogenesis, the birth and integration of new neurons from adult neural stem cells, is a striking form of structural plasticity and highlights the regenerative capacity of the adult mammalian brain. Accumulating evidence suggests that neuronal activity regulates adult neurogenesis and that new neurons contribute to specific brain functions. The mechanism that regulates the integration of newly generated neurons into the pre-existing functional circuitry in the adult brain is unknown. Here we show that newborn granule cells in the dentate gyrus of the adult hippocampus are tonically activated by ambient GABA (gamma-aminobutyric acid) before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA, the major inhibitory neurotransmitter in the adult brain, initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic chloride ion content. Conversion of GABA-induced depolarization (excitation) into hyperpolarization (inhibition) in newborn neurons leads to marked defects in their synapse formation and dendritic development in vivo. Our study identifies an essential role for GABA in the synaptic integration of newly generated neurons in the adult brain, and suggests an unexpected mechanism for activity-dependent regulation of adult neurogenesis, in which newborn neurons may sense neuronal network activity through tonic and phasic GABA activation.

Figure 1

Development of newborn DGCs in the adult mice. a, Confocal images of new DGCs (GFP⁺, green) at different stages. Shown are projections (top) and confocal images of immunostaining (bottom) for doublecortin (DCX, red) and NeuN (blue) with orthogonal views to confirm the co-localization of GFP and DCX or NeuN. Scale bars: 20 μm. b–d, Synaptic integration of newborn DGCs. Shown are sample recording traces from GFP⁺ DGCs under whole-cell voltage-clamp (V_m = −65 mV). Tonic currents shown are continuous recordings before and after adding bicuculline (100 μM, blue). Evoked PSCs shown are averaged responses from 5 consecutive stimuli before (black) and after (blue) adding bicuculline (10 μM) or CNQX (50 μM), as indicated. Scale bars: 20 pA and 25 s (b); 10 pA and 50 ms (c); 10 pA and 20 ms (d).

Figure 2

Nature of GABA-induced activation in newborn DGCs in the adult brain. a, Resting membrane potentials (V_rest) and GABA-reversal potentials (E_GABA) of GFP⁺ DGCs. Values represent mean ± s.e.m. Numbers associated with symbols refer to the number of cells examined. b, Retrovirus mediated co-expression of GFP and shRNAs specific for NKCC1, but not a control shRNA (shRNA-DsRed), reduced NKCC1 expression and had no effects on KCC2 expression in newborn DGCs (7 dpi). Shown are confocal images of GFP (green) and immunostaining of NKCC1 or KCC2 (red), DCX (blue) and DAPI (gray), respectively. Arrows point to GFP⁺ DGCs. Scale bar: 20 μm. c, V_rest and E_GABA in shRNA-NKCC1⁺ newborn DGCs. Similar as in (a). d, Tonic GABA currents in newborn DGCs (7dpi) recorded under gramicidin perforated patch or break-in whole-cell recording (V_m = −65 mV). Blue lines indicate the addition of bicuculline (100 μM). Scale bars: 10 pA and 10 s. Values in the bar graph represent mean ± s.e.m. (n = 6, * p < 0.01, ANOVA).

Figure 3

Synaptic integration of newborn DGCs in the adult brain. a–c, Formation of GABAergic synaptic inputs by GFP⁺ DGCs. Shown in (a) are sample traces of evoked PSCs recorded under whole-cell voltage-clamp (V_m = −65 mV, 5 mM Kyn) before and after the addition of bicuculline (10 μM). Scale bars: 20 pA and 100 ms. Also shown are the percentages of GFP⁺ DGCs with detectable GABAergic PSCs, mean amplitude of GABAergic PSCs (b), mean frequency and peak amplitude of GABAergic SSCs recorded at 28 dpi (c). Numbers associated with symbols refer to the number of cells examined. Values represent mean ± s.e.m. (* p < 0.01, ANOVA). d–f, Formation of glutamatergic synaptic inputs by GFP⁺ DGCs. Same as in (a–c), except that the recordings were carried out in the presence of bicuculline (10 μM). Blue lines indicate the addition of CNQX (50 μM). Scale bars: 10 pA and 40 ms.

Figure 4

Dendritic development of newborn DGCs in the adult brain. a, Confocal three-dimensional reconstruction of dendrites of control or shRNA-NKCC1⁺ DGCs (14 dpi). Scale bar: 20 μm. b, Quantification of the total dendritic length and branch number of newborn DGCs. Each symbol represents data from a single control (empty) or shRNA-NKCC1⁺ (solid) DGC at 14 dpi. Dots along the X-axis represent mean values. (*: p < 0.01, Kolmogorov-Smirnov test). c, Sholl analysis of dendritic complexity of GFP⁺ DGCs (14 dpi). Values represent mean ± s.e.m. (n = 27; *: p < 0.05, Student’s t-test).