Activity-dependent regulation of inhibitory synapse development by Npas4

Abstract

Neuronal activity regulates the development and maturation of excitatory and inhibitory synapses in the mammalian brain. Several recent studies have identified signalling networks within neurons that control excitatory synapse development. However, less is known about the molecular mechanisms that regulate the activity-dependent development of GABA (gamma-aminobutyric acid)-releasing inhibitory synapses. Here we report the identification of a transcription factor, Npas4, that plays a role in the development of inhibitory synapses by regulating the expression of activity-dependent genes, which in turn control the number of GABA-releasing synapses that form on excitatory neurons. These findings demonstrate that the activity-dependent gene program regulates inhibitory synapse development, and suggest a new role for this program in controlling the homeostatic balance between synaptic excitation and inhibition.

Figure 1. Npas4 expression is regulated by neuronal activity in vitro and in vivo

a, Immunostaining showing Npas4 protein is induced in rat hippocampal neurons (7 DIV) by depolarization (50 mM KCl, 2 h, right). p-CREB, CREB phosphorylated at Ser 133. b, Western blot showing Npas4 is selectively induced by membrane depolarization (50 mM KCl, 7 DIV rat hippocampal neurons), but not by BDNF (50 ng ml⁻¹), NT3 (50 ng ml⁻¹), NT4 (50 ng ml⁻¹), forskolin (10 μM), NGF (100 ng ml⁻¹), EGF (100 ng ml⁻¹), PDGF (100 ng ml⁻¹), CNTF (100 ng ml⁻¹) or IGF-1 (100 ng ml⁻¹). Induction is prevented by pretreatment with the Ca²⁺ chelator EGTA (5 mM, 10 min). c, Western blot showing Npas4 (7 DIV rat hippocampal neurons) is transiently induced by membrane depolarization (50 mM KCl, 30 min). d, Basal Npas4 expression increases as neurons mature, presumably because of increased endogenous spontaneous activity: compare immunostaining of 7 and 14 DIV rat hippocampal neurons. e, Western blot showing stimulation of primary hippocampal neurons (14 DIV) with bicuculline (50 μM, 2 h) increases Npas4 expression levels. This is prevented by pretreatment with nimodipine (5 μM, 1 h) or EGTA (5 μM, 5 min) and reduced by pretreatment (1 h) with antagonists to NMDA receptors (100 μM AP5) or AMPA receptors (50 μM CNQX). f, g, Mice dark-reared for one week (P21–P28) and then stimulated with strobe lights have greater Npas4 expression levels in the visual cortex than their dark-reared littermates, but there is no difference in the hippocampus. Light stimulation was applied for 2 h for immunocytochemistry analysis (f) or 1 h for Npas4 mRNA quantification (g). Significance was determined using a one-tailed paired t-test, *P < 0.05. Data are shown as mean ± s.e.m.

Figure 2. Npas4 regulates the number of GABAergic synapses in cultured hippocampal neurons

a, Npas4-RNAi, but not control-RNAi, reduces the expression of Npas4 in primary hippocampal neurons. Cultures were transfected at 6 DIV and stimulated with bicuculline (50 μM, 2 h) at 14 DIV. b, The number of GABAergic synapses is significantly reduced by Npas4-RNAi, as illustrated by two representative rat hippocampal neurons. Cultures were co-transfected (6 DIV) with GFP and either Npas4-RNAi (top) or control-RNAi (bottom). Cultures were subsequently immunostained (25 DIV) with antibodies against GAD65 (blue) and GABA_A-γ2 (red). c, Quantification of the normalized density of co-localized GABA_A-γ2 and GAD65 puncta in 14 DIV rat hippocampal neurons transfected with vector control, Npas4-RNAi or control-RNAi. d, Separate quantification of perisomatic and dendritic GABA_A-γ2 and GAD65 puncta measured in c. e, Npas4-minigene increases the density of co-localized GABA_A-γ2 and GAD65 puncta. f, Separate quantification of perisomatic and dendritic GABA_A-γ2 and GAD65 puncta measured in e. See Methods for details of data normalization and error propagation. Significance was determined using multifactorial analysis of variance. *P < 0.05; **P < 0.005; ***P < 0.0005. Data are presented as mean ± s.e.m. from three(c,d)or four (e,f)independent experiments; total numbers of neurons analysed (n) are indicated.

