Figure 1. Depolarization-Induced Expression of Npas4 in Primary Neuron Cultures. Primary cultured embryonic mouse cortical neurons after 8 DIV were silenced by blocking action potentials with TTX or mildly depolarized with KCl for 45 minutes. Mild depolarization engaged Ca2+-mediated signaling mechanisms to induce Npas4 transcription and protein expression. Neuronal cell bodies and dendrites are labelled with an anti-Map2 Ab from Sigma (HM-2) and Npas4 was labelled with the Activity Signaling Npas4 Ab.

Figure 2. Npas4 Ab Specificity Demonstrated with Npas4 KO Mice. We used WT and Npas4 KO mice to validate specificity. Npas4 was originally ‘discovered’ by Mehrdad Shamloo due to large changes in expression under ischemic conditions. Indeed, photothrombotic shock increased Npas4 immunoreactivity two hours after treatment only on the affected ipsilateral cingulate cortex. Western blots show that there is a dramatic increase in Npas4 immunoreactivity on the ipsilateral (R) side. In Npas4 KO animals there is no trace of a signal demonstrating the specificity of the antibody. (Courtesy of the Shamloo Lab, Stanford University)

Figure 3. NPAS4 expression in the mouse cortex.
Npas4 expression with and without behavioral training: Immunohistochemical staining (A) and Western blots analysis (B). Western blot analysis of Npas4 expression following cortical injection with sgRNA targeting Npas4 (C). (Courtesy of Chen Lab, University of Ottawa)

Figure 4. iDISCO Whole Intact Brain 3D Immunostaining. Mice were either left in their home cage or underwent a contextual fear conditioning task (top). After 1 hour mice were perfused and whole brains went through a modified iDISCO tissue clearing protocol. Intact whole brain 3D imaging of Npas4 immunoreactivity was conducted using the Translucence Mesoscale Imaging System and the ZEISS Lightsheet Z.1 microscope. (Courtesy of Ricardo Azevedo, Translucence Biosystems)

Figure 5. Depolarization of primary mouse cortical neurons induces NPAS4 expression. Immunostaining of NPAS4 and β-III-tubulin (Tuj1) in primary mouse cortical neurons (DIV12) before and after treatment with 20 mM KCl for 45 minutes. Neurons are labeled with β-III-tubulin (β3-Tubulin (TU-20) CST) and NPAS4 (Activity Signaling). (Courtesy of Katharina Meyer and King-Hwa Ling - Harvard Medical School, Boston)

Figure 6. Antibody Validation in Dissociated Mouse Hippocampal Cultures. Activity of neuronal networks was driven by blocking inhibitory synaptic transmission with the GABAA receptor antagonist Bicuculline. A) As expected, no immunoreactivity is seen with the anti-Npas4 Ab in unstimulated cultures, whereas after driving excitatory synaptic activity a strong signal can be seen. B) In unstimulated control cultures, no Npas4 immunoreactivity is present, however, overexpression of mouse Npas4 after AAV infection results in a prominent Npas4 band.  (Courtesy of Anna Hertle, David Brito and Ana Oliveira, Institute for Neurobiology, Heidelberg University)

Figure 5. Immunostaining of Nuclei in Histological Sections of Mouse Primary Visual Cortex after Insect Hunting. Mice were allowed to hunt insects for 10 minutes in an open field. After resting in the home cage in the dark for 1 hour, mice were anesthetized and transcardially perfused. Brains were then dropped into 2% PFA overnight at 4°C, sectioned to 30μm and then immunostained. Sequential sections from one such mouse is shown above with Npas4 staining results shown top left and C-Fos staining shown top right. A DAPI nuclear stain for each section is shown below. There are consistently more C-Fos(+) than Npas4(+) nuclei in the primary visual cortex of mice with insect hunting experience. (Courtesy of  Jennifer Hoy - University of Nevada, Reno)

Figure 7. NPAS4 Antibody validation with Western blot in Primary Hippocampal Culture. The expression of NPAS4 protein was assessed in the 14-day matured cultures with the activity signaling NPAS4 Ab. Cells were incubated with a specific agent that was predicted to cause less or no expression of the NPAS4 gene as compared to the untreated culture. As observed, protein expression could not be detected in treated cells. A 1:1000 dilution was used. (Courtesy of Laube Lab, Technical University of Darmstadt)

Figure 8. Stress-Induced Expression of Npas4 In Adult Mice. Mice were subjected to the forced swim test for 10 minutes and sacrificed with CO2 exposure after 90 minutes. Conversely, control mice were left in their home cage. Brains were then fixed in 4% PFA for 48 hours and sectioned to 50 μm. Acute stress exposure induced Npas4 expression in different brain areas, as shown in the amygdala, the cortex and the hippocampus. (Greta Visintin, Giovanni Morelli and Laura Cancedda, Istituto Italiano di Tecnologia, Genoa)