PMC

(A) Expression of Npas4 in CA3 of Npas4^-/- mice using HSV-Npas4.
(B) Npas4, but not ΔNpas4, restores expression of c-Fos in vivo.
(C) Expression of Npas4, but not ΔNpas4, drives activity of PI_BDNF independent of KCl depolarization. n = 4 cultures. *p ≤ 0.001.
(D) Npas4^-/- mice with Npas4 injected into CA3 freeze at similar levels to Npas4^+/+ mice injected with GFP 1hr and 24hrs after training. CA1 injection of Npas4 or CA3 injection of ΔNpas4 did not reverse the memory deficit. *p < 0.001.

(A,B) Npas4^-/- and Npas4^+/+ littermates exhibit similar freezing during the training session (A) and 5 min after training (B). p = 0.879.
(C,D) 1h (C) and 24h (D) after CFC Npas4^-/- mice freeze at a significantly lower level than Npas4^+/+ littermates. *p ≤ 0.001.
(E) 24h after auditory delay conditioning, Npas4^-/- and Npas4^+/+ mice exhibit similar freezing during a tone memory test. p = 0.859.

(A) Localization of Pol II to PI_BDNF and c-Fos enhancer region E2 is dependent on Npas4. No change is observed in Pol II binding at the c-Fos or β-actin promoter.
(B) Depolarization-induced activity of PI_BDNF reporter is abolished in the absence of Npas4, but the c-Fos promoter reporter is unaffected. n = 4 cultures. *p < 0.001.
(C) qPCR analysis of ChIP samples from seized Npas4^-/- and Npas4^+/+ littermates. Pol II binding to PI_BDNF (*p ≤ 0.008, n=7/genotype) and c-Fos E2 (*p ≤ 0.048, n=6/genotype) is diminished in Npas4^-/- mice relative to Npas4^+/+ littermates. No change is observed in Pol II binding at the c-Fos promoter (p = 0.333, n = 6/genotype).

(A) Upper panel: Npas4 expression is increased only in CA3 and to a lesser extent in dentate gyrus after CFC. Lower panel: c-Fos expression is induced in all subregions of DH after CFC. Right hand column: seizure induces Npas4 and c-Fos in all subregions of hippocampus.
(B) Western blot analysis of Npas4 and c-Fos expression in DH at various times after CFC. n = 5 mice/condition. Values are plotted relative to peak timepoint. *p < 0.04.
(C) Selective deletion of Npas4 in CA3 or CA1 three days after injecting HSV-Cre.
(D) Selective deletion of Npas4 in CA3 impairs long term CFC memory formation. *p < 0.001.

(A) Conditional deletion of Npas4 in CA3 results in loss of c-Fos expression.
(B) Dramatic loss of depolarization-induced IEG expression by acute deletion of Npas4 in cultured mouse neurons. n = 3 cultures. *p < 0.001.
(C) The activity of PI_BDNF reporter is abolished in the absence of Npas4, while P_Npas4, CRE, and MRE reporters show similar induction in the presence or absence of Npas4. n = 4 cultures. *p < 0.001.
(D) ChIP experiments showing that under depolarized conditions Npas4 binds to PI_BDNF and enhancer II of c-Fos (c-Fos E2). No binding is observed at the c-Fos promoter, the β-actin promoter, or a negative control region.

(A, B) Npas4 mRNA (A) and protein (B) in cultured hippocampal neurons is selectively induced by membrane depolarization and dependent on Ca²⁺ influx. Induction of Npas4 mRNA does not require new protein synthesis. n = 4 cultures.
(C) Npas4 is rapidly induced after contextual fear conditioning (CFC). Separate groups of mice were sacrificed 5min (n = 8), 30min (n = 9-11), 1hr (n = 6), or 4.5hr (n = 5) after CFC and compared to naive home cage mice (HC, n = 10). Values are plotted relative to peak timepoint.
(D) Experimental scheme and behavioral outcomes of CFC and control conditions. C+S: context + shock; C: context exposure; S: immediate shock; HC: home cage.
(E) Npas4 mRNA is selectively induced by context learning. C+S: n = 8-10; C: n = 8; S: n = 8 and HC: n = 10. All groups were sacrificed 30min after training. *p < 0.001.