Figure 3. Npas4 regulates GABAergic synapse development in organotypic hippocampal slices

a, Representative mIPSCs recorded from CA1 pyramidal neurons in organotypic hippocampal slices biolistically co-transfected with GFP and either vector control, Npas4-RNAi or Npas4-minigene. b, Cumulative distributions of mIPSC inter-event intervals and amplitudes recorded from neurons transfected with vector control, Npas4-RNAi or Npas4-minigene. c, Mean ± s.e.m. of data from b. mIPSC inter-event intervals: 2986.3 ± 105.7, 3803.0 ± 136.9 and 1776.9 ± 75.1 ms; amplitudes: 31.5 ± 1.1, 25.7 ± 0.8 and 34.1 ± 1.3 pA; for vector control, Npas4-RNAi and Npas4-minigene, respectively. d, Cumulative distributions of mIPSC inter-event intervals and amplitudes recorded from Npas4^flx/flx neurons co-transfected with GFP and either vector control or Cre recombinase. e, Mean ± s.e.m. of data from d. mIPSC inter-event intervals: 684.2 ± 31.2 and 917.4 ± 37.7 ms; amplitudes: 27.5 ± 0.7 and 27.9 ± 0.6 pA; for vector control and Cre, respectively. Total numbers of neurons analysed in each condition (n) are indicated in c and e. *P < 0.05; ***P < 0.001.

Figure 4. Npas4 has no effect on excitatory synaptogenesis but affects excitatory/inhibitory balance in neural circuits

a, The number of excitatory synapses is not affected by Npas4-RNAi. Quantification of the normalized density of co-localized PSD95 and synapsin1 puncta in 14 DIV rat hippocampal neurons transfected with vector control, Npas4-RNAi or control-RNAi. b, Separate quantification of PSD95 and synapsin1 puncta measured in a. c, Npas4-minigene has no effect on the density of excitatory synapses. Quantification of co-localized synapsin1 and PSD95 puncta is shown. d, Separate quantification of PSD95 and synapsin1 puncta measured in c. In a–d, data are presented as mean ± s.e.m. e, Cumulative distribution of mEPSC inter-event intervals and amplitudes recorded from Npas4^flx/flx neurons co-transfected with GFP and either vector control or Cre. f, Mean ± s.e.m. of data from e. mEPSC inter-event intervals: 2581.4 ± 104.1 ms and 2140.5 ± 79.7 ms; amplitudes: 12.1 ± 0.4 and 12.4 ± 0.3 pA; for vector control and Cre, respectively. g, Cumulative distribution of mEPSC inter-event intervals and amplitudes recorded from neurons transfected with GFP and either vector control or Npas4-minigene. h, Mean ± s.e.m. of data from g. mEPSC inter-event intervals: 2686.4 ± 89.3 and 3320.7 ± 117.2 ms; amplitudes: 14.3 ± 0.3 and 12.0 ± 0.3 pA; for vector control and Npas4-minigene, respectively. Total numbers of neurons analysed in each condition (n) are indicated. a, b, Three independent experiments; c, d, four independent experiments. *P < 0.05; ***P < 0.001.

Figure 5. Npas4 controls a program of gene expression that regulates GABAergic synapses

a, Hierarchical clustering of 327 probe sets (270 putative Npas4 target genes) based on their expression profiles using dChip. The expression level of each probe set is normalized to a mean of 0 and a standard deviation of 1. Expression values are displayed within the range [−3, 3] with levels above, equal to or below the mean displayed in red, white and blue, respectively. Dark red represents 3 or higher, and dark blue −3 or lower. b, Biological functions of 270 putative Npas4 target genes based on Gene Ontology information provided by Affymetrix (http://www.affymetrix.com). c, BDNF expression is reduced by Npas4-RNAi (1.9 ± 0.11-fold reduction, P < 0.01, one-tailed paired t-test). Mean ± s.e.m. from three independent experiments is shown, and each data point was generated by averaging the hybridization intensity of two BDNF probe sets (1422168_a_at and 1422169_a_at). d, BDNF levels are consistently reduced in neurons from Npas4^−/− mice compared with their wild-type littermates (58.46 ± 6.49% decrease, 95% confidence interval 30.5–86.4%). Cortical cultures prepared from Npas4^+/+ and Npas4^−/− littermates (7 DIV) were stimulated with KCl (55 mM), and BDNF mRNA levels were measured by quantitative reverse transcriptase PCR using primers in BDNF coding region. Three littermate pairs from three different litters were analysed; data (mean ± s.e.m.) from one representative pair is shown. e, Npas4 interacts directly with BDNF promoters I and IV in an activity-dependent manner, as shown by chromatin immunoprecipitation. The Npas4 promoter is used as a positive control. Data are normalized to a control region on chromosome 3 and are presented as mean ± s.e.m. from five independent experiments.

Figure 6. Knockdown of BDNF partially attenuates the ability of the Npas4-minigene to elevate GABAergic synapses

a, Cumulative distributions (left) and mean ± s.e.m. (right) of mIPSC inter-event intervals in neurons transfected with BDNF-RNAi (4086.1 ±140.7 ms) or BDNF-RNAi 1Npas4-minigene (3257.9 ± 117.7 ms). b, Cumulative distributions (left) and mean ± s.e.m. (30.1 ± 0.9 and 27.1 ± 0.7 pA, BDNF-RNAi versus BDNF-RNAi 1Npas4-minigene, right) of mIPSC amplitudes. Total numbers of neurons analysed in each condition (n) are indicated. ***P < 0.001.